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Beauty, A Social Construct: The Curious Case of Cosmetic Surgeries | Juniper Publishers

Juniper Publishers-Open Access Journal of Dermatology & Cosmetics

Authored by Vandana Roy

Abstract

In this article we deconstruct the social norm of beauty and cosmetic beauty treatment, an issue that is seldom discussed in medical circles and is often lost to popular rhetoric. In doing so, we also reflect on the institutionalized system of social conditioning.

A Historical Perspective

Cosmetic surgery, as with reconstructive surgery, has its roots in plastic surgery (emerging from the Greek word ‘plastikos’, meaning to mold or form). The practice of surgically enhancing or restoring parts of the body goes back more than 4000 years. The oldest accounts of rudimentary surgical procedures is found in Egypt in the third millennia BCE. Ancient Indian texts of 500 BCE outline procedures for amputation and reconstruction. The rise of the Greek city-states and spread of the Roman Empire is also believed to have led to increasingly sophisticated surgical practices. Throughout the early Middle Ages as well, the practice of facial reconstruction continued. The fifth century witnessed a rise of barbarian tribes and Christianity and the fall of Rome. This prevented further developments in surgical techniques. However, medicine benefited from scientific advancement during the Renaissance, resulting in a higher success rate for surgeries. Reconstructive surgery experienced another period of decline during the 17th century but was soon revived in the 18th century. Nineteenth century provided impetus to medical progress and a wider variety of complex procedures. This included the first recorded instances of aesthetic nose reconstruction and breast augmentation. Advancements continued in the 20th century and poured into present developments of the 21st century.

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Desires and Demands in Contemporary Times

In recent years, the volume of individuals seeking cosmetic procedures has increased tremendously. In 2015, 21 million surgical and nonsurgical cosmetic procedures were performed worldwide. In the United Kingdom specifically, there has been a 300% rise in cosmetic procedures since 2002. The year 2016 witnessed a surge in the number of such treatments with the United States crossing four million operations. Presently, the top five countries in which the most surgical and nonsurgical procedures are performed are the United States, Brazil, South Korea, India, and Mexico. Such demand can be viewed from different perspectives. At one end it is a product of scientific progress, growing awareness, economic capacities and easier access and on the other, something on the lines of a self-inflicted pathology. This article dwells on the latter and attempts to address a deep-rooted problem of the social mind.

Lessons from History

History is witness to a number of unhealthy fashion trends, many of which today appear extremely irrational and even cruel. Interestingly, the common thread connecting all of them is the reinforcement of social norms and stereotypes. Forms of socialization which lie at the intersection of race, class and gender-based prejudices. To elaborate, hobble skirts and chopines restricted women’s movement and increased their dependence on others. Corsets deformed body structures, damaged organs and led to breathing problems. The Chinese practice of binding women’s feet to limit physical labor was regarded as a sign of wealthiness. Dyed crinolines and 17th century hairstyles made people vulnerable to poisoning and fire related injuries. Usage of makeup made of lead and arsenic, eating chalk and ‘blot letting’, reflected a blatantly racist obsession with white and pale skin. Lower classes faked gingivitis to ape tooth decays of the more privileged who had access to sugar. Furthermore, other practices like tooth lacquering, radium hair colors, mercury ridden hats, usage of belladonna to dilate pupils and even men wearing stiff high collars, all furthered societal expectations and notions of class superiority. Till the 1920’s, there was rampant usage of side lacers to compress women’s curves. Even today many ethnic tribes continue with practices which inflict bodily deformations. In the urban context as well, trends like high heels, skinny jeans and using excessive makeup dominate the fashion discourse. Cosmetic procedures are the latest addition to the kitty.

The Social Dilemma

What is it that leads the ‘intelligent human’ of today to succumb to archaic and regressive notions of beauty? What motivates them to risk aspects of their lives to cater to selflimiting rules of ‘acceptance’? The surprising part is that this anomaly is often placed in the illusory realm of ‘informed consent’. In common parlance, ‘to consent’ implies voluntary agreement to another’s proposal. The word ‘voluntary’ implies ‘doing, giving, or acting of one’s own free will.’ However, when the entire socio-cultural set up and individual attitudes validate certain behaviors, there is very less space left for an alternate narrative. Let alone free will.

Pierre Bourdieu once argued that nearly all aspects of taste reflect social class. Since time immemorial, societal standards of beauty have provided stepping stones to social ascent and class mobility. Better ‘looking’ individuals are considered to be healthier, skillfully intellectual and economically accomplished in their lives. Such an understanding stems from well entrenched stereotypes in complete disregard of individual merit and fundamental freedoms. An inferiority complex coupled with external pressures and self imposed demands, subconsciously coerce individuals into a vicious cycle of desire or rejection. Active and aggressive media has played a key role in forming societal perceptions of what is attractive and desirable. In addition, lifestyle changes reflect an image obsessed culture, reeking of deep-rooted insecurities. At the root of a submissive and conformist attitude lies a subconscious mind lacking selfesteem and self-worth. People continue to look for remedies in the wrong places. The only difference is that corsets and blot letting have given way to surgeries and cosmetic products. The biggest question is, how have ideas otherwise seen as deviant, problematic and inadequate retained control over minds of millions of individuals?

A Gendered Culture

‘Beauty’ is understood as a process of ongoing work and maintenance, its ‘need’ unfairly tilted towards the fairer sex. History has demonstrated the impact of dangerous beautification practices on women. Contemporary ideals aren’t far from reaching similar outcomes. Today, there is a powerful drive to conform to the p*rnographic ideal of what women should look like. There has been a growth in the number of adolescents who take to cosmetic surgeries to become more ‘perfect’. In many countries, the growth of the “mommy job” has provoked medical and cultural controversies. Presumably there is an underlying dissatisfaction which surgery does not solve. Furthermore, where does the disability dimension fit in here? What happens to the ‘abnormal’ when the new ‘normal’ itself is skewed? For those with dwarfism and related disorders, new norms become even more burdensome.

The massive pressure to live up to some ideal standard of beauty, particularly for women, reeks of patriarchal remnants of a male dominated society. This kind of conformity further nurtures objectification and sexualization, reducing women to the level of ‘chattel’ to feed the male gaze. There is a also a power struggle at play where biased standards help maintain the unequal status quo. Today, there is idolization of celebrities, beauty pageants and advertisem*nts by cosmetic companies over sane medical advice. They set parameters of size, color and texture to be followed by the world at large. Moreover, people who deviate from such norms are made to feel stigmatized or ostracized from social spheres. The existence of male-supremacist, ageist, hetero sexist, racist, class-biased and to some extent, eugenicist standards reflect a failure of society as a whole. It is thus high time that we revisit and deconstruct skewed standards of beauty.

Mind Over Matter: Psychological Dimensions

Culturally imposed ideals create immense pressure of conformity. Consequently, they have been successful in engendering insecurities via their influence on perception of self and body image. Such perceptions often become distorted and discordant with reality, leading to serious psychological disorders. One such disorder is the body dysmorphic disorder (BDD). This is a psychiatric disorder characterized by preoccupation with an imagined defect in physical appearance or a distorted perception of one’s body image. It also has aspects of obsessive-compulsiveness including repetitive behaviors and referential thinking. Such preoccupation with self-image may lead to clinically significant distress or impairment in social and occupational functioning. With reference to cosmetic surgeries, patients with BDD often possess unrealistic expectations about the aesthetic outcomes of these surgeries and expect them to be a solution to their low self-confidence. Many medical practitioners who perform cosmetic surgery believe themselves to be contributing towards construction of individual identity as well. The notion that beauty treatments can act in much the same way as psychoanalysis has led countries like Brazil to open its gate of cosmetic procedures to lower income groups. This happens while the country continues its battles with diseases like tuberculosis and dengue. The philosophy behind such ‘philanthropy’ is that ‘beauty is a right’ and thus should be accessible to all social groups. While on one hand we may applaud such efforts of creating a more ‘egalitarian’ social order, on the other hand it is hard to overlook the self-evident undercurrents of social prejudice and capitalistic propaganda.

Medicalization of Beauty

Traditional notions of beauty embody a kind of hierarchy and repression which alienate individual agency and renders them as powerless victims. Such is the societal pressure which normalizes cosmetic procedures and subverts serious health effects. These include adverse effects due to cosmetic fillers like skin necrosis, ecchymosis, granuloma formation, irreversible blindness, anaphylaxis among others. Other dangers like heightened susceptibility to cancer and increased suicide rates. However, patients are often unaware of the risks which are hidden behind a veil of expectations and reassurances. Furthermore, quackery and inadequate standards such as lack of infection control also compound the problems of this under regulated field.

Role of Stakeholders

At the heart of any successful social transformation lies the power of united will and collective action. Thus, the consolidated and sustained effort by all stakeholders is the key to realizing an ecosystem conducive to tackle negative social norms. At the outset, government regulation is needed with respect to cosmetic procedures and the cosmetics industry. These regulations should encompass all private and public avenues and should also work against misleading advertising. Spreading awareness is the key to a better informed society. The state should fund and run specialized awareness sessions pertaining to psychological problems and aid mechanisms, gender sensitization as well as those aiming at spiritual and introspective personal development of individuals. NGO’s, medical professionals, academicians and members of the civil society, must come together to eradicate forms of social discrimination which undermine social institutions and individual agency around the world. This would help facilitate discussion, data collection, coalition building, and action that may eventually lead to behavioral changes.

Aesthetic surgery today seems to be passing through an ethical dilemma and an identity crisis. And rightly so for it strives to profit from an ideology that serves only vanity, bereft of real values. Nevertheless, there are exceptional cases where medical-aesthetic inputs have been vital in restoring morale by subverting stigmatization.

The Way Ahead

Beauty is unfair. The ‘attractive’ enjoy powers gained without merit. The perfectionist in humans seeks outward validation of external beauty over inner virtues. Scientific progress and an increase in human expertise to manipulate natural phenomena has paved the way for these desires to become a reality. There is no denying that advances in plastic and reconstructive surgery have revolutionized the treatment of patients suffering from disfiguring congenital abnormalities, burns and skin cancers. However, the increased demand for aesthetic surgery falls short of a collective psychopathology obsessed with appearance. This article expresses trepidation about such forms of social consciousness that first generates dissatisfaction and anxiety and then provides surgery as the solution to a cultural problem.

We have to work towards a social order which embraces people as they are and facilitates free choice, individual liberty and informed decision making. This is particularly pertinent when these decisions work towards framing cultural perceptions and expectations for millions around the world. We should open our hearts to diversification of beauty and aesthetic. Let our entertainment, fashion, capital and media revolve around heterogeneity of ideologies and cultures. In the words of Eleanor Roosevelt, “No one can make you feel inferior without your consent”. So, let us all come together and create a better society. A society, where principles of justice, equity, good conscience and humanity override primitive and archaic ideologies of naive men. A society where individual will be truly free and, discourse a product of informed thought.

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The Effects of Anti-Fibrosis Drugs on the Self-Regulation of Alveolar Macrophages and Interstitial Macrophages in the Pathological Process of Pulmonary Fibrosis | Juniper Publishers

Juniper Publishers-Open Access Journal of Gastroenterology & Hepatology

Authored by Huaqiang Zhai

Abstract

Pulmonary fibrosis is a devastating condition with many kinds of inducements. Alveolar macrophages (AMs) and interstitial macrophages (IMs) are two subsets of macrophages which involve in the inflammatory reaction of pulmonary fibrosis. Traditional Chinese medicine has good effects on the treatment of pulmonary fibrosis. This review is aimed at summarizing the different roles of alveolar macrophages and interstitial macrophages in the pathological process of pulmonary fibrosis progression and anti-fibrosis drugs related to the self-regulation mechanism of these two kinds of cells, furthermore to explain the multiple mechanisms of traditional Chinese medicine for treating pulmonary fibrosis from the self-regulation of macrophages. Searches for keywords “alveolar macrophages”, “interstitial macrophages”, “pulmonary fibrosis” and “IPF” were performed in biomedical databases. The reported incidences of AMs and IMs in the pathological process of pulmonary fibrosis are involved in this article, and the influences of traditional Chinese medicine on the cells are summarized.

A comprehensive analysis of the literature confirms that alveolar macrophages appear to be better equipped for their antimicrobial task, whereas interstitial macrophages have more capacity for immune regulatory functions. Disease-modifying therapy for pulmonary fibrosis based on intervening the macrophages self-regulation relates to chemokines and cytokines. Traditional Chinese herbs for pulmonary fibrosis have different targets on the two kinds of cells. Alveolar macrophages and interstitial macrophages could be used to explore the pathological process and clinical medicine of pulmonary fibrosis, and they are conducive to interpret the multiple mechanisms of TCM for pulmonary fibrosis.

Keywords: Alveolar macrophages; Interstitial macrophages; Pulmonary fibrosis; Chemokines; Cytokines; Traditional Chinese medicine

Abbrevations: AMs: Alveolar Macrophages; IMs: Interstitial Macrophages; IPF: Idiopathic Pulmonary Fibrosis; TCM: Traditional Chinese Medicine; BALF: Bronchoalveolar Lavage Fluid; PDGF: Platelet-Derived Growth Factor; TNF-α: Tumor Necrosis Factor-Α; IFN: Interferon; TGF-β1: Transforming Growth Factor-Β1; CTGF: Connective Tissue Growth Factor

Introduction

Pulmonary fibrosis is a kind of chronic progressive interstitial lung disease. Idiopathic pulmonary fibrosis (IPF) is a common and devastating pulmonary fibrosis with no powerful drugs. Data showed the incidence of IPF seems to be increasing rates of 3-9 cases per 100000 a year, although varies worldwide [1]. The median survival was 2.5 to 3.5 years after the diagnosis of IPF [2]. The major clinical features of pulmonary fibrosis are symptoms of dyspnea, wheezing, dry cough, etc. and eventually leading to respiratory failure and death [3]. Current researches about pulmonary fibrosis more focus on anti-inflammatory, immunity, cytokines, genetic factors, etc. [4]. A small number of experiments were conducted at the level of molecules and gene [5-7]. The etiology of pulmonary fibrosis is varied, with an array of triggers including allergens, chemicals, radiation and environmental particles [4]. Such triggers are believed to impair the tight regulation of inflammation and repair, leading to excess production of collagen by fibroblasts and the subsequent formation of scar tissue [8]. However, the cause of IPF remains unclear [9].

It is vital that tissue architecture is restored to regain normal function. The early lesions of pulmonary fibrosis mainly appear in the alveolar walls accompanied by a large number of proliferated lung fibroblasts, causing extracellular matrix deposits in alveolar and interstitial excessively [10]. Then the repaired fibrous tissues result in structural disorder and replace the normal tissues [11]. The ultimate outcome is irreversible pulmonary fibrosis [12,13]. An important factor of pulmonary fibrosis occurrence is the function and number of AMs and IMs [14]. Analysis of the different roles of AMs and IMs in the pathological process of pulmonary fibrosis in the field will provide an explanation of the multiple mechanisms for treating pulmonary fibrosis with TCM.

Roles of AMs and IMs in the Pathological Process of Pulmonary Fibrosis

AMs and IMs are getting more and more attention in the treatment of pulmonary fibrosis. The basic functions of macrophages in the immune defence system are phagocytic and bactericidal activities, which is vital in resisting the invasion of pathogenic microorganisms. It is reported that AMs appeared to be better equipped for their antimicrobic task. Whereas IM, although also having antimicrobic potential, show a more pronounced capacity of immunoregulatory functions [15]. As the sole cell population exposed to air, AMs having phagocytic activity of clearing extraneous particles, which is the first barrier of the lung. AMs get damaged after phagocytizing the pathogenic particles, further cause lung damages. IMs in the lung tissue also play a very important role in inflammation and local immune defense reaction of the lung injury, which is the second barrier of the lung (Figure 1).

The form and number of AMs and IMs in the pathological process of pulmonary fibrosis

AMs and IMs are two critical subsets of macrophages. Pulmonary macrophages consist of AMs in the surface and internal of alveolar space, and IMs in pulmonary interstitial next to alveolar septa or next to bronchi and vessels. Normally, there are significant differences between AMs and IMs in morphology, structure, phenotype and function [16]. Under the light microscope, the diameter of AM is about (12.1±1.16) μm which is larger than that of IM with about (9.3±1.1) μm. AM is round or irregular while IM is round and uniform relatively in shape and size. AM HE staining shows the cytoplasm is pink with loose nucleus deviate to the side of the cytoplasm in an oval shape and the nuclear-cytoplasmic ratio is less than 50%. The surface of the AM is uneven with many protrusions and the cells contain more intracellular lysosomes. IM, HE is staining shows that the cytoplasm is dark red, with dense nucleus shape irregularly and the nuclear-cytoplasmic ratio is greater than 50% [17].

The surface of the IM is smooth with almost no protrusions and the cells contain fewer primary and secondary intracellular lysosomes. Differences between AMs and IMs in morphology and structure determine their differences in functions [18]. IMs only accounts for 3%-5% of the total number of various cells throughout the lung tissue, so the isolation of IM is totally a difficult task. Over 90% of the rat bronchoalveolar lavage fluid (BALF) samples are AMs, and a variety of cells including macrophages, neutrophils, endothelial cells and lymphocytes can be obtained by enzymatic dispersal of the lung tissue. Separated by the lymphocyte separation liquid and purified by adherent cell culture, 90% of the cells in culture liquid are IM cells. The number of AMs and IMs in lung tissues under normal physiological conditions is less than that of fibrosis pathological conditions. Studies have found that the number of AMs in BALF reached the highest level on the 7th day after bleomycin injection, and the number and HE stain intensity of positive AMs have reached its peak as well [19]. And AMs increased significantly in the BALF on the 14th and 30th day after Pingyangmycin injection, which is consistent with the pro-inflammatory and pro-fibrotic effect of AMs. The number of AMs on the 14th day of Pingyangmycin injection is more than that on the 30th day, which may relate to the serious inflammatory injury during the first 14th days. The number of AMs will continue to develop on the 30th day with the sustained progress of pulmonary fibrosis [20].

The cell function of AMs and IMs in the pathological process of pulmonary fibrosis

AMs and IMs in the lung are distributed in two different anatomical sites, which result in their different functions [21,22]. Located in the airways and alveoli, AMs are the only cell population exposed to air, which contributes to the first barrier of the lung to clear inhaled microorganisms during breathing [23]. The morphology of the permanent AMs in alveoli is changeable, while the AMs in normal lung tissues are similar to the mature macrophages of other organizations. The microvilli of normal AMs distribute on the cell surface uniformly. The cytoplasmic volume of normal AMs is large with a large number of lysosomes, phagosomes and enzymes [24]. Besides, normal AMs produce cell recruitment factors, active nitrogen and other factors, and result in macrophages and other inflammatory cells gathering. During acute inflammatory periods, AMs become larger and containing quantities of peroxidase with a diameter of 12μm, while they become mature and larger during chronic progress period with a diameter of 14-40μm. The cytoplasmic membrane of AMs under the electron microscope is irregular

The cytoplasm contains many mitochondria and lysosomes, and the nucleus is more leaflike. The expression and release of membrane receptors in AMs depend on different states. The main role of AMs is to kill pathogenic microorganisms, phagocytize water-soluble poorly organic particles, release inflammatory mediators, present antigens, and express different cytomembrane receptors at the same time. Many studies suggest that AMs are the key cells causing early biological effects on lung [25]. In the initial process of lung injury, cellular infiltration can also be found in AMs from the observation of lung tissue by biopsy. The degree of cellular infiltration is closely related to the degree of pulmonary fibrosis, which confirms that AMs have the function of promoting the inflammatory response. In the process of promoting inflammatory infiltration, it also increases lung damage and causes excessive repair of the tissues, then leads to fibrosis.

IMs also play a very important role in lung inflammation and local immune defence reaction. The IMs are a type of macrophages in the interstitial connective tissue forming the second line of the lung defence, which may have a greater immune function with its special location. But the location of IM can also make it difficult in extracting the cells. IMs in pulmonary interstitial contact with the extracellular matrix and other interstitial cells tightly thus affects pulmonary interstitial metabolism. IMs are often acquired from the pulping lung tissue digested by collagenase, but the separation methods reported in the literature are quite different. IMs have their own unique function and morphology compared with AMs. They have a smaller diameter of 6.6μm, a more folded cell membrane, irregular nucleus and larger nucleoplasm ratio. And the non-specific lipid stain results of them can be positive, less positive or negative.

The filar pseudopod of IMs cell can be seen under the electron microscope. The cytoplasmic membrane of IMs is pleated, and still not extended to its characteristics after cultivating for 24h. Because of the difficulty in getting IMs, more papers focus on the study of AMs. However, recent studies have shown that IMs may have a more important role in lung injury. As the important cells, IM’s ability to phagocytize particles, produce oxygen radicals and chemotactic complement is weaker than that of AM [15]. But IMs express more MHC II and they are more efficient in stimulating the proliferation of T cells [26].

The phagocytosis and secretory function of IMs are like that of AMs. Although the Fc receptor-dependent cellular phagocytosis capability of IMs is like that of AMs, the Fc receptor-independent cellular phagocytosis capability of IMs is lower than that of AMs significantly. So that IMs show more capable of respiratory immune responses. Existed in pulmonary interstitial, IMs can release inflammatory mediators and cytotoxic mediators, which may be a more direct cause of lung tissue damage [27]. There has also been reported that IMs make more sense in the progression of pulmonary fibrosis than AMs in biological behaviours and active effects [28]. More experiments should be carried out to confirm the function of IMs.

The relationship between AMs and IMs in the pathological process of pulmonary fibrosis

AMs and IMs are two critical subsets of macrophages, which are critical in the pathological process of pulmonary fibrosis process. From the perspective of cells occurrence, AMs are the ultimate development status of macrophages which come from the source of bone marrow monocytes. Blood monocytes grew into AMs in the development of end-stage, while IMs may be the transitional stage of that process [18]. But there are great differences between AMs and IMs in antigen presenting, cytokines secreting and other immune functions. And the effects of endotoxin attacks on them are discrepant. All the differences suggest IMs are not simply the precursor of AMs.

Evidence also shows that platelet-derived growth factor (PDGF) secreted by AMs cannot enter the alveolar interstitial through the epithelia completely. Therefore, it may be concluded that AMs located in the alveolar may have a stronger function of phagocytosis, and IMs in the interstitial are primarily related to the immune regulation. In vitro studies, Adamson found that short fibers (<1μm) and long fibers (> 20μm) asbestos could stimulate macrophages to produce the Fibroblast Growth cytokines. In vivo studies also found that AMs phagocytized almost all short fibers which did not enter the pulmonary interstitial without pulmonary fibrosis lesion when mice inhaled short asbestos fibers. While IMs were activated when the long fiber deposited on the bronchi furthered into the pulmonary interstitial and caused effectors accumulate in pulmonary interstitial. Then fibroblast multiplied quickly, eventually led to the formation of pulmonary fibrosis [29].

The Self-Regulation Mechanism of AMs and IMs in the Pathological Process of Pulmonary Fibrosis

Pulmonary fibrosis is characterized by the accumulation of fibroblasts. The deposition of inflammatory cells such as macrophages, lymphocytes, and granulocytes are also the hallmark of pulmonary fibrosis. The mechanisms responsible for the migration of fibroblasts and inflammatory cells to the lung in pulmonary fibrosis are not known, but cytokines and chemokines are essentially considered [30]. The self-regulation mechanism of AMs and IMs includes two parts: Firstly, the inflammatory factors stimulate macrophages and other immune cells to secrete chemokines [31], which allow AMs, IMs and other inflammatory cells to accumulate and release a large number of cytokines. Secondly, the release of cytokines stimulates myofibroblast, leading to the accumulation, proliferation and activation of fibroblasts, then pulmonary fibrosis occurs. By further activating signal transduction pathways at the same time, cytokines stimulate inflammatory cells to generate new cytokines and the process cycles [32] (Figure 2).

Chemokines assist in the migration and invasion ability of AMs and IMs

The CC family and CXC family are two big families of chemokines involving the pulmonary fibrosis. The transfer of fibroblasts and inflammatory cells in the lung requires the participation of various chemokines/chemokine receptors such as CXCL12/CXCR4, CCL21/CCR7, CCL2/CCR2, CCL3/CCR5 [32]. Many biological functions of macrophages play their roles in their cytomembrane mediated action. The cytomembrane receptors of AMs and IMs correspond with their specific binding ligand, triggering the cell signalling pathways. The binding force of the receptor on the cell surface and the number of receptors on the surface of the cytomembrane can affect the signal transduction [33,34]. Phagocytosis is one of the important functions of macrophages, and the interaction between effector cells and the membrane of the target cells is a key factor in macrophages to kill tumor cells [35].

Cell migration is an important reason for adjusting the number of cells. Monocyte chemoattractant protein-1 (MCP- 1) and macrophage inflammatory protein-1α (MIP-1α) (also known as CCL2 and CCL3) in-vivo are important chemokines participated in the chemotaxis of the cells, the main chemotactic cells and macrophages are concentrated in the inflammatory regions [36]. Studies have found that inhibiting the activity of MCP-1 and MIP-1α in the early pulmonary fibrosis can reduce the accumulation of alveolar macrophages [37]. MCP-1mRNA in the lung tissue of rats can be found mainly expressed in alveolar macrophages, airway epithelial cells in Bleomycin-induced pulmonary fibrosis rats [38]. Thus, MCP-1 alters the population of alveolar macrophages through recruitment of blood monocytes into the luminal airspace. MIP-1α can also mobilize the bone marrow monocytes into myeloid precursor cells and enhance the infiltration of macrophages in inflammation areas [39].

According to the general reasoning, the reason for the increase of AMs in the alveoli and IM in the pulmonary interstitial is likely to be under the chemotaxis of chemokines, blood monocytes cells infiltrate or migrate to the inflammatory sites and grow into the macrophages. The increased extent of AMs varies depending on the stage of the pulmonary fibrosis, while IMs increase more obviously in the early period. In vitro study of the macrophages migration and invasion chemotaxis of MCP-1 shows that MCP-1 can assist in the migration and invasion ability of macrophages. And the chemotactic efficiency of macrophages related to the concentration of MCP-1 [40]. The growing number of macrophages with enhanced invasion capability can contribute to the pathological process of pulmonary fibrosis.

AMs and IMs secrete cytokines to regulate fibroblasts and regulate cytokines secretion

Macrophages are gathered and activated under the chemotaxis of chemokines. AMs secrete platelet-derived growth factor (PDGF) and transform growth factor beta (TGF-β), which can promote the accumulation, proliferation and activation of fibroblasts in lung injury area, causing the extracellular matrix accelerating the synthesis. It can also increase fibroblast by increasing the transfer and adhesion of osteopontin, resulting in pulmonary fibrosis [41]. AMs secrete tumor necrosis factor-α (TNF-α), interferon (IFN), transforming growth factor-β1 (TGF-β1) and other active media that can anti-pathogenic microorganisms strongly, which can effectively kill the pathogens invaded the body. IMs have lower ability to produce these cytokines than AMs, while the ability to secrete cytotoxic medium and interleukin (IL) are high, which cause more direct damages to lung tissue and show higher MHCII class antigen expression activity and stronger supplementary ability.

Cytokines regulate fibroblasts and macrophages involved in a variety of signal transduction pathways such as the MAPK pathway, JAK-STAT pathway, Smad pathway, PI3K-Akt pathway, NF-κB pathway, etc [42]. TGF-β1, a powerful fibrosis cytokine, rely on Smads and MAPK signal transduction pathway to regulate lung fibroblasts to muscle fibroblasts, produce a large number of extracellular matrix proteins, and lead to pulmonary fibrosis [43]. IL, TGF-β1, IFN involved in the JAK-STAT pathway to regulate the proliferation of fibroblasts by regulating the gene expression. At the same time, cytokines further activate the signal transduction pathways and stimulate AMs and IMs to produce new cytokines. TNF-α stimulates MAPK/ERK pathway, increases transcription of the TGF-β1 Gene and stabilizes TGF-β1mRNA to induce the expression of TGF-β1 [44,45]. On the other hand, TNF-α activates NF-κB pathway to accelerate the new TNF-α secretion of macrophages [46]. The upgraded cytokines create new biological effects.

Disease-Modifying Therapy for Pulmonary Fibrosis Based on Intervening the Self-Regulation of AMs and IMs

Clinical medicinal therapies of pulmonary fibrosis including symptom-focused therapy and disease-modifying therapy. Randomised controlled trials show that various therapies (eg, prednisolone and azathioprine, acetylcysteine, and warfarin) were ineffective or harmful, but disease-modifying therapies with nintedanib and pirfenidone are effective [47]. Both drugs are currently approved worldwide. Thus, therapies focusing on modifying pulmonary fibrosis are promising.

AMs and IMs primarily regulate the pulmonary fibrosis with cytokines TNF-α and TGF-β. Clinically recommended medicines for the treatment of pulmonary fibrosis are related to these cytokines. TNF-α can induce the adsorption of inflammatory cells and promote the occurrence of the inflammatory response. It can also regulate the production of other cytokines, deposit collagen, and promote the proliferation of fibroblasts, which plays an important role in the progress of pulmonary fibrosis. TNF-α inhibitors include etanercept, infliximab and adalimumab [48]. TGF-β can cause direct and effective stimulation to collagen synthesis, which makes an important impact on the development of pulmonary fibrosis. It takes action by enhancing the activity of connective tissue growth factor (CTGF), promoting the formation of muscle fiber cell type, as well as producing collagen and proteoglycan. Its action mechanism is associated with stimulating the Smads protein pathways. Clinical antifibrotic drugs Pirfenidone can reduce the proliferation of fibroblasts and inhibits collagen synthesis through regulation of TGF-β [49]. And some under-developmental TGF-β inhibitors such as GC1008, BG00011 and STX-100 can take actions by inhibiting the TGF-β pathway [50].

The NF-κB and MAPK pathways are important pathways to cytokine upregulation. The inhibition of these two pathways can reduce cytokines cycle and ease the fibrosis reaction. SP100030 is a type of underdeveloped NF-κB pathway inhibitor. Experiments showed that SP100030 inhibit the protease isomerized to block NF-κB pathway and reduces the degree of inflammation and pulmonary fibrosis. Current p38MAPK inhibitors under clinical trials include BIRB796, SB203580, TAK715, etc. BIRB796 shows good inhibition of all p38 MAPK isoforms in-vitro and in vivo [51].

There are no clinical chemokine inhibitors in the treatment of pulmonary fibrosis. Experiments showed that fluorofenidone (a me-too drug of pirfenidone) can inhibit the expression of MCP- 1 and TNF-α in mice macrophages induced by dead cells, which show the anti-inflammatory action. And fluorofenidone has the influence on NF-κB and MAPK pathway as well [52]. CNTO 888 (Carlumab), an under-developmental human anti-CCL2 antibody, is a monoclonal antibody that binds and neutralizes CCL2, can reduce the migration of macrophages [53]. Binding of CCL2 to its receptor CCR2 triggers chemotaxis. Inhibition of the CCR2 can reduce macrophages migration and accumulation in experimental models [54].

The Effect on AMs and IMs of TCM Treatment of Pulmonary Fibrosis

Large quantity of experiments showed that TCM had an influence on the expression of cytokines such as TNF-α, TGF-β, PDGF, CTGF, HGF, INF-γ, etc. [55]. Studies have found that macrophages are main cells which secrete tumor necrosis factor (TNF-α) in bleomycin-induced pulmonary fibrosis [56]. Experimental results of the buyanghuanwu decoction effect on the AMs of pulmonary fibrosis rats were also found the model group had higher levels of TNF-α than that of the control group. The levels of TNF-α in the model group reached its peak on the 7th day, and fell on the 28th day, with higher levels than the control group. The result showed that TNF-α has an important role in the pathological process of pulmonary fibrosis. The experiment showed that buyanghuanwu decoction can inhibit AMs of lung fibrosis rats to release TNF-α. So we can conclude that the mechanism for buyanghuanwu decoction preventing pulmonary fibrosis may be associated with its function of suppressing the TNF-α releasing of AMs [57]. The level of TNF-α in AMs supernatant was measured in each group of different periods during the Lignstrazine study of on pulmonary fibrosis. The results indicated that Lignstrazine could inhibit the release of TNF-α in AM of bleomycin-induced pulmonary fibrosis rats. It can be inferred that the mechanism of Lignstrazine treating pulmonary fibrosis is related to TNF-α release inhibition of AMs [58].

In the study of discussing the mechanism of Pneumonia Mixture (containing ephedra 60g) in the treatment of pulmonary fibrosis, researchers found that after the formation of bleomycin-induced pulmonary fibrosis in rats, TGF-β1 mRNA expressed in AMs and IMs in the model group increased more significantly than that in the control group. Therefore, we can infer that the mechanism of Pneumonia Mixture interfering the pulmonary fibrosis process is possibly associated with the reduction of the TGF-β1 expressing in AMs and IMs. The main components of Pneumonia Mixture are Astragalus, leeches, Polygonum cuspidatum, etc. Combined treatments of these drugs in all aspects of pulmonary fibrosis remediation inhibit the aggregation of inflammatory cells and reduces the synthesis of TGF-β1, as well as reduces abnormal tissues repairing and fibrosis progression [59]. The pathological process of lung diseases including pulmonary fibrosis is called “Xuansu disordered in lung” according to TCM theory. Ephedra and Schisandra are commonly used in the treatment of lung diseases.

Our laboratory has completed ephedra and Schisandra drugcontaining serum on normal rat AMs and IMs secrete TGF-β1. In vitro experiments found that ephedra, Schisandra could inhibit the TGF-β1 secretion of AMs and IMs. Ephedra-containing serum has a stronger inhibition of AM secretions compared to ephedracontaining serum, whereas Schisandra-containing serum has a stronger inhibition of IM secretions compared to Ephedracontaining serum [60]. The results showed that ephedra and Schisandra may have different influences on pulmonary fibrosis. Ephedra may have a major role in the acute inflammation period while Schisandra may have a major role in pulmonary fibrosis period.

Many experiments also elucidated the mechanism of treating pulmonary fibrosis from the regulation of chemokines. Xuejun Li showed the concentration of MIP-1α and MCP-1 in the plasma of the Panax Notoginseng, the treating group began to rise on the 3rd day and reached the peak on the 7th day in rats of bleomycininduced pulmonary fibrosis, then gradually decreased [61]. The concentration of both MIP-1α and MCP-1 in the plasma of the treating group had been significantly lower than that of the model group since the 7th day. Zhanshuai Song used Yifei-Huaxian granules in the treatment of paraquat-induced pulmonary fibrosis in rats showed the concentration of MCP-1 reached the peak on the 7th day and kept lower than the model group all through the experiment [62]. The result suggests that Chinese medicine reduces the migration of fibroblasts and inflammatory cells by suppressing the macrophages to express MIP-1α and MCP-1, thereby reducing the degree of pulmonary fibrosis.

Conclusion

In conclusion, AMs and IMs are vital regulatory macrophages in the pathological process of pulmonary fibrosis. Because of the anatomical site and cell function of AMs and IMs, anti-fibrosis drug options related to them have higher targeting ability than anti-inflammatory therapy drugs. And AMs and IMs can also be used to explore the multiple mechanisms of traditional Chinese medicine treatment for pulmonary fibrosis.

TCM has made great progress in the experimental study of pulmonary fibrosis prevention. Drugs involving enriching yin and nourishing the blood, clearing up heat and toxin, fortifying the spleen to boost qi, invigorating blood to dissolve stasis, supplementing the lung to boost the kidneys, relieving a cough and panting and many other aspects, meanwhile, the effective components of Chinese medicine provide a larger space and treatment basis for the clinic. Single herb showed good results in fighting against pulmonary fibrosis. At the same time, traditional Chinese medicine decoctions and Chinese patent medicines can have significant effects on pulmonary fibrosis [63,64]. Overall, the mechanisms explanation of the Chinese medicines on pulmonary fibrosis is still in its infancy.

The current studies were largely restricted to animal experiments rather than clinical studies, which focused on anti-inflammatory, immune and cytokines without systematic measurements and standards [65]. Because of the complicated compositions of single Chinese herbs, there is no clear scientific research basis for the effective compositions in the treatment of pulmonary fibrosis. And some therapeutic mechanisms are built on the hypothesis with a lack of rigorous experimental verification. Small samples, different clinical diagnosis and efficacy evaluation criteria result in the no convincing results. Although the research on the molecular and genetic level has been increasing in recent years, it is still limited to a few kinds of Chinese medicines. In this context, some experiments from the perspective of AMs and IMs in the pulmonary fibrosis rats and the influence of some common herbs for lung diseases on the AMs and IMs by using drug-containing serum were conducted. Thus, we can explore the pathological process of pulmonary fibrosis and explain the multiple mechanisms of TCM to guide clinical medication better, which is conducive to the succession and development of traditional Chinese medicine.

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Different Effects of Olive Leaf on Purine Metabolizing Enzymes of Human Gastric Tissues in Vitro | Juniper Publishers

Juniper Publishers-Open Access Journal of Cancer Therapy & Oncology

Authored by Hikmet Can Çubukçu

Abstract

Olive leaf (Olea europaea leaf) is a natural food source known to have anticarcinogenic, antiproliferative and anti-inflammatory effects in different types of tissues. Adenosine deaminase, 5’nucleotidase and xanthine oxidase are enzymes playing part in purine metabolism including salvage pathway. In the present study, it is aimed to investigate possible inhibitory effects of aqueous extract of olive leaf on different purine metabolizing enzyme activities in benign and malign human gastric tissues. Fouteen cancerous and 14 adjacent noncancerous human gastric tissues were surgically removed from patients underwent surgical operation. Olive leaf extract- treated and - not treated tissues were analyzed in vitro for adenosine deaminase , 5’-nucleotidase and xanthine oxidase activities.

Our results showed that aqueous extract of olive leaf inhibited adenosine deaminase activity significantly in cancerous gastric tissue (p=0.000) and 5’-nucleotidase activity in non cancerous gastric tissue (p=0.001). However, no significant differences were found between tissue xanthine oxidase activities. Results indicate that aqueous extract of olive leaf may exhibit anti-cancer activites by inhibiting adenosine deaminase and 5’nucleotidase in gastric tissues.

Keywords: Olive leaf; Cancer; Adenosine deaminase; 5’-nucleotidase, Xanthine oxidase; Oleuropein; Apigenin; Luteolin; Quercetin; Tyrosol; Hydroxytyrosol; Caffeic acid; Ferulic acid; p-Coumaric acid; Cancer

Introduction

Cancer is increasingly becoming a worldwide public health problem. 14.1 million new cancer cases and 8.2 million cancer deaths were reported in 2012 worldwide. It is expected that by 2025, 20 million new cancer cases are diagnosed each year.The most common cancer types are lung , breast, and colorectal cancer respectively [1]. Gastric cancer is the fourth most common cancer and second most common cause of cancer deaths worldwide [2]. While radiotherapy and chemotherapy are used to treat these cancers, severe side effects can be seen in some patients. Recently natural and herbal remedies have taken attention owing to their represented ability to treat some diseases like cancer. Natural products can be used not only to treat cancer but also to prevent it [3]. Smoking cessation, fruit and vegetable intake, reducing salt intake, Helicobacter pylori eradication can help prevent from gastric cancer [4]. Olea europaea is an evergreen tree which belongs to Oleaceae family . The plant is cultivated widely in Mediterranean basin [5]. While the fruits and the oil are consumed for nutrition, olea europaea leaf has been used as a folk remedy for centuries.

Studies have shown that olive leaf has antiproliferative, apoptotic, antiatherosclerotic, antioxidant, antidiabetic, antiHIV and antifungal properties. Olive leaf contains several phenolic compounds like oleuropein, apigenin, luteolin, quercetin, tyrosol, hydroxytyrosol, caffeic acid, ferulic acid. The potential health benefits of olive leaf have mainly attributed to these bioactive substances [6]. Adenosine deaminase (ADA) is an enzyme involved in purine metabolism which deaminates adenosine and deoxyadenosine to inosine and deoxyinosine respectively. It plays an important role in differentiation of the lymphoid system. ADA deficiency related to severe combined immunodeficiency disease (SCID) .Therefore ADA inhibitors are used to treat lymphoproliferative disorders as an immunosuppressive therapy [7].

5’nucleotidases are important enzymes for maintaining nucleotide pools which dephosphorylate nucleoside monophosphates to nucleosides and inorganic phosphates. Nucleoside triphosphates necessary for maintaining vital cellular processes. Since 5’-nucleotidases are responsible for degradation of nucleoside monophosphates, they can regulate cellular energy homeostasis by changing nucleoside triphosphate to monophosphate ratio [8].

Xanthine oxidase (XO) is involved in purine metabolism catalyzing the oxidation of hypoxanthine to xanthine, and xanthine to uric acid [9]. It generates superoxide radicals and hydrogen peroxide during oxidation [10]. These reactive oxygen substances may contribute to various diseases like cancer [9]. It has also been reported that XO may be a crucial therapeutic target for some diseases like gout, cancer, inflammation and oxidative damage [11]. The present study aims to clarify possible proposed anticarcinogenic effects of aqueous olive leaf extract with regard to purine metabolizing enzyme activities of human gastric tissues in vitro.

Methods

Fourteen cancerous and 14 adjacent noncancerous human gastric tissues were obtained from patients by surgical operation. After cleaned by saline solution, fresh surgical specimens were stored at -80 °C until analysis. Before analysis procedure, specimens were first hom*ogenized by DIAX 900 (Heidolph, Kelhaim, Germany) in saline solution (20 %, w/v). The hom*ogenates were centrifuged at 5000 rpm for 30 min by a Harrier 18/80 centrifuge (MSE, London, UK) to remove debris. Then, clear supernatant fractions were taken for enzymatic analysis. Aqueous extract of olive leaf (Olea europaea leaf) was prepared at concentration of 10 % (w/v) in distilled water. Tissue hom*ogenates were treated with aqueous extract of olive leaf for 1 hour.

Enzyme activities were measured in the specimens with and without olive leaf extract spectrophotometrically by using Helios alpha Ultraviolet/Visible Spectrophotometer (Unicam, Cambridge, UK). Protein concentration in the samples was measured by the method of Lowry, and adjusted to equal concentrations [12]. ADA activities were measured by Giusti method. The method is based on spectrophotometric measurement of a blue colored dye occurred after the reaction of ammonia (product of adenosine deamination) with phenol nitroprusside and alkaline hypochlorite solution [13]. Xanthine oxidase activities were evaluated by measuring uric acid formation from xanthine at 293 nm [14], and 5’-nucleotidase activities were performed by determination of liberated phosphate at 680 nm as described previously [15].

Statistical evaluations between groups were made by using Mann-Whitney U test, and p values lower than 0.05 were evaluated significant. All statistical calculations were performed by using SPSS statistical software (SPSS for Windows, version 11.5)

Results

ADA, 5’-NT and XO activities are shown in the Table 1, and p values in the Table 2. It has been observed that aqueous extract of olive leaf inhibited adenosine deaminase in malign gastric tissue (p=0.000), and 5’-nucleotidase in benign gastric tissue (p=0.001) significantly. However, no significant differences were found between tissue xanthine oxidase activities. Although ADA activities in the treated benign tissues, and 5’-nucleotidase activities in the treated malign tissues were lower than those in the non- treated tissues, they were not significant statistically (p=0.067, p=0.062). In addition, we found no meaningful differences between benign and malign tissue enzyme activities.

Discussion

Natural remedies have been used from ancient times till now in conventional Eastern medicine. It has been known that more plant consumption reduces the incidence rates of cancer. Phenolic compounds are those of the plant ingredients that represent anticancer properties [16]. Olive leaf contains various phenolic compounds including oleuropein, ligstroside aglycone, oleuropein aglycone, quercetin, isorhamnetin, rutin, catechin, gallocatechin, apigenin, luteolin, tyrosol, hydroxytyrosol, gallic acid, p-coumaric acid, caffeic acid and ferulic acid that contribute to anti-carcinogenic, antioxidant, anti-inflammatory and antimicrobial effects [17].

Researchers have demonstrated that hydroxytyrosol-rich extract of the olive leaf can inhibit human breast cancer cell growth owing to cell cycle arrest in the G0/G1 phase [18]. Oleuropein and its semisynthetic peracetylated derivatives have been documented for its antiproliferative and antioxidant effects on human breast cancer cell lines [19]. Olive leaf extract’s antigenotoxic, antiproliferative and proapoptotic activities on human promyelocytic leukemia cells were previously reported [20]. Further researchers have shown that dry olive leaf extract possesses strong anti melanoma potential by reducing tumour volume, inhibiting proliferation,causing cell cycle arrest [21]. Studies have also demonstrated gastroprotective activity of olive leaf [22] and antioxidant effects on ethanol-induced intestinal mucosal damage [23].

ADA is responsible for adenosine and inosine breakdown. Inhibition of ADA blocks the deamination of purine nucleotides, and as a consequence accumulation of ADA substrate, 2-deoxyadenosine inhibits ribonucleotide reductase. This process leads to a reduction of nucleotide pool, and limits DNA syntesis [24]. Phosphorylation of deoxyadenosine results in deoxyadenosine triphosphate production. Deoxyadenosine triphosphate and deoxyadenosine both inactivate S-adenosinehom*ocysteinase [25] and affect cellular methylation of some substances like proteins, DNA and RNA [26]. There are many studies as to the ADA activation on different pathologic conditions. Inhibition of ADA was found to reduce intestinal inflammation in experimental colitis [27]. A study on human gastric cancer cell line has also shown that extracellular adenosine induces apoptosis [28]. It is known that chronic inflammation predisposes to gastric cancer [29]. Adenosine reflects its metabolic function by its four G-protein coupled receptors. Adenosine A2A reseptor activation possesses antiinflammatory effects on various conditions [30].

In the present study, aqueous extract of olive leaf was found to inhibite adenosine deaminase in malign gastric tissue (p=0.000), significantly. Inhibition of ADA can promote adenosine accumulation, and therefore it not only induces apoptosis but also exhibit anti-inflammatory effects on gastric cancerous tissue. 5’nucleotidases are responsible for degradation of nucleoside monophosphates. Until now, 7 types of human 5’-nucleotidases have been identified [8]. One of them is ecto-5’-nucleotidase that is also known as CD73. Studies have implied that ecto-5’- nucleotidase regulates proliferation, migration and invasion of cancer cells in vitro, tumor angiogenesis and tumor immune evasion in vivo [31]. Nucleoside analogues are used as both anticancer and antiviral agents. These drugs inhibit DNA syntesis by its active substances. Studies have shown that enhancing nucleotidase activity can cause anticancer drug resistence by inhibiting nucleoside analogue activation [8]. Moreover, Lu et al have reported that CD73 expression in malign gastric tissues is higher than benign gastric tissues. This study has also indicated that CD 73 overexpression is related to differentiation of tumour, depth of invasion, stage and metastasis [32]. However in the present study, no meaningful differences were found between 5’nucleotidase enzyme activities of benign and malign tissues. Furthermore results of the present study show that aqueous extract of olive leaf inhibits 5’-nucleotidase in benign gastric tissue (p=0.001) significantly. Although 5’-nucleotidase activities in malign tissues treated with olive leaf extract were found to be lower than those in the untreated tissues, the differences were not howevr significant from statistical points of wiew.

Xanthine oxidase is the last enzyme in purine degradation which converts purines to uric acid and hydrogen peroxide. Hydrogen peroxide is one of the reactive oxygen species. Although hydrogen peroxide can play a part in oxidative damage of DNA, and promotes malignant transformation, some studies have shown that this substance is able to kill cancer cells at higher concentrations [33].It has been reported that olive leaf extract inhibits xanthine oxidase activity in vitro [34] . However in our study, we found no significant differences between tissue xanthine oxidase activity values.

Our results show that olive leaf extract inhibits adenosine deaminase activity in malign gastric tissue significantly, but does not affect xanthine oxidase activity. It seems quite reasonable that accumulation of adenosine can exert anti-carcinogenic properties by inducing apoptosis and by anti-inflammatory effects. Additionally, inhibition of ADA and 5’nucleotidase can deplete nucleotide pool which is very important for new DNA synthesis. This study reveals preliminary information about different effects of olive leaf on purine metabolizing enzymes of benign and malign gastric tissues. Therefore, further in vivo studies should be conducted to clarify possible anti-carcinogenic effects of olive leaf.

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Severity of Hip Displacement in Relation to Subtypes and Motor Function in Cerebral Palsy- Role of Hip Surveillance | Juniper Publishers

Juniper Publishers-Open Access Journal of Orthopedics and Rheumatology

Authored by Kunju PAM

Abstract

Background: Hip dislocation in children with cerebral palsy (CP) is a common and often over looked problem by the treating pediatricians. Though it can be diagnosed early by using radiographs, knowledge about the standardized methodology and need for periodic surveillance is lacking among primary care pediatricians. Hip surveillance by X-ray pelvis can identify early hip dislocation and it is shown that early intervention may prevent the need for surgery [1].

Methods: The study was done in a tertiary care hospital as a onetime radiological evaluation of children with CP between the age group of 4-9 yrs. One hundred and one children with CP formed our study population.Clinical evaluation for details regarding CP type and assessment of motor ability by gross motor function classification system (GMFCS) was done. A hip X-ray was done for calculation of, migration index for establishing or ruling out hip displacement. Migration percentage (MP) in relation to CP subtypes and GMFCS grades were done.

Results: There were 48 boys and 53 girls (mean age 4.80 years). 12 children were Gross Motor Function Classification System (GMFCS) level 5, while 26 were GMFCS level 4. Out of 36 hemiplegic CP only one had MP > 40. out of 6 children with spastic quadriplegia, 5 (83%) had MP > 40%. Spastic diplegic and choreoathetotic subtypes showed MP >40% in 9 out of 43 and 7 out of 16 respectively.According to the gross motor function classification system, GMFCS level I had no child with MP > 40%. Whereas 50% of children in GMFCS level IV and V had MP > 40% compared to only 4.76% in GMFCS I and II put together.

Conclusion: All the children in this study did not undergo a hip X-ray prior to this study. 22 out of 101 children had severe degree of hip displacement. The maximum number of hip displacements was seen in children with spastic quadriplegia; Spastic diplegic and choreoathetotic subtypes showed intermediate risk of hip displacement and hemiplegia had very low risk. According to the gross motor function classification system,GMFCS level I had no child with MP > 40%. Whereas 50% of children in GMFCS level IV and V had MP > 40%. The study showed the relationship between the CP subtypes and the severity of the motor involvement. It also emphasized the need for early hip surveillance.

Keywords: Hip dislocation; Cerebral palsy; Lateral Displacement; Hip surveillance

Introduction

In children with spastic cerebral palsy reduced activity of the hip abductor muscles in comparison to the spastic adductors leads to diminished growth of the greater trochanter of femur results in pathologic deformities of the hips-femoral anteversion and coxa valga antetorsa [2]. If untreated, dislocation of the hip typically occurs at age 2–7 years with a maximum at the age of 6 years. The incidence of hip displacement in cerebral palsy is related to the severity of involvement; varying from 1% in children with spastic hemiplegia, up to 75% in those with spastic quadriplegia [2,3]. So periodic evaluation of hip function is essential for early intervention and preventive measures.

Hip surveillance is defined as: “The process of monitoring and identifying the critical early indicators of hip displacement” [4].Hip displacement refers to the displacement of the femoral head laterally out of the acetabulum and is measured using a migration percentage (MP). Hip subluxation refers to hip displacement where the femoral head is partially displaced from under the acetabulum while hip dislocation refers to hip displacement where the femoral head is completely displaced from under the acetabulumn [5,6].Hip surveillance is important, to prevent morbidity of spastic hip disease-The aim of the management in children with spastic hip displacement is to maintain flexible, well-located and painless hips with a symmetrical range of movement. The key to achieving this goal is early identification and intervention.

Periodic hip surveillance also helps to reduce the need for extensive surgical procedures which is highly specialized area of orthopedics which may not be available in every center. So primary care pediatrician has a role for hip surveillance and timely referral.

Patients and Methods

The study was done in a pediatric neurology department of a tertiary care hospital as a onetime radiological evaluation of children with CP between the age group of 4-9 yrs prior to the referral to orthopedics. One hundred and one children between the age group of 4-9 yrs. with the diagnosis of CP formed our study population.A pediatric neurologist and physiotherapist in the department examined the children and completed an assessment form. Clinical evaluation for details regarding CP subtype and assessment of motor ability by gross motor function classification system (GMFCS) [6] was done. Winters, Gage, Hicks (WGH) gait type was determined, in addition to inquiring regarding pain during history taking. Orthopedic consultations done whenever required.

Radiographic Examination

Decision for referral for surgery depends on the degree of displacement of the femoral head and acetabular dysplasia. The migration percentages as described by Reimers and the acetabular index described by Hilgenreiner are the conventional measurements of displacement of the hip and acetabular dysplasia in young children with cerebral palsy.Radiographic assessment consists of measurement of migration percentage (MP) from a supine AP pelvis radiograph with standardized positioning [7] (Figure 1). Reimers Hip Migration Percentage is the percentage of body width of femoral capital epiphysis displaced out of the acetabulum (which falls lateral to perkins line) [8].Measurement of migration percentage of femoral head was done as given in the (Figure 2).

In the adult or older child, where the triradiate cartilages are fused and therefore inapparent, the inferior margin of the pelvic teardrop is used instead.The acetabular angle using Hilgenreiner’s line should be less than 28°at birth. The angle should become progressively shallower with age and should measure less than 22° at and beyond 1 year of age.

Present study an anteroposterior (AP) pelvic radiograph at the time of first visit was done. Any decrease in the range of movement at the hip or presence of scoliosis was a definite indication for further detailed radiological examination & immediate referral. In the present study 101 children were assessed between 4 and 9 years of age. Children with MP > 33% and > 40% were compared in relation to those with MP below these limits. Migration percentage (MP) in relation to CP subtypes and GMFCS grades were done.

Results

There were 48 boys and 53 girls (mean age 4.80 years). Distribution of Cerebral Palsy sub types were as follows. Hemiplegic 36 (35.64%), Quadriplegic 6(5.94%), Diplegic 43(42.57%) and Choreo athetotic 16(15%). 12 children were Gross Motor Function Classification System (GMFCS) level 5, while 26 were GMFCS level 4. Results of hip displacement by radiography as measured by MP in relation to CP subtypes and motor severity are presented in (Tables 1&2) and (Figure 3).

Only one child out of 36 children with spastic hemiplegia developed MP > 40%. The maximum number of hip displacements was seen in children with spastic quadriplegia, where 5 of 6 children (83%) had MP > 40%. Spastic diplegic and choreoathetotic subtypes showed intermediate risk of hip displacement (9 out of 43 and 7 out of 16 respectively had MP >40%). In the present study onset of hip displacement could not be assessed as hip surveillance was not done in a periodic basis. Figure 4 shows x-ray hip of 4-year-old with very minimal displacement (MP 33.33%) and Figure 4 shows severe hip displacement in an 8-year-old child.

According to the gross motor function classification system, GMFCS level I had no child with MP > 40%. Whereas 50% of children in GMFCS level IV and V had MP > 40% compared to only 4.76% in GMFCS I and II put together.

Discussion

The natural history of spastic hip disease of CP is progressive lateral displacement of the hip secondary to spasticity and muscle imbalance in the major muscle groups around the hip. Displacement may progress to severe subluxation, secondary acetabular dysplasia, deformity of the femoral head, dislocation and painful degenerative arthritis [4,5]. The long-term effects of dislocation of the hip can be disastrous for individual patients leading to pain and loss of the ability to sit comfortably in up to 50% of cases [6]. Other problems include difficulty with perineal care and personal hygiene, pelvic obliquity and scoliosis, poor sitting balance and loss of the ability to stand and walk [7-11].

A hip is usually considered to be subluxed,if the migration is equal to or greater than 33%. Reimers [17] found that among normal, the 90th gentile for migration percentage at four years was 10% with spontaneous migration of less than 1% per year. An unstable migration percentage is when progression is greater than or equal to 10% over 1 year [12-16]. Present study has shown that even a single radiological evaluation could identify hip displacement in children after the age of 4 yrs. Majority of (5 out of 6) quadriplegic CP, had severe type of hip displacement compared to hemiplegic CP (1 out of 36). Compared to other bilateral types of CP diplegia had lower rate of hip displacement (9 out of 43). This may be because of the less motor function impairment. So GMFCS may be a better predictor for early prediction of hip structural impairment. It is seen that there is direct correlation between the GMFCs class and severe hip displacement. According to the gross motor function classification system, GMFCS level I had no child with MP > 40%. Whereas 50% of children in GMFCS level IV and V had MP > 40% compared to only 4.76% in GMFCS I and II put together.

Subtyping of CP may have a role in predicting occurrence of severe hip displacement as shown by the almost complete occurrence in quadriplegic CP. However, a mere clinical examination and subtyping will not help in identifying severe hip disease in other type of CP. So, a systematic analysis of GMFCS is required for intensified screening of hip dysfunction. Moreover, as described in various guide lines periodic hip surveillance is mandatory for better ambulation and avoidance of surgery. This can be attained by early intervention measures. Figure 4 itself shows the importance of early surveillance. AACPDM - (American Academy for Cerebral Palsy and developmental medicine) recommends following schedule of hip surveillance (Table 3).

Conclusion

Need for hip evaluation in children with CP is emphasized by this study. All the children in this study did not undergo a hip Xray prior to the study. 22 out of 101 children had severe degree of hip displacement. The maximum number of hip displacements was seen in children with spastic quadriplegia and hemiplegia had very low risk. According to the gross motor function classification system, GMFCS level I had no child with MP > 40%. Whereas 50% of children in GMFCS level IV and V had MP > 40 %. The study showed the relationship between the CP subtypes and the severity of the motor involvement in producing hip displacement. Referral to an orthopedic surgeon with experience in treating hip displacement in children with CP is recommended when there is presence of hip pain on history and/or physical examination. Periodic hip surveillance is mandatory for early detection of hip displacement. When the migration percentage is greater than 30% and/or there is less than 30 degrees of hip abduction with or without other findings, referral to an orthopedic surgeon is recommended [1,17].

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Overview of Simmondsia chinensis (Jojoba shrubs) Cultivation and Propagation Methods | Juniper Publishers

Juniper Publishers-Open Access Journal of Agricultural Research & Technology

Authored by Waleed Fouad Abobatta

Abstract

Jojoba plants (Simmondsia chinensis) is an excellent plant for development marginal lands, Jojoba propagate by different vegetative methods including (stem cuttings, grafting, air-layering, root cutting and tissue) or direct seed propagation. Jojoba cultivated for it is valuable oil, this oil used in various industries such as pharmaceutical products, cosmetics, produce biodiesel fuel as well as biodegradable lubricants, jojoba considered as a new solution of bio-fuel in coming era.

jojoba has a deep-rooted system can extract water, therefore, it is adapted to drought and salinity conditions, it can persevere where several conventional crops cannot survive, so, it is growing and produce economic crop in marginal lands and offer promise for cultivation under harsh conditions. This work aimed to explain important of jojoba, propagation methods and some usage of jojoba oil.

Keywords: Simmondsia chinensis; Propagation methods; Jojoba oil; Biofuel

Introduction

Jojoba Simmondsia chinensis (Link) (pronounced hohoba) is an evergreen perennial shrub, jojoba has numerous names in several regions of the world, such as bucknut, coffee nut, goat nut, pig nut and lemon leaf [1]. In recent years, there has been considerable interest in development arid and semi-arid lands by promoting crops which can tolerate these conditions such as Jatropha curcas [2], and jojoba (Simmondsia chinensis) [3], Jojoba is a multipurpose crops, and have a potential use for rehabilitation of the marginal lands, also, Jojoba is a high-value shrub and a promising cash crop, it is considered a source for the provision of income to the pastoral communities [4], it is produces high quality oil used in cosmetics, lubricant industry, pharmaceuticals and electronics [5].

Jojoba could tolerant drought and salinity conditions, it is adapted to the aridity conditions, also, it is grown and produce economic crop in marginal lands, jojoba has deep-rooted system can extract moisture from a wide area of soil and, it can persist where several conventional crops cannot survive; it offers promise for agriculture in harsh conditions. jojoba produce small seeds, which has liquid wax considered the nearby vegetarian oil to sperm whale in value, the main usage for jojoba oil mostly in the manufactories pharmaceutical products, cosmetics, and used as lubricants in the motor industry, besides that, it is use to produce bio-fuel. Nowadays, jojoba is cultivated as an economic plant in various regions of the world like Egypt, Argentina, Australia, India, Mexico, Peru, Kenya, Brazil, South Africa, Costa Rica, Paraguay, Chile, Iran, and Israel, due to its high commercial value [6].

Botanical

Jojoba Simmondsia chinensis is the sole genus (Simmondsia), in family Simmondsiaceae; which contain the only species in this genus (jojoba-Simmondsia chinensis) (Link) Schneider. Jojoba is a perennial evergreen dioecious with male and female plants, depends on wind for successful pollination [7]. It is a shrub or small tree variable in height from 0.5 to 3 meters (Figure 1) with dense round crown, also, jojoba has many main stems and sometimes it is growing desperate subjected to its environment.

Jojoba leaves have an oval shape, with 2-3cm long and 1.0 -1.5cm broad, it is the leaves are opposite, thick waxy grey-green in color (Figure 2), also, jojoba plant has a deep root system can reach more than 15m depth [8], deep-rootedness habit helps it to reach to tap water from deep aquifers for its existence under dry conditions and also to extract the leached nutrients from the deep layers of the soil which plant use to survive under harsh conditions. In the origin jojoba natural life prolongs to more than 100 years and may extend up to 200 years.

Flowering and fruiting

Jojoba is dioecious plants with separate male and female plants, flower buds growing on the fresh vegetative shoots, it is formed in the axils of the leaves during the warm period of growth, jojoba flowers are greenish-yellow, with 5-6 sepals without petals, the female flowers grown and bloom single auxiliary (sometimes twice flowers) at alternate nodes, it has light green color with long pedicles, the male flowers are bigger than female one with yellow color and grow in bunched contain 7 or more flowers at nodes [9], generally there are no female petals or scent to attract insects, jojoba pollinated by wind. In the northern hemisphere flowering began from March to May depend on fulfillment of chilling requirements and winter rains.

Chilling requirements

Due to jojoba native in arid area, it needs low chilling requirements (average from 15- 20 °C) for short period about a month for break flower dormancy to start flower induction in jojoba [10], therefore, exposure flower buds to enough cold units and fulfillment of their chilling requirements during winter improve the flowering and total yield of jojoba plants [9].

Soil type

Jojoba is grown in a different type of soil, but preferable cultivated jojoba in well drainage light soils, sandy or gravely soils, with a pH ranged from 5 to 8.5 [11], soil pH not an very determined factor for jojoba cultivation, Jojoba prefers very sandy soils with low organic matter (Figure 3). However, in clay soil drainage is critical element for jojoba growth and productivity, also, water-logged in heavy clay soils may be execute plants, it is need well water penetration, therefore, jojoba grew and flowering slower in heavy soil than light soils, also, preferable avoiding cultivated jojoba in heavy clay soil [6].

Jojoba nutrition

Poor management of the existing Jojoba bushes has led to low production of seeds (0.5-2kg/bush) through severe abortion of flowers and pods. This is mainly through competition for nutrients, water and space [12]. Jojoba grows on soils of marginal soil fertility, fertilization of field plots with nitrogen and phosphorus improved plant growth and increase seed production. In marginal lands preferable added some organic fertilizers for plants (about 1kg compost with ¼ kg of super phosphate and ¼ kg of sulfur/adult tree) from mid of November till January, low nutrition of jojoba trees reduce seed production through abortion of fruit set and drops of fruits before maturity [13].

Water requirement

Jojoba is native to the arid zone of Sonoran desert with harsh climatic conditions, therefore, Jojoba plant is drought resistant, the water requirements for jojoba is very low compared to other crops like maize and grape, it is needs little water for survival, the ideal growth and economic productivity of jojoba require rainfall average of 254 to 380mm annually, consequently, under low rainfall conditions (254mm or less) supplementary irrigation is required particularly during establishment the orchards and the first year after cultivation and to get healthy trees in arid areas [6], also, jojoba could tolerate soil salinity more than other crops, so, treated sewage water and saline water could be used to irrigate jojoba plants.

Economic consideration dictates that supplementary irrigation is essential for a healthy tree and sustained high annual crop in arid and marginal areas. Irrigation water supply with relatively good drainage stimulated more luxuriant vegetation and good flower production and, thus, good seed yield.

Climate

Jojoba naturally grows in marginal areas with rainfall ranging between 220-350mm yr-1, jojoba cultivated when soil temperatures are 20 °C, and the optimum temperature average for growth and productivity between 27-33 °C [14], but jojoba flowering require low temperature about 15-20 °C for a month at least to break flower dormancy, from other side, jojoba tolerates high temperatures without negative effects on plant growth up to 54 °C (122 ˚F).

Jojoba cannot resist frosts below -3 °C for long time, particularly during flowering stage, cold weather could destroy flowers completely, new flushes and terminal portions of young fruit set, however, new plants cannot tolerate excessive cold temperature during early seedling development and may extinguish whole plantations, and however, mature plants could withstand during cold weather than the new establishment plant , therefore, temperature may be the most critical factor in growing jojoba.

Propagation

Jojoba could propagate by different way, vegetative propagation including (stem cuttings, grafting, air-layering, root cutting and tissue) or seedy propagation technique [15].

3.8.1. Vegetative propagation methods: Vegetative propagation use to avoiding high male to female ration in jojoba farms, and use determined plants ratio, there is various advantages of using vegetative propagation methods in commercial jojoba plantations like they produce genetically uniform plant, and early fruiting [16], it is allow growers to decreasing cultivation expenses, use the proper ratio of female to male plants according to cultivation plans, and plants start produce seed crop earlier than seedy plants. There are various asexual techniques used for jojoba propagation, such as;

a. Stem cuttings: Rooting of stem cuttings is the main adopted technique used for vegetative propagation, dormant stage considered the best time to take stem cutting to increase successive ratio, (Figure 4), due to high carbohydrate to nitrogen ratio (C/N ratio) in this period, also, preferable use stem cuttings with 4-5 nodes [17], beside that rooting hormones like NAA or IBA improve root formation [18], also, semi-hardwood cuttings , and single-node cuttings ( double-eye or Single –eye) could use under controlled conditions.

b. Grafting: Grafting technique of jojoba has various advantages particularly in seedy cultivation by using the deep root system of male plants as rootstock grafted with Scions of mature female plants previously known as high yield with good fruit quality properties, August and September considered proper timing for Veneer grafting [19], also, preferable use scions from mature wood, grafting help to survive plants under stress conditions (drought, salinity, and malnutrition) by use scion of desirable plants which will allow predictable plant vigour and high crops, also reducing juvenile stage of jojoba plants particularly males plants which produce seeds in two years approximately [20].

c. Tissue culture: tissue culture technique provides opportunities for the production of thousands of finest plants from the selected trees, Jojoba plants from tissue culture grow more vigorously than other seedlings (Figure 5), regularly, shoot tip or nodal segment used as explant source, could conceivably provide thousands of new true to type plantlets per year [21], but till now jojoba propagation by tissue culture still in research field only.

d. Roots cuttings: This method has not been used commercially for some reasons like shortage of plant materials as sources of cuttings; also, this technique need controlled conditions facilities to produce new plants and it is slowly proceeding for commercial production.

Seed propagation method: seed propagation is easier and low-cost methods but there are negative effects like produced more male plants than female plants ( average 1:1 up to 5:1 male to female plants), this cause low crop production due to more than 50% of plants are nonproducing, also, the females start production in the fourth or fifth year, and there are many female plants are produce very low crop with low quality [22], also, there are heterogeneity differences between seedy plants which affect growth uniformity, physiological characteristics, yield and early bearing [23], therefore, after flowering grower must remove exceed males and the poorer female plants from the farm and replanting new plants which delay economic production.

Yield

There have been considerable yield differences between seedy trees and asexual seedlings in total production and seed quality. Jojoba fruits are green capsules 1-2cm long, with three-angled at a corn-shaped ovoid, capsule partly enclosed at the base by the sepals, the fruits turn to brown color on maturation period.

The mature seed is a hard oval, or slightly spherical, brown in color (Figure 6) and contains oil (liquid wax) content of 40-50% approximately, there is variation between seeds in shape, size, and weight of seeds, regarding farm management practice [24].

Harvesting

Jojoba shrubs regularly start flowering and seed production in the second or third year after planting [10&14], and seeds ripen during the summer, however, seedy cultivation starts production from the fourth or fifth year.

In marginal lands jojoba are harvested manually, in other regions seed collected manually and mechanically after they have fallen to the ground, jojoba seeds could remain for a long time on the ground without negative effects, unless there are rodents are active in the cultivation area, so, it needs quick harvested to avoid losing the crop seeds.

Oil Content: Jojoba oil is a soluble wax; it includes esters of high molecular weight monounsaturated fatty acids and alcohol and various components of sterols and vitamins [25], Jojoba oil could transform to hard white wax by hydrogenation, it has a very long straight-chain wax ester (C36-C46) and not a triglyceride [26], this oil has unique properties to use in various industrial and medicinal uses, this characteristics of gave more attention to this shrubs.

Jojoba uses

Jojoba considered versatile plants, and it is a promising cash crop for the poor communities, also, have a potential use for rehabilitation particularly in marginal regions, Jojoba is a proper plant to combat and prevent desertification in arid regions such as in India (Thar desert area) [27], and in Egypt around 6 October city [5].

Concerning jojoba oil there are numerous applications in pharmaceuticals like a number of skin and scalp disorders, skin emollient [28], anti-acne, anti-psoriasis [29], anti-inflammatory [30], and anti-hypercholesterolemia [31], also, it is used in cosmetics products and in various fields like polishing, and gardening applications, from another side, jojoba oil is used to produce a biodiesel fuel, and as biodegradable lubricants, it is a new solution of fuel in coming era [32]. Also, Jojoba meal could use for producing bioenergy [33], or other useful products like animal feed (Figure 7).

Conclusion

Jojoba shrubs is a proper plant to sustainable development for the poor area and rehabilitation marginal regions, jojoba grown in different types of soil and resist drought and salinity conditions more than other plants, it is easily propagated by both vegetative and seedy methods, also, there are a few pests and diseases affect jojoba shrubs productivity. Jojoba oil considered raw materials for various industries in pharmacology and cosmetics production, also, it provides a new source for biofuel and biodegradable lubricants; however, jojoba meal used for animal feed production or bioenergy and other useful products. Therefore, Jojoba has a promising future for developed dry and marginal lands, it is offers an excellent opportunities in various sectors.

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An Investigation of the Physicochemical and Thermal Characteristics of Consciousness Energy Healing Treated L-Cysteine | Juniper Publishers

Juniper Publishers-Open Access Journal of Nutrition & Food Science

Authored by Snehasis Jana

Abstract

L-cysteine is an essential amino acid that helps in improving the overall health status of humans. The objective of the study was to determine the impact on the physicochemical and thermal properties of L-cysteine after the Trivedi Effect®-Consciousness Energy Healing Treatment using modern analytical techniques. The control sample was kept without any treatment, while the Biofield Treatment was given to the treated sample remotely by a renowned Biofield Energy Healer, Gopal Nayak. The particle size values were significantly reduced by 11.96%(d10), 9.01%(d50), 4.92%(d90), and 7.66% [D (4, 3)], thus, the specific surface area was significantly increased by 14.28% of the treated L-cysteine compared to the control sample. The peak intensities and crystallite sizes were altered ranging from -86.73% to 456.65% and -4.91% to 451.22% respectively, however, the average crystallite size was significantly increased by 87.58% in the treated sample compared to the control sample. The latent heat of decomposition for the 1st and 2nd peak of the treated sample was increased by 12.77% and 5.42% respectively, compared with the control sample. The weight loss was increased by 9.35%; however, the residual weight was significantly reduced by 85.32% in the treated sample compared to the control sample. The maximum thermal degradation temperature of the treated sample was increased by 2.87% compared to the control sample. Hence, the Biofield Energy Treated L-cysteine might show better solubility, dissolution, bioavailability, and be more thermal stability in the pharmaceutical formulations, which would be more efficacious in the treatment of diabetes, cancer, psychosis, and seizures compared to the control sample.

Keywords: L-cysteine; The Trivedi Effect®; Energy of consciousness healing treatment; Complementary and alternative medicine; PSA; PXRD; DSC; TGA

Introduction

Cysteine is an essential amino acid that contains sulphur, which allows it to get bonded and maintain its structure within the body [1]. Cysteine is considered an essential as it is the basic building block of the glutathione formation (mother antioxidant) and used by the body to produce taurine, i.e., also an amino acid [2]. L-cysteine had several other uses that help in improving the overall health status of humans. One of its supplements, N-acetyl-L-cysteine (NAC), is used for improving the level of glutathione within the body, as the right glutathione level supports the functioning of brain, lungs, and immunity, and helps in liver detoxification [3]. L-cysteine also acts as a scavenger that fights with the free radicals of the body. Such radicals cause cellular damage in the body by the process of oxidative stress; therefore, L-cysteine preserves the glutathione in the body and improves the antioxidant capacity [4,5]. The supplements containing L-cysteine are also used to improve the immunity in postmenopausal women; prevent the side effects from toxic chemicals and drug reactions; treat infertility in men having the poor quality of sem*n; improve the digestive capacity; slow the aging process [6-9]. Other functions of L-cysteine in the body includes balancing the blood sugar levels, relieving the symptoms of bronchitis or chronic obstructive pulmonary disease (COPD), and treating some psychiatric disorders [10-12]. The physicochemical properties of amino acids, such as L-cysteine, play important role in its biological performance within the body. Thus, efforts were made by several researchers to improve such physicochemical profile of the compound that ultimately affects their biological activities, such as improving the solubility and absorption by reducing the particle size, increasing the surface area, or modifying the crystal morphology [13,14]. In this scenario, the Biofield Energy Treatment is known for its significant impact on various properties of living and non-living objects [15,16].The Biofield energy is a unique phenomenon that involves the traditional as well as the contemporary models of energy medicine and is used as the Complementary and Alternative Medicine (CAM) to fight against various diseases and disorders [17-19]. The Biofield Energy Healing is an Energy therapy that is used in wide population of the USA and also accepted by the National Centre for Complementary and Alternative Medicine (NCCAM) along with Ayurvedic medicine, traditional Chinese herbs and medicines, naturopathy, essential oils, homeopathy, yoga, meditation, massage, acupuncture, acupressure, Tai Chi, Qi Gong, deep breathing, special diets, relaxation techniques, aromatherapy, guided imagery, healing touch, Reiki, chiropractic/osteopathic manipulation, movement therapy, hypnotherapy, pilates, Rolfing structural integration, mindfulness, cranial sacral therapy, and applied prayer [20]. Thus, an expert human can harness energy from the universal and can transmit it to any living organism(s) or non-living object(s) around the globe. Similarly, the Trivedi Effect®-Consciousness Energy Healing Treatment has shown remarkable impacts in the field of agriculture science [21,22], microbiology [23-25], metals and ceramics [26,27], livestock [28], biotechnology [29,30], and human health and wellness [31- 33]. Moreover, the impact of The Biofield Energy Treatment is also evident on the physicochemical and thermal properties of various organic and pharmaceutical compounds [34-36]. Thus, this study was done with the objective to determine the effect of the Biofield Energy Healing Treatment (the Trivedi Effect®) on the physicochemical and thermal properties of L-cysteine with the help of several analytical techniques such as particle size analysis (PSA), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA)/differential thermogravimetric analysis (DTG).Thus, this study was also aimed to analyse the impact of the Biofield Energy Healing Treatment (The Trivedi Effect®) on the physicochemical and thermal properties of ascorbic acid by using various analytical techniques such as, particle size analysis (PSA), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA)/ differential thermogravimetric analysis (DTG), and differential scanning calorimetry (DSC).

Materials and Methods

Chemicals and reagents

L-cysteine was purchased from Alfa Aesar, USA. All other chemicals used during the experiments were of analytical grade available in India.

Consciousness energy healing treatment strategies

In this study, the test compound used was L-cysteine, which was divided into two parts, i.e., control and treated. The control sample was not given the Biofield Energy Treatment; however, the treated part of L-cysteine received the Energy of Consciousness Healing Treatment by the renowned Biofield Energy Healer, Gopal Nayak (India), and considered as the Biofield Energy Treated sample. The process of Biofield Energy Treatment involves keeping the sample under the standard laboratory conditions and then the Biofield Energy Healer provided the Trivedi Effect®-Energy of Consciousness Healing Treatment to the sample, remotely, for 3 minutes through the Unique Energy Transmission process. The control L-cysteine was further subjected to a “sham” healer under the similar laboratory conditions, who did not have any knowledge about the Biofield Energy Healing Treatment. The control and the Biofield Energy Treated L-cysteine samples were kept in similar sealed conditions and further characterized by using PSA, PXRD, DSC, and TGA/DTG techniques.

Characterization

Particle size analysis (PSA): The particle size analysis of the L-cysteine was conducted on Malvern Mastersizer 2000, from the UK with a detection range between 0.01 μm to 3000 μm using wet method [37,38]. The sample unit (Hydro MV) was filled with a dispersant medium (sunflower oil), and the stirrer operated at 2500rpm. PSA of L-cysteine was performed to obtain the average particle size distribution. Where d (0.1) μm, d (0.5) μm, d (0.9) μm represent particle diameter corresponding to 10%, 50%, and 90% of the cumulative distribution. D (4,3) represents the average mass-volume diameter, and SSA is the specific surface area (m2/g). The calculations were done by using software Mastersizer Ver. 5.54.The percent change in particle size (d) for at below 10% level (d10), 50% level (d50), 90% level (d90), and D (4,3) was calculated using the following equation 1:

Where dcontrol and dTreated a re the particle sizes (μm) at below 10% level (d10), 50% level (d50), and 90% level (d90) of the control and the Biofield Energy Treated samples, respectively. The percent change in surface area (S) was calculated using the following equation 2:Where Scontrol and STreated are the surface area of the control and the Biofield Energy Treated L-cysteine, respectively.

Powder X-ray Diffraction (PXRD) Analysis: The PXRD analysis of L-cysteine was performed with the help of RigakuMiniFlex-II Desktop X-ray diffractometer (Japan) [39,40]. The Cu Kα radiation source tube output voltage used was 30 kV and tube output current was 15 mA. Scans were performed at room temperature. The average size of individual crystallites was calculated from XRD data using the Scherrer’s formula (3):

Where k is the equipment constant (0.94), G is the crystallite size in nm, λ is the radiation wavelength (0.154056 nm for Kα1 emission), β is the full-width at half maximum (FWHM), a nd θ is the Bragg angle [41]. The percent change in crystallite size (G) of L-cysteine was calculated using the following equation 4:Where Gcontrol and GTreated are the crystallite s ize o f t he control and the Biofield Energy Treated samples, respectively.

Differential Scanning Calorimetry (DSC): The DSC analysis of L-cysteine was performed with the help of DSC Q200, TA instruments. Sample of 1-5 mg was loaded to the aluminium sample pan at a heating rate of 10oC/min from 30°C to 350°C [37, 38]. The % change in melting point (T) was calculated using the following equation 5:

Where Tcontrol and TTreated are the melting point of the control and treated samples, respectively.The percent change in the latent heat of fusion ( ΔH) was calculated using the following equation 6:Where ΔHcontrol and ΔHTreated are the latent heat of fusion of the control and treated L-cysteine, respectively.T

hermal Gravimetric Analysis (TGA)/ Differential Thermogravimetric Analysis (DTG): TGA/DTG thermograms of L-cysteine were obtained with the help of TGA Q50 TA instruments. A sample of 5 mg was loaded to the platinum crucible at a heating rate of 10°C/min from 25°C to 1000°C with the recent literature [37, 38]. The % change in weight loss (W) was calculated using the following equation 7:

Where Wcontrol and WTreated are the weight loss of the control and the Biofield Energy Treated L-cysteine, respectively. The % change in maximum thermal degradation temperature ( Tmax ) (M) was calculated using the following equation 8:Where Mcontrol and MTreated are the Tmax values of the control and the Biofield Energy Treated L-cysteine, respectively.

Results and Discussion

Particle size analysis (PSA)

The particle size analysis of the control and the Biofield Energy Treated L-cysteine samples corresponding to d10, d50, d90, and D (4, 3) was done (Table 1) to determine the impact of the Biofield Energy Treatment on the particle size distribution of L-cysteine. It revealed that the particle size distribution of the Biofield Energy Treated sample at d10, d50, d90, and D (4, 3) was significantly reduced by 11.96%, 9.01%, 4.92%, and 7.66%, respectively compared to the control sample.Such reduction in the particle sizes of the Biofield Energy Treated sample after the Biofield Energy Treatment resulted in 14.28% increase in the specific surface area of the Biofield Energy Treated sample (0.016 m2/g), in comparison to the untreated L-cysteine sample (0.014 m2/g). Several types of research were conducted nowadays that correlate the particle size and surface area of the compound with its solubility and dissolution profile [42,43]. Moreover, the reduced particle size and increased surface area of the compound are known for improving the solubility, absorption, and bioavailability performance in the body [44]. Thus, it is assumed that the Biofield Energy Treated L-cysteine might show better solubility and dissolution rate within the body that ultimately enhances its bioavailability in comparison to the untreated sample.

Powder X-ray diffraction (PXRD) analysis

Figure 1 shows the diffractograms of the control and the Biofield Energy Treated L-cysteine samples; and both contains sharp and intense peaks, thereby indicating their crystalline nature. The analysis regarding the changes in the peak intensities and the crystallite sizes of the Biofield Energy Treated sample in comparison to the control sample was done and presented in Table 2. The analysis of the diffractograms revealed alterations in the Bragg’s angle of the characteristic peaks of the Biofield Energy Treated sample as compared to the control sample. Also, the highest peak intensity (100%) was observed at 2 equal to 37.13° in the control sample; while in the Biofield Energy Treated sample at 2 equal to 24.91°. The significant alterations were observed in the peak intensities and the crystallite sizes of the Biofield Energy Treated L-cysteine sample as compared to the control sample. The Biofield Energy Treated sample showed changes in the peak intensities ranging from -86.73% to 456.65%; while the crystallite sizes were altered ranging from -74.91% to 451.22%, compared to the control sample. Such changes corresponding to the characteristic diffraction peaks of the Biofield Energy Treated sample showed that the crystallinity and crystalline structure of L-cysteine sample might get altered after the Biofield Energy Treatment as compared to the untreated sample.The average crystallite size of the Biofield Energy Treated sample was 1252.22 nm, which was increased by 87.58% compared to the control sample (667.56 nm). The significant alterations in the crystal morphology and the crystallinity of the compounds after the Biofield Energy Treatment were reported previously in various studies. They reported the occurrence of such changes based on the alterations in their peak intensities and crystallite sizes of the Biofield Energy Treated compounds that might indicate the formation of a novel polymorph of the compound [45,46]. Thus, the Biofield Energy Treated L-cysteine showed significant changes in the peak intensities and crystallite size corresponding to the characteristic peaks that might take place as a result of the new polymorph formation of L-cysteine. The literature reported the improved bioavailability profile of drug as a result of the physical modifications, i.e., alteration in the crystal habit of the drug [47]. Hence, the Biofield Energy Treated L-cysteine might show improved bioavailability as compared to the untreated sample.

Differential scanning calorimetry (DSC) analysis

The DSC thermograms of the control and the Biofield Energy Treated sample are shown in Figure 2, that are further analysed to determine the melting and other thermal behaviours of both the sample [48]. The literature reported that L-cysteine got decomposed instead of sublimation during its thermal heating. It was mentioned that the peak in DSC thermogram was present at the same temperature as the drop in the TGA thermogram. Thus, the DSC peak temperature coincides with the TGA drop thereby, indicated the process of decomposition in place of melting during the heating of L-cysteine [48,49].There were two peaks present in the thermograms of the control and the Biofield Energy Treated sample. The results revealed the alterations in the decomposition temperature of the 1st and 2nd peak of the Biofield Energy Treated sample by -2.51% and 0.46%, respectively in compared to the control sample. The latent heat of decomposition ( decomposition ΔH ) for 1st and 2nd peak of the Biofield Energy Treated L-cysteine sample was significantly increased by 12.77% and 5.42%, respectively, compared to the control sample (Table 3).The results indicated that the Biofield Energy Treated sample started decomposing 4.6º earlier temperature as compared to the control sample, which might be due to some changes in the particle size and crystallization structure of the L-cysteine [48,50] after the Biofield Energy Treatment. Thus, the Biofield Energy Treated sample showed an increase in thermal degradation in comparison to the untreated L-cysteine sample.

Thermal gravimetric analysis (TGA)/ Differential thermogravimetric analysis (DTG)

The weight loss of the L-cysteine samples during the thermal degradation was analysed from the TGA thermograms (Figure 3) of the control and the Biofield Energy Treated sample. Also, the degradation profile of both the samples were observed like the reported literature [49]. The analysis of both the thermograms showed that the total weight loss of the control sample was 90.12%; whereas it was observed as 98.55% for the Biofield Energy Treated L-cysteine sample. Thus, the total weight loss during the thermal degradation of the Biofield Energy Treated sample was increased significantly by 9.35% that resulted in 85.32% decrease in the residual mass compared with the control sample (Table 4). Hence, the thermal degradation of the Biofield Energy Treated sample was increased after the Biofield Energy Treatment in comparison to the untreated sample.The DTG data (Table 4) (Figure 4) of the Biofield Energy Treated sample showed the maximum thermal degradation temperature ( Tmax ) a t 2 25.72 ° C, w hich w as i mproved b y ∼ 6 °C (2.87%) as compared to the Tmax of the control sample (219.42 °C). Hence, the overall TGA/DTG studies revealed that the thermal stability profile of the Biofield Energy Treated L-cysteine sample was altered after the Biofield Energy Treatment as compared with the untreated sample.Go toConclusionThe study results suggested that there was a significant impact of the Trivedi Effect®-Consciousness Energy Healing Treatment on various properties of the Biofield Energy Treated L-cysteine sample related to their physicochemical and thermal profile. The PSD data showed that the particle size values of the Biofield Energy Treated sample were significantly reduced at d10, d50, d90, and D (4, 3) by 11.96%, 9.01%, 4.92%, and 7.66%, respectively, compared to the untreated sample. The specific surface area of the Biofield Energy Treated L-cysteine was significantly increased by 14.28% in comparison to the untreated L-cysteine. The PXRD peak intensities of the Biofield Energy Treated L-cysteine were observed to be changed ranging from -86.73% to 456.65% and crystallite sizes showed alterations ranging from -74.91% to 451.22% as compared to the control sample. The average crystallite size of the Biofield Energy Treated sample was significantly increased by 87.58%, as compared to the control L-cysteine sample. The DSC thermograms of the control and the Biofield Energy Treated sample showed two peaks in their thermograms that are related to the decomposition of L-cysteine sample during heating. However, the corresponding to 1st and 2nd peak were also observed to be significantly increased by 12.77% and 5.42%, respectively, compared to the control L-cysteine sample. The TGA/DTG data indicating that the Biofield Energy Treated sample showed 9.35% increase in total weight loss of the L-cysteine sample that causes a significant 85.32% reduction in the residue amount compared to the untreated sample. However, the of the Biofield Energy Treated sample was increased by 2.87% compared to the control sample. Thus, the overall data showed the reduced particle sizes and increased surface area of the Biofield Energy Treated sample with alterations in the crystal structure, which might increase the solubility, absorption, and bioavailability of L-cysteine within the body. Also, the results indicating significant changes in the thermal degradation and stability profile of the Biofield Energy Treated L-cysteine sample compared to the untreated sample. Therefore, the Trivedi Effect®-Consciousness Energy Healing Treated L-cysteine might improve the properties that might ensure its better performance and therapeutic response against various diseases, i.e., diabetes, cancer, psychosis, and seizures.

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An Expedient One-Pot Synthetic Protocol to the Preparation Of Isoxazolo and Pyrazolo Annulated Analogues of Benzoxazepino Condensed Carbazoles and Azacarbazoles of Medicinal Interest | Juniper Publishers

Juniper Publishers-Open Access Journal of Organic & Medicinal Chemistry

Authored by Nirupama Mishra

Abstract

A facile one-pot synthetic protocol for the preparation of isoxazolo and pyrazolo annulated analogues of the benzoxazepine condensed carbazoles and azacarbazoles from the corresponding enolic ethers, α, β-unsaturated ketones, oxoketene dithioacetals and dimethylaminomethylene ketones derived from benzoxazepino condensed oxocarbazoles and oxoazacarbazoles has been described. Compounds were realized from the Japp-Klingemann reaction of the diazonium salt of 2-aminodibenzo [b,f] [1,4]oxazepine with 2-hydroxymethylideno cyclohexanone and N-benzyl-3-hydroxymethylidenopiperidin-4-one followed by Fischer indolization of the obtained hydrazone with kent’s acid. The synthesized heterocyclic compounds were characterized by IR, NMR and MS spectral data and in vitro antimicrobial activity of the synthesized compounds was screened against the standard bacterial and fungal strains. Some of these benzoxazepine derivatives have been found to display excellent antimicrobial activity against bacteria and fungi.

Keywords:Japp-Klingemann Reaction; 2-Aminodibenzo [b,f] [1,4] Oxazepine; Fischer Indolization; Enolic Ether; Chalcone; oxoketenedithioacetals; dimethylaminomethylene ketone; oxocarbazoles and oxoazacarbazoles.

Introduction

Benzoxazepine is one of the privileged heterocyclic template in medicinal chemistry, dibenz[b,f ][1,4]oxazepine (CR) is an important incapacitating agents of this class which is also more potent and less toxic than widely used other riot control agents [1,2]. Analogues of 11H-dibenz [b,e] azepine and dibenz [b,f] [1,4] oxazepine (tear gas) are used as human transient receptor potential ankyrin 1 (TRPAI) antagonists [3]. Sintamil, a new dibenzoxazepine derivative has been extensively studied during the last decade for anti depressant action with fairly good results in animals and human beings [4-6]. N-substituted dibenz [b,f] [1,4] oxazepin-11(10H)-ones have been reported to exhibit antidepressant [6], calcium antagonist [7], anticancer [8-10] and squalene synthase inhibiting activities [11]. Dibenz [b,f] [1,4] oxazepines are found in many physiochemically active compounds. Numerous derivatives have been prepared and evaluated for PGEs antagonists and analgesic activities [12] (Figure 1).Condensed heterocyclic systems containing carbazole, azacarbazole, azepine, benzoxazepine, isoxazole, pyrazole etc. represent such pharmacophoric scaffolds which have a wide range of biological applications such as analgesic, anti inflammatory, anti-bacterial, antifungal and anticancer activity [1-8, 13-20]. Carbazoles, azacarbazoles (including pyridocarbazoles) show impressive cytotoxic activity and form interesting targets in synthesis since such structures have potential for the development of anticancer drugs [5-8]. Coupled with this, encouraging findings of anticancer and antileukemic activity of triazolo and pyrrolo condensed benzoxazepines which was hailed as a major step forward in the battle against cancer [9-11], stimulated many eyes to examine the versatility of this nucleus in combating this disease. It firmly affirmed the observation that incorporation of additional rings onto the benzoxazepine core tended to exert a profound influence in conferring novel biological activities in this molecule. Another class of heterocycles which exhibit similar versatility in providing pharmacophoric scaffolds in medicinal field are the isoxazoles and pyrazoles whose derivatives have emerged as potent materials useful as potential anti HIV agents [21- 25]. In view of the prodigious range of activities of the above molecules, it was considered of interest in the present work to develop the bioactive materials incorporating benzoxazepine nucleus on one side of carbazole or azacarbazole core and isoxazole and pyrazole nucleus on its other side, on this premise that their presence in tandem in the same molecular framework could contribute significantly to the biological activity in the resulting molecules. Here in, in this communication, we report the applications of facile synthetic protocols which have allowed an easy incorporation of isoxazole and pyrazole motifs on to the benzoxazepine condensed carbazole nucleus.In pursuance of our interest in the biological potential of isoxazolo and pyrazolo annulated analogues of benzoxazepine condensed carbazoles and azacarbazoles 15-26 shown in scheme-1 [26-29], these were required to be obtained. On brainstorming in the direction of finding easy routes to their formation, we envisioned that these could possibly be readily available from the corresponding enolic ethers 7, 8, α,β- unsaturated ketones 9, 10, oxoketenedithioacetals 11, 12 and dimethylaminomethyleneketone 13, 14, which in turn, in principle be readily formed from the carbocyclic ketones derived from the benzoxazepino condensed oxocarbazoles and oxoazacarbazole 5 and 6 respectively on their reaction with-HCOOEt, NaOEtC6H5CHOCS2, CH3I (base)dimethylformamide dimethylacetal (DMF-DMA).These intermediates have been known to undergo reaction with the bidentate nucleophiles to provide a very convenient synthetic entry to the five, six, and seven membered heterocycles [30-33]. In accord to their versatility in synthesis the procedures reported in the literature were applied on 5- and 6 to obtain 7-14, whose treatment with hydroxyl amine hydrochloride, and hydrazine hydrate afforded the corresponding isoxazole and pyrazole annulated analogues of benzoxazepine condensed carbazole and azacarbazoles 15-26 respectively (scheme-1).The potential of 5- and 6 in the above synthesis prompted us to explore the feasibility of their preparation through an expedient route from the easily accessible starting materials. A perusal of literature on the synthesis of oxocarbazoles revealed that the most recorded method of their preparation is use of the Japp-Klingemann reaction on the corresponding aryl amines, followed by Fischer indolization of the obtained hydrazone in acid, to give the corresponding tetrahydrocarbazol-4-one derivative [34,35]. This methodology was applied on the diazotized 2-amino-dibenzo [b,f] [1,4] oxazapine (2) which was allowed to condense with 2-(hydroxy-methylideno) cyclohexanone (3) and 3-(hydroxymethylideno)-N-benzyl-piperidin-4-one (4) (which were prepared in situ from the reaction of cyclohexanone or N-benzyl-4-piperidone with ethyl formate in presence of sodium ethoxide) followed by cyclization in Kent acid (AcOH+HCl 20:5 ml v/v) to give 5 and 6 respectively (scheme-1) (Figure 2).2-Amino-dibenzo [b,f] [1,4] oxazepine (2) was obtained by the reduction of the corresponding 2-nitroderivative. The later was in turn realized through a reported procedure [36] which consisted of the base catalyzed cyclocondensation of o-aminophenol with 2-chloro-5-nitrobenzaldehyde. The formation of 5 and 6 is believed to take place through the cyclocondensation of the corresponding phenyl hydrazone derivative (scheme-2) which results from the reaction of 2 with 2-hydroxymethylideno cyclohexanone (3) or N-benzyl- 3-hydroxymethylidenolpiperidin-4-one (4). The reaction is suggested to proceed through the sequence of reactions shown in scheme-2 (Figure 3).In order to find out the biological potential of synthesized compounds, antimicrobial activity of compound 15, 17, 18, 19, 21, 22, 23, 25 was studied towards four bacterial Escherichia coli (MTCC119), Bacillus subtilis (MTCC7419), Pseudomonas aeruginosa (MTCC1688) and Staphylococcus aureus (MTCC9886) using ciprofloxin as standard and two fungal species A flavus (MTCC871) and A.niger (MTCC282) using fluconazole as standard antifungal agent by disc diffusion method. By following a reported method by Kumar et al. [37] with some modifications. Antimicrobial activity of indicated compounds was recorded as zone of inhibition in mm and results are summarized in (Table 1). Results showed that majority of the compounds display varying degree of inhibition against the tested microorganisms. In general the potency against gram positive is higher than gram negative. Some tested compound showed remarkable antibacterial and antifungal activities. Compound 22 inhibited both gram positive and gram negative bacterial strains but show less efficiency towards fungal strains. Instead, compound 25 showed activity against both bacterial and fungal strains. Compound 21 showed excellent antifungal activity against both fungal strains comparable to standard drug Fluconazole.In summary, an efficient methodology for easy access to the pyrazolo and isoxazolo annulated analogues of carbazole and azacarbazole fused benzoxazepines has been described. The application of Japp-Klingemann reaction on 2-aminodibenzoxazepine and 2-hydroxymethylideno cyclohexanone/3-hydroxymethylideno-N-benzyl-4-piperidone followed by Fischer indolization of the obtained hydrazone with acid provided a very convenient synthetic entry to the oxocarbazole and oxoazacarbazole fused benzoxazepine. The later formed a very facile substrate in providing the subsequent annulation of this nucleus with pyrazole and isoxazole through the reaction of the corresponding enol ethers, enones, oxoketene dithioacetals and dimethylaminemethylene ketones with hydrazine hydrate and hydroxylamine hydrochloride respectively. Eight selected compounds from this series were evaluated for antimicrobial activity showing moderate to excellent antibacterial and antifungal activity.

Experimental

All reagents were purchased from HIMEDIA, Aldrich, Fluka and used directly without further purification. The melting points of all compounds were recorded in open glass capillaries on a VMP-D melting point apparatus and uncorrected. 1H-NMR spectra were recorded on Bruker-F-300 (300 MHz) spectrometer using CDCl3 as solvent and trimethylsilane (TMS) as internal reference and chemical shifts were expressed in δ (ppm). Mass spectra were recorded on model Clarus-600 Perkin Elmer; IR spectra were recorded on FTIR-9050S. CE (SCHIMADZU). Elemental analyses were carried out using a PERKIN ELMER CHNS/O analyzer series-II Model 2400. Reactions were monitored through TLC using benzene and methanol as eluent 2,3-dihydo-1H-benzo-[2,3] [1,4] oxazipino [6,7-b] carbazol-4 (5H) -one (5).

Preparation of hydrazone

A solution of 2-amino- dibenzo [b,f] [1,4] oxazepine 1 (525 mg. 2.5 mmol) in aqueous HCl (5ml conc. HCl and 2ml water) was treated with a cold saturated solution of sodium nitrite (650 mg in 5ml of water) while the temperature was kept 0-5°C, It was then added drop wise to ice cold mixture containing 3-hydroxymethylidene cyclohexanone 3 (945 mg 7.5 mmol), sodium acetate trihydrate (810 mg, mmol), methanol (40ml), water (20ml) over a period of 5 hr with stirring. This content was allowed to stand for further 5h and the resulting solid mass was filtered, dried and recrystallized from ethanol to give the hydrazone, which was used in next step without further purification.

Cyclization of hydrazone

A solution of hydrazone (792 mg, 2.5 mmol) in Kent acid (a mixture of acetic acid (20ml) and hydrochloric acid (5ml.) was refluxed on an oil bath at 120-130 oC for 30 min. the content was cooled and poured to cold water with stirring. A yellow solid was obtained which was purified by column chromatography to give 2,3-dihydo-1H-benzo-[2,3] [1,4] oxazipino [6,7-b] carbazol-4 (5H) -one 5, Yield 532 mg (67%), cream-white, crystalline powder, mp 310-311oC. IR spectrum, ν, cm-1: 3110 (C-H str. ArH), 1710 (C=O), 1600 (C=N), 1480 (C=C), 1150 (C-N), 1110 (C-O-C). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.91 (1H, s, NH); 8.39 (1H, s, N=CH); 7.78 (1H, s, H Ar); 7.47 (1H, s, H Ar); 7.36 (4H, m, H Ar); 3.0 (2H, t, J = 6.1, CH2); 2.58 (2H, t, J = 6.1 CH2); 2.11 (2H, m, CH2). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 302.10 [M]+ (65). Found, %: C 75.12; H 4.63; N 9.21. C19H14N2O2. Calculated, %: C 75.48; H 4.67; N 9.27/9.21. Similarly compound oxazipino derivative 6 was prepared. Yield 64%, cream-white, crystalline powder, mp 322-324 oC. IR spectrum, ν, cm-1: 3120 (C-H); 1720 (C=O); 1600 ((C=N); 1440 (C=C); 1150 (C-N); 1110 (C-O-C). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.91 (1H, s, NH); 8.39 (1H, s, N=CH); 8.19 (1H, s, H Ar); 7.55 (1H, s, H Ar); 7.28 (8H, m, H Ar); 4.51 (2H, s, CH2); 3.39 (2H, t, J = 6.1, CH2); 2.63 (2H, t, J = 6.1, CH2); Mass spectrum (ES+, 70 eV), m/z (Irel, %): 393.15 [M]+ (47). Found, %: C 75.94; H 4.90; N 10.60. C25H19N3O2. Calculated, %: C 76.32; H 4.87; N 10.68.3-(ethoxymethylene)-2,3-dihydro-1H-benzo[2,3][1,4] oxazepino[6,7-b]carbazol-4(5H)-one (7).To a solution of sodium ethoxide (1300 mg) in dry benzene (10ml) at 0 oC, a solution of ethyl formate (520 mg 1.75mmol) in dry benzene (3 ml) was added. To this mixture 2,3-dihydo- 1H-benzo-[2,3][1,4]oxazepino[6,7-b]carbazol-4(5H)-one 5 (302 mg, 1 mmol) in benzene (10ml) was then added and the mixture was stirred for 4h at room temperature and allowed to stand overnight. It was then diluted with cold water, acidified with dil. HCl and extracted with ether. The solid mass obtained was recrystallized from ethanol to give 7, Yield 210 mg (70 %), light-yellow, crystalline powder, mp 318-320 oC. IR spectrum, ν, cm-1: 3180 (C-H str. ArH); 1700 (C=O); 1660 (C=C); 1550 (C=N); 1590 (C=C); 1200(C-O-C). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.91 (1H, s, NH); 8.39 (1H, s, N=CH); 7.78 (1H, s, HAr); 7.47 (1H, s, HAr); 7.36 (4H, m, HAr);; 6.99 (1H, s, CH); 4.49 (2H, q, J = 6.9, CH2); 3.1 (2H, t, J = 6.1, CH2); 2.82 (2H, t, J = 6.1, CH2); 1.21 (3H, t, J = 7.5, CH3). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 359.14 [M]+ (48). Found, %: C 73.37, H 5.02, N 7.76. C22H18N2O3. Calculated, %: C 53.73/; H 5.06; N 7.82.Similarly 8 was prepared from 6. Yield 260 mg (66%), paleyellow, crystalline powder mp 314-316 oC. IR spectrum, ν, cm-1: 3150 (C-H); 1730 (C=O), 1630 (C=C); 1610 (C=N); 1480 (C=C str.); 1220 (C-O-C). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.91 (1H, s, NH); 8.39 (1H, s, N=CH); 8.19 (1H, s, H Ar); 7.84 (1H, s, H Ar); 7.28 (8H, m, H Ar); 7.55 (1H, s, H Ar); 7.11 (1H, s, CH); 4.71 (2H, s, CH2); 4.49 (2H, q, J = 6.9, CH2 ); 3.73 (2H, s, CH2); 1.21 (3H, t, CH3). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 449 [M]+ (36). Found, %: C 74.80; H 5.11; N 9.01. C28H23N3O3. Calculated, %: C 74.82; H 5.16; N 9.35..3 - b e n z y l i d e n e - 2 , 3 - d i h y d r o - 1 H - b e n z o [ 2 , 3 ] [1,4[oxazepino[6,7-b]carbazol-4(5H)-one (9). A mixture of 2,3-dihydro-1H-benzo-[2,3][1,4]oxazepino[6,7-b]carbazol- 4(5H)-one 5 (453 mg. 1.5 mmol), benzaldehyde (265 mg. 2.5 mmol) and fused sodium acetate (300 mg. 3.7 mmol) in glacial acetic acid was refluxed for 5h. The reaction mixture was cooled in ice water. The resulting solid was filtered and washed with water, dried and recrystallized from ethanol to give pure oxazepino derivative 9, Yield 300 mg (yield 66%), brawn, crystalline powder, mp 326-328 oC. IR spectrum, ν, cm-1: 3200 (C-H str. ArH); 1680 (C=O); 1600 (C=N); 1400 (C=C), 1350 (CN); 1000 (C-O-C). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.91 (1H, s, NH); 8.39 (1H, s, N=CH); 7.78 (1H, s, H Ar); 7.50 (5H, m, H Ar); 7.47 (1H, s, H Ar); 7.36 (4H, m, H Ar); 7.15 (1H, s, CH); 3.1 (2H, t, J = 5.9, CH2); 2.82 (2H, t, J = 6.1, CH2). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 390.8 [M]+ (63). Found, %: C 79.64; H 4.61; N 7.12. C26H18N2O2. Calculated, %: C 79.98; H 4.65; N 7.18.Similarly 24 was prepared from 12. Yield 837 mg (68%), light-brawn, crystalline powder, mp 260-263 oC. IR spectrum, ν, cm-1: 1630 (C=C); 1600 (C=N); 1220 (C-O-C); 1100 (C-H), 900 (C-O-N); 700 (C-S). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 11.8 (1H, s, NH); 8.3 (1H, s, CH); 8.1 (1H, s, H Ar); 7.6 (1H, s, H Ar); 7.3 (4H, m, H Ar); 7.2 (5H, m, H Ar); 4.7 (2H, s, CH2); 4.3 (2H, s, CH2); 2.5 (3H, s, CH3). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 464 [M]+ (71). Found, %: C 69.38; H 4.31, N 12.82; S 6.56. C27H20N4O2S. Calculated, %: C 69 80; H 4.34; N 12.06; S 6.90.3-(methylthio)-1,4,5,15-tetrahydrobenzo[2,3][1,4] oxazepino[7,6-b]pyrazole[3,4-a]carbazole (25). Hydrazine hydrate (1-.5 ml) and bis(methylthio)methylene compound 11 (405 mg. 1mmol) was taken in 10 ml ethanol and refluxed for 3h. The resulting residue was poured in water and extracted with ethyl acetate, washed with saturated solution of sodium bicarbonate and dried over anhydrous sodium sulfate. Removal of the solvent gave pyrazole derivative 25, Yield 259 mg (64%), pale-yellow, crystalline powder, mp 288-289 oC. IR spectrum, ν, cm-1: 3300 (NH str. pyrazole); 3000 (C-H str. ArH); 1590 (C=N); 1570 (C-H str. ArH); 1200 (C-O-C); 1150 (C-N); 700 (C-S). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 12.3 (1H, s, NH); 11.8 (1H, s, NH); 8.3 (1H, s, CH); 7.9 (1H, s, H Ar); 7.4 (1H, s, H Ar); 7.3 (4H, m, H Ar); 2.8 (4H, m, CH2); 2.5 (3H, s, CH3). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 372.44 [M]+ (62). Found, %: C 67.24; H 4.28; N 14.73; S 8.21. C26H16N4OS. Calculated, %: C 67.7; H 4.33; N 15.05; S 8.61.Similarly 26 was prepared from bis(methylthio)methylene compound 12. Yield 330 (66 %), pale-yellow, crystalline powder, mp 274-275 oC. IR spectrum, ν, cm-1: 3320 (NH str. pyrazole); 3050 (C-H), 1590 (C=N); 1570 (C=C); 1210 (C-O-C); 1150 (C-N str.); 720 (C-S). 1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 12.3 (1H, s, NH); 11.8 (1H, s, NH); 8.3 (1H, s, CH); 8.1 (1H, s, H Ar); 7.6 (1H, s, H Ar); 7.3 (4H, m, H Ar); 7.2 (5H, m, H Ar); 4.7 (2H, s, CH2); 4.3 (2H, s, CH2). Mass spectrum (ES+, 70 eV), m/z (Irel, %): 463.15 [M]+ (86). Found, %: C 69.58; H 4.52; N 14.82, S 6.54. C32H23N5OS. Calculated, %: C 69.95; H 4.56; N 15.11; S 6.92.The authors are grateful to the Head, Sophisticated Analytical Instrument Facility (SAIF), CDRI Lucknow India for providing spectral data of the compounds. We are are thankful to the Department of Science and Technology, New Delhi (India), for granting project to “Banasthali Centre of Education for Research in Basic Sciences” under their CURIE (Consolidation of University Research for Innovation and Excellence in Women Universities) program.

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In Vitro Topoisomerase II Inhibitory and Apoptotic Activities of Novel 3,5 Disubstituted Thiophene-2-carboxylates | Juniper Publishers

Juniper Publishers-Open Access Journal of Organic & Medicinal Chemistry

Authored by Kanchugarakoppal S Rangappa

Abstract

Topoisomerases (topoII) are crucial enzymes involved during DNA replication, repair and transcription. Recent studies have shown topo II as an interesting target for cancer therapy. In the current study we have synthesized and characterized few thiophene derivatives and determined their antiproliferative activity and apoptosis studies in cancer cell lines. Biochemical analysis showed that thiophene derivatives inhibit the alpha isoforms of topoisomerase II and hence these molecules can be considered as a potential anticancer drug.

Keywords: Thiophene-2-carboxylates; Aldol condensation; Topoisomerase II; Nalm6 cells; Cytotoxicity

Introduction

Thiophene derivatives represent an important class of sulfur containing heterocycles that is present in numerous bioactive natural products and pharmaceutically active agents [1,2]. Thiophene derivatives are well explored as cytotoxic agents and recognized as a promising topoisomearse I inhibitor [3,4]. From the literature survey, one may presume that thiophene derivatives can bind to various DNA structures and therefore may interfere with DNA associated enzymes. Topoisomerase are considered as one of the important targets for thiophene derivatives. Cancer cells are highly metabolically active due to their proliferative nature. Each time when a cell reolicate first replication of DNA takesplace and for which role of topoisomerases is very important [5].In eukaryotes, Topoisomerase II enzyme (Topo II) is highly over expressed in robustly dividing tumor cells, and plays an important role in DNA replication, nuclear genome maintenance and transcription [6-8]. Hence, this nuclear enzyme is an attractive target for cancer chemotherapy. Nearly 50% of all cancer therapeutics contains at least one entity targeted to htopo II [9-11]. All the topoisomerase II (topo II) -targeted anticancer drugs are clinically used for their antitumor properties. Broad range of topoII inhibitors are normally classified as topo II poisons and catalytic inhibitors [12]. Topoisomerase inhibitors have played a significant role in chemotherapy. The most extensively used topoisomerase II inhibitors include etoposide, doxorubicin, daunorubicin, mitoxantrone, teniposide and amsacrine. However, it is observed that unfortunately, treatment with some of the topo II inhibitors failed after early effective therapy. Therefore, there is need for the development of novel topoisomerase II targeted drugs, with the goal of disabling current boundaries.

Results and Discussion

ChemistryOne of the important synthetic approaches for the preparation of substituted thiophene derivatives is from using functionalized polarized ketene dithioacetals as precursors [13]. However, most of the reported methods suffer from one or the other disadvantages, mainly formation of mixed alkylation products during one pot sequential alkylation with different alkyl halides. Later, selective sequential alkylation has been achieved by Asokan et al. [14], but this method is limited to introduce only alkyl thio group in the 2-position of the thiophene derivatives. Further, Ila et al. [15] have developed sequential one-pot three component route to tri- & tetra substituted/annulated thiophenes, this method produced comparatively low yields of thiophenes. In continuation of our synthetic efforts [16], we set out to identify the possible mild conditions under which, the intermediate β-alkyl thioenone would undergo intramolecular aldol condensation with synthetically useful rate. Initially we have started the synthesis of ethyl 3,5-disubstituted thiophene-2-carboxylate derivatives starting directly from 1,3-monothio-β-diketones in one pot operation. The monothio-β-diketone 3a was selected as a model substrate for optimizing the reaction condition for the formation of thiophene TH1 in the presence of various bases. Sodium hydroxide in benzene: water (2:1) system with TBAB as a phase transfer catalyst at room temperature was found to be better reaction condition for the synthesis of TH1 with 92% yield. With the optimized protocol in hand we went on to synthesize different thiophene derivatives (TH2- TH5). Thus the 1,3-monothio-β-diketones bearing electron donating and electron withdrawing substituents have also gave the considerable product yield. The probable mechanism for the formation of thiophene looks to be simple, treatment of 1,3-monothio-β-diketones with ethyl bromo acetate in-situ generates β-alkyl thioenone intermediate which underwent in-situ intramolecular aldol condensation to afford ethyl 3,5-disubstituted thiophene-2-carboxylate derivatives in good yield (Table 1) (Scheme 1).Scheme 1: Synthetic route to tri substituted thiophene derivatives.Biological studiesEffect of TH1 on various human cancer cell lines: The activity of newly synthesized thiophene compounds were checked in various human cancer cell lines such as Molt4, Nalm6, K562 and HEK 293T for their cytotoxic activity. The MTT assay results confirmed that the tested compounds inhibit the cell proliferation after 48h at different concentrations. Importantly, TH1 inhibited the growth of Molt4, Nalm6 and K562 cells in dose dependent manner with IC50 value of 68.9, 69.2 and 82.1μM respectively. Furthermore TH1 didn’t inhibit the growth of normal cells such as Human epithelial kidney (HEK293T) cells with IC50value of 141.2μM. These results suggested that TH1 inhibits the growth of human cancer cells.TH1 causes the accumulation of the cells in the SubG1 phase of the cell cycle in human leukemic cells: In order to assess the influence of TH1 on cell cycle, Nalm6 cells were treated with TH1 and examined the effect after 48h treatment of TH1 (25, 50, 75 & 100 μM) onNalm6 cells induced concentration dependent increase in SubG1 population of cell cycle and it was prominent at 100μM. This data indicated that TH1 could promote cell death through apoptosis without affecting to cell cycle arrest. The bar graph representing different stages of cell population (Figure 1A&1B) of TH1 treated cells appeared to induce cell death in concentration dependent manner in Nalm6 cells.Live dead assay for TH1 on Nalm6 cells: We carried out live dead cell assay using calcein and propidium iodide staining to confirm the cell death induced by TH1 on Nalm6 cells. Calcein stains live cells and propidium iodide stains only dead cells because of the damaged cell membrane. The 48 h treatment of TH1 showed increase in propidium iodide stained (single and double positive) cells in a dose dependent manner (50, 75 and 100 μM) and decreased number of calcein-AM stained cells which further confirming the cytotoxic effect caused by TH1 on Nalm6 cells (Figure 2A & 2B).Study of mitochondrial membrane potential assay: Study of mitochondrial membrane potential (Δψm) is an important parameter in anti-cancer drug discovery pipeline. We evaluated the effect of TH1 on mitochondrial membrane potential in Nalm6 cells using JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol -carbocyanineiodide) dye. In normal mitochondria JC-1 forms aggregate and emits red fluorescence, on the other hand the dead population with low mitochondrial membrane potential dye remains in monomeric form which emits green fluorescence. Interestingly, 48h of TH1 treatment (75 and 100 μM) lead decrease in the mitochondrial membrane potential in Nalm6 cells which was evident in the increased green fluorescence (Figure 3A&3B). This suggested that participation of mitochondrial apoptotic mechanism in induction of cell death upon treatment with TH1 in Nalm6 cells.Action of TH1 leads to induction of apoptosis: The increase in subG1 population and cell death which were evident in cell cycle analysis and live dead assay induced by TH1 on Nalm6 cells lead us to detect the mode of the cell death induced by TH1. Hence we stained the Nalm6 cells which are treated with TH1 (48 h, 75 μM) with annexin-FITC and propidium iodide to study the different types of apoptotic cell populations. The externalization of phosphatidylserine (PS) in living cells is a hallmark of apoptosis. Soon after apoptosis is induced, PS is translocated from the inner leaflet of the plasma membrane to the outer leaflet. We observed increase of early, late apoptotic cells (8.1%, 24.7%) at 75μM compared to DMSO treated control (5.7%, 3.6%) respectively (Figure 4A&4B). Results revealed that the Compound TH1 induced cell death in Nalm6 cells by inducing apoptosis.Inhibition of human DNA topoisomerase II: It was evident that TH1 is the active potent compound in tested panel in ex vivo studies. While additional studies are necessary to develop a further understanding on mechanism of action of the TH1 compound. Majority of pharmacological agents bind to DNA and exhibit their effect through interference with the activity of topoisomerase [17,18]. The interaction with cellular macromolecules is the basis for the selective anticancer activity for any class of heterocyclic compounds. Thiophene derivatives are one among them which demonstrated drugs selective cytotoxicity and caused severe disturbance of the cell cycle and inhibited topoisomerases which related to DNA binding activity. Hence, we performed topoisomerase assay using human topoisomerase II. Furthermore we analyzed the effect of TH1 on human topoisomerase II alpha mediated relaxation assay. TH1 compound showed significant inhibition of topoisomerase II alpha activity from 50 μM onwards. VP16 (Topo II inhibitor) were used as positive control (Figure 5). Our results showed that inhibitory activities of topoisomerase II enzyme.

Conclusion

In summary, we report step-wise synthesis and biological evaluation of new thiophene derivatives. Among synthesized molecules TH1 showed enhanced cytotoxicity in leukemic cell lines. Preliminary results about mechanism of action of compound suggested that it is having better capacity to induce apoptosis and substantial effect on topoisomerase II activity. These data revealed that significant correlation in the cytotoxicity. All of these above mentioned considerations tempted us to identify the capability of thiophene-2-carboxylates of generating DNA interactive species that lead to their anticancer activity.

Experimental Section

Reactions were monitored by TLC using precoated sheets of silica gel G/UV-254 of 0.25 mm thickness (Merck 60F254) using UV light for visualization. The melting points were determined on Selaco melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on an NMR spectrometer operating at 400 and 100 MHz, respectively, using the residual solvent peaks as reference relative to SiMe4. Mass spectra were recorded using high resolution mass spectrometry. Infrared spectra were recorded on Shimadzu FT-IR model 8300 spectrophotometer.Ethyl 3,5- disubstituted thiophene-2-carboxylates (TH1-5).Monothio-β-diketones (3a-e) were synthesized by the reaction between different acetophenones (1a-e) with dithioesters (2a-c) [19,20]. To a stirred solution of monothio- β-diketones 3a-e (1.0 mmol), 10% aq. Na OH (2.0 m mol) and TBAB (catalytic) in benzene (10 volume), ethylbromoacetate (1.0 m mol) was added drop wise at 0oC. The reaction mixture was brought to room temperature and further stirred for 4 h. Reaction was monitored by TLC and after the completion of the reaction, mixture was diluted with water and extracted to ethylacetate. The organic layer was washed with brine solution and dried over anhydrous sodium sulphate, filtered and solvent was evaporated under reduced pressure to get a crude product which was purified by silica gel column chromatography using hexane ethylacetate as eluent to get pure product.Ethyl 3,5-diphenylthiophene-2-carboxylate (TH1)Off white solid (92%): mp 67-69 0C; Rf 0.5 (2:8 EtOAc : Hexane); IR (KBr, Cm-1) 2976, 1668, 1602, 1462, 1435, 1365, 1269, 1131, 819, 764, 657, 501; 1H NMR (400 MHz, CDCl3) δ 7.67-7.65 (d, J = 7.2 Hz, 2H, ArH), 7.51-7.48 (m, 2H, Ar H), 7.44- 7.36 (m, 6H, ArH), 7.29 (s, 1H, C4H), 4.27-4.22 (q, J = 7.2 Hz, 2H, OCH2CH3), 1.27-1.23 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 161.0, 135.3, 135.2, 133.4, 131.0, 129.8, 129.0, 127.9, 127.3, 127.0, 124.7, 121.5, 119.9, 60.3, 14.1; HRMS (ESI) m/z Cal cd for C19H16O2S [M + Na]+ 331.0871, found 331.0895.Ethyl5-(3,4-dimethoxyphenyl)-3-(4-(trifluoromethyl) phenyl)thiophene-2-carboxylate(TH2)Pale yellow solid (86%): mp 108-110 0C; Rf 0.35 (2:8 EtOAc : Hexane); IR (KBr, Cm-1) 2988, 2849, 1678, 1627, 1461, 1299, 1152, 1098, 845, 739, 634, 538; 1H NMR (400 MHz, CDCl3) δ 7.66- 7.64 (d, J = 8.0 Hz, 2H, ArH), 7.60-7.58 (d, J = 8.0 Hz, 2H, ArH), 7.24 (s, 1H, C4H), 7.114-7.110 (d, J = 1.6 Hz, 2H, ArH), 6.90-6.88 (d, J = 8.0 Hz, 1H, ArH), 4.24-4.22 (q, J = 8.0 Hz, 2H, OCH2CH3), 3.93 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 1.26-1.22 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 149.0, 132.8, 132.8, 129.0, 127.9, 127.8, 125.7, 125.19, 125.15, 125.13, 125.11, 122.1, 120.0, 119.7, 109.4, 109.2, 61.5, 55.99, 55.95, 14.0; HRMS (ESI) m/z Calcd for C22H19F3O4S [M + Na]+ 459.444, found 459.0854.Ethyl3-(4-methoxyphenyl)-5-phenylthiophene-2- carboxylate (TH3)Off white solid (89%): mp 91-930C; Rf 0.4 (2:8 EtOAc : Hexane); IR (KBr, Cm-1) 2978, 1661, 1603, 1462, 1437, 1365, 1268, 1134, 819, 764, 657, 505; 1H NMR (400 MHz, CDCl3) δ 7.65-7.63 (d, J = 8.0 Hz, 2H, ArH), 7.45-7.32 (m, 5H, ArH), 7.25 (s, 1H, C4H), 6.94-6.92 (d, J = 8.0 Hz, 2H, ArH), 4.27-4.21 (q, J = 7.2 Hz, 2H, OCH2CH3), 3.83 (s, 3H, OCH3), 1.28-1.23 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 160.6, 145.3, 140.0, 133.4, 131.0, 129.5, 129.0, 127.9, 127.6, 127.0, 126.7, 121.5, 114.9, 60.8, 55.4, 14.1; HRMS (ESI) m/z Calcd for C20H18O3S [M + Na]+ 361.0977, found 361.0994.Ethyl5-(4-methoxyphenyl)-3-m- tolylthiophene-2- carboxylate (TH4)Yellow solid (90%): mp 97-990C; Rf 0.35 (2:8 EtOAc : Hexane); IR (KBr, Cm-1) 2999, 2843, 1674, 1479, 1448, 1297, 1256, 1242, 1019, 802, 729, 533; 1H NMR (400 MHz, CDCl3) δ 7.71-7.69 (m, 2H, ArH), 7.61-7.59 (m, 2H, ArH), 7.56-7.54 (d, J = 8.0 Hz, 2H, ArH), 7.24-7.22 (d, J = 8.0 Hz, 2H, ArH), 7.21 (s, 1H, C4H), 4.27- 4.22 (q, J = 7.0 Hz, 2H, OCH2CH3), 2.39 (s, 3H, Ar-CH3), 1.28-1.24 (t, J = 7.2 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 161.0,151.8, 137.4, 135.7, 134.6, 133.9, 130.2, 127.4, 127.5, 126.6, 126.3, 126.0, 121.5, 114.4, 60.2, 55.3, 21.4, 14.1; HRMS (ESI) m/z Calcd for C21H20O3S [M + Na]+ 375.1133, found 375.1156.Ethyl3 - (furan-2-yl)-5-(4-methoxyphenyl) thiophene-2-carboxylate (TH5)Pale yellow solid (85%): mp 101-1030C; Rf 0.3 (2:8 EtOAc : Hexane); IR (KBr, Cm-1) 2988, 2842, 1671, 1485, 1467, 1278, 1237, 1186, 1029, 828, 745, 521; 1H NMR (400 MHz, CDCl3) δ 7.63 (br s, 1H, ArH), 7.60-7.58 (d, J = 8.0 Hz, 2H, ArH), 7.559- 7.551 (d, J = 3.2 Hz, 1H, ArH), 7.24 (s, 1H, C4H), 6.93-6.91(d, J = 8.0 Hz, 2H, ArH), 6.509-6.505 (d, J = 1.6 Hz, 1H, ArH), 4.36- 4.31 (q, J = 8.0 Hz, 2H, OCH2CH3), 3.83 (s, 3H, OCH3), 1.39-1.35 (t, J = 8.0 Hz, 3H, OCH2CH3); 13C NMR (100 MHz, CDCl3) δ 160.5, 159.1, 148.8, 141.0, 135.5, 126.3, 123.6, 122.9, 116.7, 114.4, 111.5, 109.3, 106.1, 60.5, 55.1, 14.0; HRMS (ESI) m/z Calcd for C18H16O4S [M + Na]+ 351.0769, found 351.0812.BiologyAll chemical reagents were obtained from SRL, India, and Sigma-Aldrich, USA. Culture medias were from Sera Laboratory International limited (West Sussex, UK), FBS and Pen Strep were (from Gibco BRL, USA). Topoisomerase IIα enzyme (Topo GEN Inc USA).Cell lines and cultureHuman cell lines, K562, HEK 293T were purchased from National Center for Cell Science, Pune, India. Cells were grown in RPMI 1640, MEM and DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U/mL of Penicillin, and 100 μg of streptomycin/mL and incubated at 37°C in a humidified atmosphere containing 5% CO2. Nalm6 and Molt4 cells were cultured and maintained as described [21]. The cells were grown under the similar culture conditions as mentioned above.MTT assayThe antiproliferative effect of thiophene derivatives against different cancer cell lines was determined by the MTT dye uptake method as mentioned previously [22,23]. Molt4, Reh, and Nalm6 cells (5 × 104 cells/ml) were treated with different concentration of thiophene derivatives (25, 50, 75 & 100 μM) for 48 h and 100 μl cell suspensions was taken duplicates in a 96 well plate. Thereafter, 20 μ l MTT (5 mg/ml in PBS) was added and incubated for 4h at 37°C and the resulting blue crystals were dissolved upon adding 69 μl of lysis buffer (50% final concentration of N, N-dimethylformamide and 10% of sodium dodecyl sulfate, 30 min at 37°C).and subsequently the optical density (OD) was measured at 570 nm using ELISA plate reader.Cell cycle analysisThe study of DNA substance analysis in cells was performed as mentioned earlier [24]. Nalm6 cells were seeded (0.5x105 cells/ml) and treated with different concentrations of TH1 (25, 50, 75 100 μM) for 48 h. Cells were harvested, washed and fixed with 80% cold ethanol, and treated with RNase-A (50 μg/ml). Cells were stained with propidium iodide (10μg/ml) and cell cycle progression was monitored using flow cytometer (BD FACSVerse™). A minimum of 10000 cells were acquired. Results were analyzed using Flowing Software (Version 2.5) and plotted histograms.Live dead cell assayThe live dead cell assay was performed to determine the viability of cells [25]. It is a two color fluorescence assay that simultaneously determines live and dead cells. Live cells have intracellular esterases that convert non fluorescent, cell permeable Calcein acetoxymethyl (Calcein-AM). Dead cells have damaged membranes, the propidium iodide (PI) enters damaged cells and is fluorescent when bound to nucleic acids, PI produces a bright red fluorescence in damaged or dead cells [26]. Cells were treated with different concentrations of TH1 (50, 75 and 100 μM for 48 h). Cells were harvested after processed and finally acquired for live cells the excitation (max) and emission (max) are 480nm and 527/32 nm, respectively and dead cells, the excitation (max) and emission (max) are 480 nm and 586/42 nm, respectively were used (FACS Verse™, BD Biosciences, USA). Results were analyzed in FACSDIVA software and presented as dot plot. Quantification of live and dead cells was shown as bar diagram based on two independent experiments.JC-1 AssayThe study of mitochondrial permeability conversion is an important hallmark for study of apoptosis [27]. To assess the mitochondrial membrane potential, Nalm6 cells were seeded in 12 well plates (0.5 x 105 cells per ml), treated with TH1 at different concentration (75 and 100 μM). After incubation at 37°C for 48 h, cells were harvested, washed with 1x PBS and stained with (0.5 μg/ml) JC-1 dye for 30 min at 37°C. After incubation, cells were washed and re-suspended in 0.3 ml of 1x PBS and acquired in to flow cytometer (FACSVerse™, BD Biosciences, USA) using Cell Quest Pro Software. Minimum of 10,000 cells were acquired per sample and 2, 4-Dinitrophenol (2, 4-DNP) was used as positive control. Data were analyzed using Flowing Software (Version 2.5).Detection of apoptosis by Annexin V/PI stainingQuantification of apoptotic cells at the single cell level was performed by Annexin V FITC/PI (Santacruz, USA) staining. Cells were seeded in 6 well plates (0.75x105 cells/ml) and treated 75 μM TH1 for 48 h Cells were harvested and processed according to the manufactures instructions [28]. Briefly, cells were washed with 1x PBS, resuspended in binding buffer (10 m M HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) containing annexin V-FITC (0.2 mg/ml) and propidium iodide (0.05 mg/ml), incubated at room temperature for 15 min and analyzed immediately in flow cytometer (FACSVerse™, BD Biosciences, USA). A minimum of 10000 cells were acquired and analyzed the data in FACSDIVA software. Control cells were treated with equal volume of DMSO. Cells in various stages of early, late and necrotic population were plotted in dot plot and quantification was presented in bar diagram.Topoisomerase II DNA relaxation assayThe topoisomerase II assay was performed in a reaction mixture containing pBS-SK+ plasmid (Sigma Aldrich, USA), two units of recombinant human DNA topoisomerase II (Topo GENInc, USA) along with the various concentrations of test compounds [29]. Reaction was carried out at 37°C for 30 min in a relaxation buffer 1x topo II buffer (50 mMTris-Cl (pH 8.0), 10 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM dithiothreitol and 30 μgs BSA/ml) and reactions were terminated by adding 5x stop buffer containing 5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol. The DNA samples were electrophoresed on 1% agarose gel at 40 volts for 4 h with 0.5x TBE (Tris-borate-EDTA). The gels were stained for 30 min in milli Q water containing ethidium bromide (0.5 mg/ml) followed by distaining for 30 min in milli Q water and images were taken.

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Validation of the Metastatic Formula: A Method of Cervical Lymph Nodes Assessment in Oral Cancer Patients | Juniper Publishers

Juniper Publishers-Open Access Journal of Dentistry & Oral Health

Authored by Yousif I Eltohami

Abstract

Background: The Metastasis Score (MS) had been introduced in 2007 as a new method for cervical nodes assessment in oral and maxillofacial cancer patients. The metastasis score (MS) was taken from the CT scan interpretation in the preoperative assessment and was found to be reliable.

Objective: To validate and evaluate the accuracy of the metastasis score (MS); a new method for cervical lymph nodes ASSESSMENT for metastasis in oral and maxillofacial cancer patients, in comparison to histopathology results.

Materials and Method: The study was conducted in Khartoum Teaching Dental Hospital, the main oral and maxillofacial referral center, during the period 2011-2013. Clinical investigation, CT scan, the metastasis scores (MS); from the CT scan interpretation, was calculated preoperatively on 25 patients who undergone neck dissection for primary head and neck malignancy.

Results:Seven cases had a score of (0-3) and 18 cases had a score of (6-10). Twelve (48.0%) cases were positive (+ve) for neck metastasis and 13 (53.0%) cases were negative (-ve) for neck metastasis in the histopathology results. The histopathology results for the cases with metastasis score (MS) (0-3) showed (-ve) results in all the cases with an accuracy of 100% as there was no (+ve) results. For metastasis score (MS) the group (6-10) the histopathology results were (-ve) in 6 cases with an accuracy of 33.3% and it was (+ve) in 12 cases with an accuracy of 66.7%. The Sensitivity (true +ve results) and specificity (true -ve results) of this study are 100% and 53% respectively.

Conclusion: The metastasis score (MS) predicts cervical metastasis with an accuracy of 100% for the group (0-3) and with an accuracy of 66.7% for the group (6-10) as there was an incidence of false positive results; nevertheless, this group mostly present with clinically positive neck where prophylactic neck dissection is indicated.

Keywords: Shade; Compatibility; VITA; Composite

Introduction

Cancer is a generic term for a large group of diseases that can affect any part of the body. Other terms used for cancer are malignant tumors and neoplasms. It is a leading cause of death worldwide and accounted for 7.6 million deaths (around 13% of all deaths) in the year 2008. The mortality rate of it is increasing with an estimate of 13.1 million deaths by the year 2030 [1].The characteristic features in the pathogenesis of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries and which can then invade adjoining parts of the body and spread to other organs, such spread can be through lymphatic and blood vessels; this process is known as metastasis, which is the major cause of death from cancer [1].Head and neck cancer (HNC) are the sixth most common cancer globally [2]. HNC includes cancer originating in over 30 specific anatomical sites, most of them occur in the surface layers of the upper aerodigestive tract (UAT), the oral cavity, the upper part of the throat, the respiratory system (pharynx) and the voice box (larynx) [3].The oropharynx is the third commonest site among males in the developing and industrialized countries, with men affected two to three times as often as women due to alcohol and tobacco use. Alcohol, tobacco use and poor diet taken together are responsible for 90% of head and neck cancer [4]. In addition, human papilloma virus (HPV) was shown to be associated with the development of a unique papillary type of squamous cell carcinoma (SCC) within the upper aerodigestive tract, HPV-16 accounting for 90%-95% of such cases [5].In Sudan a recent study showed that oral cancer is the second most occurring cancer among all body cancers [6]. In a previous study of 261 cases of cancer, the most common pattern was intraoral squamous cell carcinoma (73.6%), with a male to female ratio of approximately 3:2 [7].Squamous cell carcinoma of the head and neck grows locally and then metastasize to cervical lymph nodes [8]. Cervical lymph nodes metastasis reduces the survival rate by up to 50% in patients with SCC of the upper aerodigestive tract [9,10]. The presence of metastatic cervical lymph nodes is very important in the prognosis and treatment planning of cancer. Cervical lymph nodes should be suggested as metastatic in patient with primary head and neck cancer and treated accordingly. However clinically palpable lymph nodes might not be metastatic and those were not detected clinically might be involved histopathological [10].Surgical treatment of these oral tumors is excision and neck dissection [8]. The difficulty in predicting the presence of metastatic disease in clinically negative necks lead to wide spread use of elective (prophylactic) neck dissection or radiation [11], increasing the risk of morbidity or even mortality for the patient [10].Pretreatment evaluation methods, palpation, ultrasonic tomography (USG) and computed tomography scan (CT scan) for the neck staging are significantly different from histopathology results and suggesting that no pretreatment study can accurately assess the requirement to histopathology [10].Computed Tomography (CT) has been available for over a decade as diagnostic tool to evaluate cervical lymph nodes since 1981 [12]. The Metastatic formula which has been suggested is an index for cervical lymph nodes assessment in oral and maxillofacial cancer patients that allows for a more proper treatment [13].In this study we aim to assess the validity of this formula among patients of oral and maxillofacial cancer at Khartoum Dental Teaching Hospital. Patients will be scored accordingly, and the score will be correlated with histopathology results.

Materials and Method

A prospective descriptive hospital-based study carried out during the period of 2010 to 2013 at Khartoum Teaching Dental Hospital, the main referral center of oral and maxillofacial cancer patients in Sudan. Clinical investigation, CT scan, the Metastasis Scores (MS); from the CT scan interpretation, was calculated preoperatively on 25 patients who undergone neck dissection for primary head and neck malignancy. Exclusion criteria were: Patient with evidence of distant metastasis (M1), surgically inoperable patient who will receive palliative care and patient who are known to be allergic to the CT scan contrast media or those cannot tolerate the contrast media. Data were collected from patients, interpretation of CT scan and histopathology reports. Data were entered in computer using the SPSS software. All statistical analysis was set at 95%cl, and all test of significance are two sided.a. Study plan: CT scan interpretation and assessment: All CT scans were taken in axial, coronal, and 3D in fine slices and were evaluated by a single radiologist for the parameters of the metastasis score (MS), named: Number of the lymph nodes per region, Shape of the lymph nodes, Presence or absence of central necrosis, and size of the lymph node.Metastasis formulaAll the above-mentioned criteria were given a score according to its metastatic possibility. The total of these scores was called metastasis score (MS) = lymph nodes number score+ lymph nodes shape score + lymph nodes necrosis score+ lymph nodes size score. Patients fulfilling the inclusion and exclusion criteria were operated in Khartoum Teaching Dental Hospital by a single expert surgeon. Excised tissues were received and sent for histopathology lab in 10% formaldehyde as follows; container of the excised primary lesion for assessment of free surgical margins and containers labeled according to excised lymph nodes level. All levels of the lymph nodes were processed and examined by single expert histopathologist for the presence or absence of malignancy. Presence of the tumor deposits in any lymph node defines the entire neck as positive for metastasis.Ethical issues: Ethical approval was obtained from the Ethical Committee at Sudan Medical Council and Research Committee Review Board at the Faculty of Dentistry University of Khartoum and from the General Directorate of Khartoum Teaching Dental Hospital. Patients were asked to participate in the study verballyResults: Twenty-five cases of oral and maxillofacial cancer patients were investigated for neck metastasis, 12 (48.0%) cases were positive (+ve) for neck metastasis and 13 (52.0%) cases were negative (-ve) for neck metastasis in a histopathology result (Table 1).The frequency for the metastasis score (MS) was taken from each case individually and from the metastasis score (MS) groups as classified by the previous study (Table 2 & 3) respectively. No patients were found to have the metastasis score (MS) 4 and 5.The histopathology results for the metastasis score group (0-3) were (-ve) in 7 cases with 100% accuracy as there was no (+ve) results. For the metastasis score (MS) group (6-10) the histopathology results were (-ve) in 6 cases with 33.3% accuracy and it was (+ve) in 12 cases with 66.7% accuracy (Table 4).The results of this study are statistically significant as P value = 0.03. The Sensitivity (true +ve results) and specificity (true - ve results) of this study are 100% and 53% respectively.

Discussion

The status of the cervical lymph nodes is the single most important prognostic factor in head and neck cancer [14]. Knowing whether metastasis is present in the neck is the corner stone in the treatment of patients. Frequently needless neck dissections are performed increasing cost and morbidity of patients. According to Van Den Brakel et al. up to 25% of patients with squamous cell carcinoma of the head and neck have exclusively micro metastasis [8]. Evidence of metastases in the neck necessitates comprehensive clearance of regional lymphatic basins. However, even if there is no evidence of lymph nodes metastases, when the risk for positive neck lymph nodes exceeds 15-20% elective neck dissection is indicated [2].CT scan has been used in the staging of head and neck tumors; evaluating local extension and neck metastasis. The criteria to consider a neck node positive have been described in several publications [8]. The present study is aimed to assess the validity of the metastasis score (MS); a new method for cervical lymph nodes metastasis, designated in a previous study by Elkulibi & Suleiman and adopted to assess oral and maxillofacial cancer patients at Khartoum Teaching Dental Hospital. The present study is designed to validate and evaluate the accuracy of the metastasis score (MS) in the assessment of cervical lymph nodes metastasis in oral and maxillofacial cancer patients.Twenty-five patients of oral and maxillofacial cancer had been investigated for cervical lymph nodes metastasis using the metastasis score (MS) and correlated with histopathology results for the presence or absence of metastasis. The findings revealed that 12 (48.0%) cases were positive (+ve) for neck metastasis and 13 (52.0%) case were negative (-ve) for neck metastasis. Two patients (8.7%) scored 0, 1(4.0%) patient scored 1, 14 (17.4%) patients scored 2, 2 (8.7%) patients scored 6, 3 (13.0%) patients scored 7, 9 (39.1%) patients scored 8 and 4 (16.0%) patients scored 9. From these findings most of the patients showed high metastasis score (MS). The authors classified the (MS) into groups according to its possibility for nodal metastasis, first group ranging from (0-3), the second group for (MS) = 4, the third group is for (MS) = 5 and the final group is for (MS) (6-10). In the present study we have 7 patients (28.0%) in the group (0-3) and 18 (72.0%) patients in the group (6-10).The resultant scores were correlated with the histopathology results with a cross tabulation test using the (SPSS) software. The results showed that the accuracy of the preoperative assessment of the metastatic status of the cervical lymph nodes in patients presenting with oral and maxillofacial cancer using the metastatic score (MS) remains superior to the previous studies and somewhat compatible with the study to be validated. It showed that the sensitivity of the assessment for neck metastasis has improved marginally. This study is consistent with the previous study in the first group (0-3), showing an accuracy of 100%.Moreover, for the Metastasis Score (MS) in the group (6-10) the histopathology results were (-ve) in 6 cases with an accuracy of 33.3% and was (+ve) in 12 cases with an accuracy of 66.7%. The histopathology findings in the surgical neck dissections provided convenient results associated with the Metastasis Score (MS) results. Our findings showed that the Metastasis Score (MS) can accurately assesses the cervical lymph nodes for metastasis in the group (0-3) with an accuracy of 100% and for the group (6-10); with an accuracy of 66.7% and an incidence of 33.3% false positive results.In general, these findings are in close agreement with the previous study, i.e. the study to be validated, although the sample doesn’t included Metastasis Score (MS) 4 and 5. Nevertheless, it suggests that CT scan when performed in the manner described and interpreted with special attention to the parameters outlined by the previous study, can provide superior information to formulate the Metastasis Score (MS).The increase in false(+ve) incidence rate in the present study than in the previous study is uncertain although similar methods were used, such as fine slices of the preoperative CT scan, image enhancement with an intravenous contrast medium and all the CT scans were assessed by a single radiologist using the currently recommended diagnostic criteria. The patients underwent surgery as soon as possible after the preoperative assessment to reduce the risk of new tumor growth influencing the results.Multiple researches had discussed the accuracy of the diagnostic techniques in assessing the cervical nodes metastasis. Techniques that in use for the assessment of cervical lymph nodes metastasis are, clinical palpation, imaging techniques such as CT, MRI, PET CT scan, SLN biopsy and ultrasound-guided fine needle aspiration cytology and all of them had been used to improve upon the results of clinical palpation alone. These diagnostic techniques showed less than 100% accuracy for neck metastasis and showed lower sensitivity and Somewhat lower specificity, thus the risk of occult disease in the neck will remain [10,15-19].Martinez-Gimeno Scoring System (MGSS) which permits a risk evaluation for neck metastases in squamous cell carcinoma of the oral cavity is a histopathology-based scoring system. On their study in 2010 the authors showed a high sensitivity of 100% and specificity of 83% in comparison to CT scan and clinical palpation [8]. Using the Metastasis Score (MS) which is a clinical method built on a CT scan as a preoperative tool for cervical lymph nodes assessment is easier than using (MGSS) which is a histopathologybased scoring system of multiple complicated parameters. In addition, necrosis of a lymph node (presence of central hypoattenuation in the lymph nodes on CT scan) is a turning point in the buildup of the Metastasis Score (MS). It had been strongly correlated with histopathology results in the previous study as its presence was the major determinant factor for lymph node metastasis histopathological, with 100% accuracy [20].The presence or absence of necrosis on the CT scan is critical, as it will add a score of 4 to the formula which will affect the final Metastasis Score (MS) and increasing the possibility for metastasis. For example, if the other parameters of the formula were 0, a positive necrosis will push the final Metastasis Score (MS) from the first group (0-3), which showed 100% accuracy for (-ve) metastasis on the histopathology, into group (MS) = 4, which showed 33% accuracy for (+ve) metastasis.This novel method of evaluating the cervical lymph nodes is easy and reliable and allows for a new way to select patients indicated for neck dissection and sparing those which are not indicated for neck dissection. In those cases, in which the probability of metastasis is low we have two options: either a wait and see policy with a close follow up of the patients; or if the patient prefers or it is not possible to do a close follow up, prophylactic neck dissection is indicated. Following this protocol, we can avoid many needless neck dissections in patients with head and neck cancer.

Limitations of the study

a. The present study was based on small sample of patients, and therefore, the incidence of false (+ve) results for the group (6- 10) may looks big when presented as a percentage and compared to the previous one.b. Poor quality CT scans will alter the scoring system as criterion of necrosis is of paramount importance for the final (MS).

Conclusion

A significant relationship was found between the metastasis score (MS) and the pathologic status of the neck with 100% sensitivity and 53% specificity. From the results of the present study the accuracy of the (MS) for predicting (+ve) histopathology results in the metastasis score (MS) group (0-3) were 100% and was 66.7% for the group (6-10) as there were false positive results. These findings agree with the findings of the previous study.

Recommendations

a. Recording of the metastasis score (MS) for any cancer patient which is easy and reliable as a preoperative assessment tool.b. Metastasis score (MS) is a postoperative reference, as both clinicians and patients will benefit from it.

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Assessment of Pharmaceutical Equivalence and In-Vitro Bioequivalence of Four Brands of Commercially Available Nifedipine Tablets in Swaziland | Juniper Publishers

Juniper Publishers-Open Access Journal of Bioequivalence & Bioavailability

Authored by Julius O Soyinka

Abstract

Background: Nifedipine is key in the management of hypertension, but also represents additional financial burden, particularly with the use of branded products. The availability of generic products permits generic substitution with a much-reduced cost of treatment. However, only generic products that offer similar bioavailability with the innovator should be considered. This study was designed to assess the pharmaceutical equivalence and in-vitro bioequivalence of generic Nifedipine tablets within Swaziland.

Objectives: Assessment of quality and in-vitro bioequivalence of four brands of Nifedipine tablets available in Swaziland in order to ascertain their safety, efficacy, stability and pharmaceutical equivalence.

Methods: Four brands of immediate release Nifedipine 20 mg tablets, coded PM, PN, EA and EN respectively, were obtained from retail pharmacy outlets and the central medical store. They were assessed for quality using the United States Pharmacopoeia (USP) guidelines. Characteristics assessed for the determination of their pharmaceutical equivalence were physical appearance, weight variation, diameter, hardness, friability, drug content and disintegration, while the determination of their dissolution rate was used in the assessment of their in-vitro bioequivalence.

Results: Sample PM, did not comply with the official specifications for weight variation, whilst all the others complied. Both samples PM and PN complied with the specifications for diameter and the thickness variation but did not pass the test for hardness. However, both samples EA and EN complied with the specification for hardness. None of the four samples complied with the specifications for both the disintegration and the dissolution rate, but they all complied for the specifications of friability and drug content.

Conclusion: Results from the study have shown that none of the brands is of acceptable quality, and the brands are not pharmaceutically equivalent. Moreover, none of the brand is likely to produce acceptable oral bioavailability, which is deduced from in-vitro bioequivalence. Thus, switching or substituting brands of Nifedipine tablets for patients should be guided by a critical assessment of the dissolution rate and other quality parameters.

Keywords: Pharmaceutical Equivalence; In-vitro Bioequivalence; Nifedipine; Bioavailability

Abbreviations: WHO: World Health Organization; BCS: Biopharmaceutics Classification System

Introduction

Hypertension is a major public health problem world-wide with its attendant high morbidity and mortality. Almost a billion of the world’s adult population exhibited signs and symptoms of hypertension with rather low detection, control and treatment rates [1]. The disease is generally managed through medication use and dietary or lifestyle changes. Nifedipine [Dimethyl-2,6-methyl-4-)2-nitrophenyl)-1,4-dihydropyridine-3, 5-dicarboxylate] (Figure 1), is a calcium channel blocking agent of the dihydropyridine type which is commonly employed in the management of systemic hypertension and angina pectoris [2]. Nifedipine has a short elimination half-life of 2-4 hours, and it is rapidly and completely absorbed over the entire gastrointestinal tract, despite its low water solubility. It is mainly administered orally in a form of both immediate release and sustained release tablet formulations, and due to the lack of major metabolic adverse effects, it is a relatively safe and well tolerable medication [3].Two or more drugs are said to be bioequivalent, when they are pharmaceutically or chemically equivalent products, and they produce comparable bioavailability characteristics in any individual when administered in equivalent dosage regimen [4]. Bioavailability is a term used in pharmacology to refer to the degree and rate at which an administered drug is absorbed by the body’s circulatory system, the systemic circulation. It determines therapeutic efficacy because it affects onset, intensity and duration of therapeutic response of a drug [5].There is a growing concern about the availability of substandard pharmaceutical products to the general public in developing countries. Such products have therapeutic as well as social and economic implications. There is little data available which points to the reasons for products being substandard, but the majority of literature reports contain no concrete evidence and assume the products to be counterfeit [6,7]. There are, however, other reasons for products being substandard, such as poor quality control during manufacture or decomposition of the active ingredient(s) [8]. The quality of drugs in lessdeveloped settings is inadequate, although concrete evidence is largely not documented. Reasons for poor quality include the widespread counterfeiting of medicines, decomposition of the active ingredient in drugs due to high temperature and humidity of storage, and poor quality assurance during the manufacture of medicinal products [8].In countries like Swaziland, where the drug control is quite inadequate, the quality of marketed drug products cannot be guaranteed. Evaluation of some of the marketed products could give an insight as to the quality of products sold & consumed and could lay basis for future corrective measures. If a drug, upon laboratory testing in accordance with the specifications it claims to comply with, fails to meet the specifications, then it is classified as a substandard drug. Substandard drugs are drug products that do not meet quality specifications set for them and they may either be genuine or counterfeit medicinal products [7]. The World Health Organization (WHO) defines a counterfeit medicine as “a medicine which is deliberately and fraudulently mislabelled with respect to identity and/or source [9]. Counterfeiting can apply to both branded and generic products and counterfeit products may include products with the correct ingredients or with the wrong ingredients, without active ingredients, with insufficient active ingredients or with fake packaging [9]. There are several reports on the presence of medicines or drugs of doubtful quality (substandard medicines) on the market in both developed and developing countries [6,7]. Consequences of substandard medicines include: reduction in bioavailability as a result of reduction in dissolution of active ingredient. In general, the high-price associated with some branded products may predispose patients to opt for generic products.In Swaziland, many generic products are in circulation, and they are often preferred by the populace because of the prevailing poor socioeconomic status. This trend has helped to curtail rising in pharmaceutical expenditure, especially in lowto middle-income countries [10]. However, generic substitution should not be based solely on the initial cost of treatment but on the overall cost effectiveness of pharmacological treatment [10]. As a result, a standard has been set for generic substitution. Interchangeability is permitted when the generic product demonstrates bioequivalence and therapeutic equivalence with the innovator.Bioequivalence of a generic product could be determined by either in-vivo or in-vitro studies. In-vivo bioequivalence studies are frequently used to establish therapeutic equivalence, but this approach is usually expensive and more rigorous and may require clinical trial or study expertise [11]. In-vitro dissolution profiles are proxies for establishing bioequivalence when the drug meets the criteria prescribed for a Biopharmaceutics Classification System (BCS) biowaiver [12]. The BCS considers three major factors: dissolution, solubility and intestinal permeability, which influence the rate and extent of drug absorption from immediate release solid oral dosage forms [12]. Biopharmaceutics Classification System class II drugs exhibit low solubility and high permeability characteristics. Their oral absorption is mostly governed by in vivo dissolution; the solubility and the dissolution rate are therefore key determinants for the oral bioavailability of these drugs. This implies that a small increase in the dissolution rate will result in a multifold increase in bioavailability [13]. Dissolution testing is an important tool employed in pharmaceutical development and in quality evaluation of solid dosage formulations [13], especially poorly soluble drugs. Dissolution testing is also used as a surrogate for in-vivo drug release and bioavailability of drugs.The pharmaceutical quality and dissolution properties of commercial Nifedipine tablet brands have been a major concern to researchers and healthcare professionals. As a result, several studies have been undertaken to assess the pharmaceutical and biopharmaceutical quality of Nifedipine tablet formulations available to the healthcare delivery system of various countries. The pharmaceutical quality evaluation of ten commercial brands of 20mg sustained release Nifedipine tablets in Nigeria showed that only four were pharmaceutically equivalent [14]. The dissolution properties of prolonged release Nifedipine tablets sampled on the Belgian market indicated that the brands were dissimilar and were therefore not interchangeable [15]. The dissolution of Nifedipine tablets produced in five different factories in China were found to comply with standards of the Chinese Pharmacopoeia but failed to meet the specifications of both the British Pharmacopoeia and the United States Pharmacopoeia [16]. Developing countries will benefit from generic products, unfortunately the resources for testing drug quality is limited.Swaziland, a landlocked country, in the southern part of Africa, is amongst the countries without a pharmaceutical regulatory body [17]. Such a body is of paramount importance as it would help to ensure a regular supply of good quality medicines. Therefore, in the absence of such a body, the probability of any infiltration of sub-standard and counterfeit medicines into the distribution chain is high. Thus, the quality of medicines entering the country is not guaranteed. Therefore, this study aimed to assess the pharmaceutical equivalence and in-vitro bioequivalence of generic formulations of immediate release Nifedipine tablets available in Swaziland.There are substantial evidences that the method of manufacture and the final formulation of the drug can markedly affect the bioavailability of the drug. In fact the World Health Organization (WHO) and all drug regulatory agencies support commercialization of generic medicines because they control costs and are irreplaceable therapeutic options in countries lacking the innovator product [18]. However, it is necessary to examine brand drug products in regard to in-vitro dissolution and in-vivo bioequivalence, and interchangeable uses with the innovator product. Therefore, in the present study in-vitro dissolution is used as a measure for bioequivalence of the four brands of Nifedipine tablets.

Materials and Methods

Nifedipine was obtained from Sigma Chemicals Co., USA. All other chemical reagents used were of pharmaceutical grade. All aqueous solutions were prepared exclusively in distilled water. Four different brands of Nifedipine 20mg immediate release tablets were obtain from retail pharmacy outlets and the Central Medical Store in Swaziland.

Assessment of parameters for pharmaceutical equivalence

Thin layer chromatographic identification: Thin layer chromatograms of pure Nifedipine (0.2 % w/v) in equal volumes of dichloromethane and methanol, and equivalent solutions of tablets obtained with ethylacetate: cyclohexane (2:3) as solvent system were compared using their Rf values and colour characteristics under ultraviolet (254nm).

Thickness and diameter: The thickness of the tablets was determined using vernier caliper and standard deviations were calculated. 10 tablets from each brand were used, and average values were calculated.

Uniformity of weight: Weight variation was determined by weighing 20 tablets from each brand individually, the average weight was calculated and the percentage variation of each tablet from the average weight of tablet was calculated [19].

Friability: The friability of the tablets was determined using 20 tablets from each brand, with a friability tester (Erweka TAR- 20) at a speed of 25rpm for 4min. The tablets were weighed before and after the friability test, and friability was determined as percent weight change [19].

Hardness: Hardness was determined by taking 10 tablets from each brand using a digital tablet hardness tester (TBH 210, Erweka) and the average of pressure (N) applied for crushing the tablet was determined [19].

Disintegration: Disintegration time was determined by taking 5 tablets from each of the brands and subjected into the disintegration tester (Manesty Tablet Disintegration Test Unit TD 75T176, England). The timer and the temperature (37 °C) were set, and Distilled water was used as the disintegrating medium. The time required to obtain complete disintegration of all the tablets was noted [19].

Drug content (Assay): 20 tablets were weighed from each of the brand, powdered and equivalent to 20 mg of Nifedipine were weighed and dissolved in sufficient quantity of methanol and make up to 100 ml with methanol. The solutions were suitably diluted with buffer solution pH 1.2 and the content of Nifedipine was estimated by measuring the absorbance in a spectrophotometer at 340nm using pH 1.2 as a blank [19].Assessment of parameter for In-Vitro bioequivalence

Dissolution rate: In-vitro dissolution rate studies were carried out using dissolution apparatus type 2 [19], in 900 ml of 0.1 M Hydrochloric acid maintained at 37±0.5 °C. The stirring speed was set at 50rpm. At predetermined time intervals a 10- ml sample was withdrawn and replaced with fresh dissolution media up to 3hrs. After appropriate dilutions, the samples were analysed by the UV spectrophotometric method using a 2-cm layer cell at 340nm. Cumulative percent of drug released was calculated and the mean of six tablets each from the different brands was used in data analysis. The dissolution profiles for the different brands of Nifedipine tablets were generated after plotting the graph of amount of Nifedipine dissolved against dissolution time. The average T70 (time for 70% of the active drug to be dissolved) and the amount dissolved at 45 minutes were obtained for each brand.

Statistical analysis: All results obtained were subjected to statistical analysis of mean and standard deviation, using Microsoft Excel 2013 version.Go toResultsImmediate release Nifedipine 20mg tablets of different brands were subjected to various evaluation tests, such as thickness, uniformity of weight, disintegration time, drug content, hardness, friability and in-vitro dissolution. As summarized in Table 1, all the brands complied with the specifications for friability, as none as a mass loss greater than 1%, and for drug content, as the drug content of all brands ranged from 96.50 to 103.70 % [19]. Table 2 showed that none of the brands complied with the specifications for disintegration and in vitro dissolution. The dissolution profiles for the four brands is illustrated in Figure 2. Only two brands, PM and PN met the specification for diameter and thickness but failed the test for hardness (Table 3). Similarly, brands EA and EN met the specifications for hardness but failed the test for diameter and thickness (Table 3). The weight variations of the tablet (Figure 1), for three of the brands were between 2.07 and 3.76% which complied with pharmacopoeia specifications, indicating the presence of an acceptable amount of drug in these brands, however, brand PM failed the uniformity of weight test [19].

Discussion

Each of the immediate release Nifedipine brands had a shelflife of three years and the tablets were analysed at least a year before their expiry dates. Pure Nifedipine had Retardation factor (Rf) value of 0.72 and a similar spot was detected in all the tablet brands of Nifedipine. This preliminary test indicated that all the tablet brands contained the active ingredient, Nifedipine. One brand, PM did not comply with the specifications for weight uniformity [19], as the weights of more than two out of twenty tablets used for the test in these brands deviated from the mean value by an amount greater than 10.0 % (Table 1). This indicated that the tablets in the batch of this brand will vary in weights outside the official limits.In the friability test as illustrated in Table 1, all the brands gave a weight loss of < 1% w/w, showing compliance with the official specification [19]. Hence, all the brands could withstand abrasion without loss of tablet integrity. Similarly, Table 1 showed that all the brands complied with the official specification for drug content of active drug content within the range 95-105 % [19]. This is an indication that all the brands when taken at the required doses are able to release the right amount of the active drug should elicit appropriate therapeutic response.It is illustrated in Table 3, that only two out of the four brands met the specifications for hardness (Brands EA and EN), and the specification for thickness and diameter (Brands PM and PN). Both these parameters have an effect on the overall disintegration time and dissolution rate of the tablets. As indicated in Table 2, none of the brands met the specification of less than 15 minutes as disintegration time for uncoated tablets [19]. The inability of these brands to disintegrate within this time limit is an important indication that the tablets will not disintegrate in the gastrointestinal tract to release their contents into the system. Similarly, none of these brands (Table 2) met the specification for in-vitro dissolution test [19], which specifies that not less than 70% w/w labelled content should dissolve at 45 minutes. The dissolution rate profile showed that all the brands did not attain 70% dissolution in 45min period of determination and consequently had low (46.64 – 60.89%) percent dissolution at 45min. Consequently, these brands may exhibit poor bioavailability profiles in-vivo as well as poor clinical performances. The disintegration time and dissolution rates have direct bearing on the bioavailability profile of tablet dosage forms as it can be used to predict the drug release pattern in-vivo [11].Bioequivalence of a generic product could be achieved either by in-vivo or in-vitro studies. In-vivo bioequivalence studies are frequently used to establish therapeutic equivalence but this approach is usually expensive and more rigorous and may require clinical trial or study expertise [20]. In-vitro dissolution profiles are proxies for establishing bioequivalence when the drug meets the criteria prescribed for a Biopharmaceutics Classification System (BCS) biowaiver [11,12]. The BCS considers three major factors- dissolution, solubility and intestinal permeability which influence the rate and extent of drug absorption from solid oral dosage forms [12]. The in-vitro and in-vivo bioequivalence correlation was launched in the ICH guidelines on Good Clinical Practice [21] and Guidance for Industry Bioavailability and Bioequivalence Studies [22], but this correlation is still debatable for some drugs, therefore, a complete and reliable bioequivalence study is the one that has data from both in-vitro and in-vivo studies.While it is a known fact that generic drugs have the advantage of reducing medical expenses, a situation which is greatly desired in low-income countries like Swaziland, it is very essential to be doubly sure that these generic drugs are of good quality, especially in terms of their safety and bioavailability. This will ensure that patients with life-threatening diseases like hypertension, are adequately treated to improve their quality of lives. This study has demonstrated that while the four different brands of immediate release Nifedipine 20mg tablets that are commercially available in Swaziland contained appropriate Nifedipine drug content, they cannot be said to be pharmaceutically equivalent, and they may demonstrate poor bioavailability, and hence inability to elicit the desired therapeutic responses in patients receiving them for the management of hypertension or angina pectoris.

Conclusion

Results from this study have shown that none of the brands is of acceptable quality, and the brands are not pharmaceutically equivalent. Moreover, none of the brand is likely to produce acceptable oral bioavailability, which is deduced from in-vitro bioequivalence. Thus, switching or substituting brands of immediate release Nifedipine for patients should be guided by a critical assessment of the dissolution rate and other quality parameters.

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