[Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal rEviEw articlE Biophytum sensitivum: Ancient medicine, modern targets Sakthivel K. M., Guruvayoorappan C. Department of Biotechnology, Karunya University, Coimbatore, India J. Adv. Pharm. Tech. Res. Abstract Research on medicinal plants began to focus on discovery of natural products as potential active principles against various diseases. Medicinal plants are very interesting, have the ability to produce remarkable chemical structures with diverse biological activities. Biophytum sensitivum is used as traditional medicine to cure variety of diseases. During the last few decades, extensive research has been carried out to elucidate the chemistry, biological activities, and medicinal applications of B. sensitivum. Phytochemical analysis have shown that the plant parts are rich in various beneficial compounds which include amentoflavone, cupressuflavone, and isoorientin. Extracts and its bioactive compounds have been known to possess antibacterial, anti-inflammatory, antioxidant, antitumor, radioprotective, chemoprotective, antimetastatic, antiangiogenesis, wound-healing, immunomodulation, anti-diabetic, and cardioprotective activity. The present review has been carried out to shed light on the diverse role of this plant in the management of various ailments facing us. Key words: Biophytum sensitivum, medicinal plants, natural products INTRODUCTION Plants are naturally gifted. In recent times, research on plants has increased across the globe owing to their immense potential to heal life-threatening diseases. A number of medicinal plants have been evaluated for their therapeutic potentials; most of them have shown their protective efects against various diseases. The secondary metabolites, especially the bioactive compounds present in the plants, provided the basis for several sophisticated traditional medicine systems like Ayurveda, Unani, Folk, and Chinese.[1] Address for correspondence: Dr. Guruvayoorappan C, Assistant Professor (SG), Department of Biotechnology, Karunya University, Karunya Nagar, Coimbatore – 641 114, Tamil Nadu, India. E-mail: immunologykarunya@gmail.com Access this article online Quick Response Code: Website: www.japtr.org DOI: 10.4103/2231-4040.97279 Due to the alarming increase in the rate of advancement of illnesses and their diagnosis, drug discovery from active constituents of the plants is gaining more importance. Drug discovery from medicinal plants involves a multifaceted approach combining botanical, phytochemical, biological, and molecular techniques. Efectiveness of the natural products, mainly the herbal extracts with their proven potential and negligible side efects in therapeutics, has already replaced the synthetically derived chemical substances as in modern allopathic medication system which is regarded as unsafe to human and environment.[2] Herbal medicines contains one or more herbal substances as active ingredients which is derived from the aerial and non-aerial parts, juices, resins, and oils of the plant either in crude state or as pharmaceutical formulation.[3,4] The chemical constituents of plant may help as cofactors for already available biologically active compounds.[5] Recently, there has been awareness on the understanding of the herbal knowledge for gaining insights into the socioeconomic and cultural components of the society. Advances in quantification and detection of herbal drug molecules provided us the better understanding of the relation between the speciic component(s) and its eicacy.[6] Just like conventional molecular drugs, the phytopharmaceuticals are involved in the development and marketing of modern herbal drugs which is clinically efective, inexpensive, and Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 83 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum globally competitive.[7] Phytomedicines (can be plants, parts of plants, and isolated bioactive compounds) have been used to treat or prevent various disorders.[6,8] Innumerable drugs from plants found worldwide have been documented in literatures, yet there continues to be untapped knowledge on the curative potential of several plants. OCCURRENCE DESCRIPTION AND B O TA N I C A L Biophytum sensitivum (L: Linnaeus) DC belonging to Division: Magnoliophyta, Class: Magnoliopsida, Order: Oxalidales, Family: Oxalidaceae, found in wet lands of tropical India, South Asia and Africa. Normally, it is present in the shades of trees and shrubs, in grass lands at low and medium altitudes. It is commonly known as Life plant (English). In India, it is also known by various vernacular names, Jhalai (Bengali), Laajjaalu, Lakshmana (Hindi), Hara muni, Jalapushpa (Kannada), Mukkuti (Malayalam), Lajwanti (Marathi), Vipareetalajjaalu, Jhulapushpa (Sanskrit), Nilaccurunki, Tintaanaalee (Tamil), Atapati, chumi, Jalapuspa, (Telugu). It has been used in traditional medicine for various ailments, especially in Indian medicine.[9,10] The lower of this plant is considered as one of the ten sacred plants which are called as Dasapushpam in tradition and culture of Kerala state in India.[11] Due to seeds remaining dormant, the cultivation of this plant has become very diicult. Shivanna et al. (2008) established a protocol to regenerate B. sensitivum through indirect and direct and somatic embryogenesis from its various explants.[12] therapeutic agent to combat life-threatening diseases lourished during the last decade. PLANT MORPHOLOGY The litle plant grows up to maximum of 20 cm and possess unbranched woody erect stem. Leaves: Leaves abruptly pinnate, lealets opposite, 6 to 12 pairs, and each lealet is up to 1.5 cm long, the terminal pair is the largest. Flower: The lowers are many and crowded at the apices of the numerous peduncles, normally yellow, white, or orange with red streak in the center of each of the ive petals. The sepals are subulate-lanceolate, striate, and about 7 mm long. Fruits: Fruits are ellipsoid capsules which are shorter than the persistent calyx.[15] PHYTOCHEMISTRY The phytochemistry of B. sensitivum showed a wide range of chemical compounds including two bilavones: cupressuflavone and amentoflavone; three flavonoids: luteolin 7- methyl ether, isoorientin and 3’-methoxyluteolin 7-O-glucoside; two acids: 4-Caffeoylquinic acid and 5-Cafeoylquinic acid which were isolated from the aerial parts of B. sensitivum.[16] It also contains 3’, 8’’-biapigenin,[17] proanthocyanidins (also known as condensed form of tannins), [18] and some phenolic compounds. [19] These compounds were isolated from the aerial part of this plant [Figure 1].[11] TRADITIONAL THERAPY Various crude extract of this plant have shown multifarious activities which includes antioxidants, anti-inlammatory, and antitumor activity. Medicinal plants become the main source for cancer drug development.[13] Pharmacological screening from leaves of the B. sensitivum showed signiicant antitumor activity in Dalton’s Lymphoma Ascites (DLA)bearing mice[14] and this represents B. sensitivum as a valuable medicinal plant with therapeutic efects. Continued research to elucidate the molecular mechanisms behind the role of antitumor activity is worthwhile. The present article aims to provide a comprehensive review on its state of knowledge about morphology, phytoconstituents, and its various biological activity of B. sensitivum as a revolutionary Tracing the history of traditional practice of B. sensitivum reveals its key action; the plant is bitter, expectorant, stimulant, and tonic in Ayurveda. It has been used as traditional medicine for several purposes. It is recommended to be used in the treatment of stomach ache, asthma, treating insomnia, convulsions, cramps, chest complaints, inflammations, tumors, and remedying chronic skin diseases. Commonly, the whole plant decoction is used for asthma and phthisis and the decoction of root is used for gonorrhea and lithiasis.[9] Speciically, the leaves are diuretic and relieve strangury and commonly known as “Nagbeli,” a folk medicine against “Madhumeha” (Diabetes mellitus), Figure 1: Structures of phytoconstituents isolated from B. sensitivum 84 Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum particularly in Eastern Nepal.[20,21] The powdered leaves and seeds were used to apply on wounds.[22] B. sensitivum is one of the plants used against snake envenomation. The whole part of plant is used to counteract the snake venom activity. [23] The major ethnopharmacological uses of B. sensitivum have been depicted in Table 1. P H A R M AC O L O G I C A L P O T E N TA L O F B. SENSITIVUM Focusing on the review of research works with various crude extract and its compounds with radioprotection, immunomodulation, antitumor, antioxidant, antibacterial, hypoglycemic, antimetastatic, antiangiogenesis, chemoprevention, anti-diabetic, anti-inflammatory are covered. The widest spectra of pharmacological activities exhibited by B. sensitivum are represented in the Figure 2. Radioprotective Action Over the past 50 years, research in the development of radioprotective agents atained an area of great signiicance due to its applications in planned radiotherapy and unplanned radiation exposure. Free-radical-induced reproductive cell death is the basis of radiotherapy, and it is clear that the main problem of this approach to ight cancer is to target the ionizing radiation to the tumor cells in order to prevent damage to healthy tissue.[24] The use of B. sensitivum as a radioprotective agent in Swiss albino mice is evaluated. The results of this study showed that B. sensitivum treatment could scavenge the free radicals generated during whole-body radiation exposure and may protect the mice from radiation-induced hemopoietic damage which is mediated through immunomodulation as well as sequential induction of IL-1β, Granulocyte colony stimulating factor (GM-CSF), and IFN-γ.[25] Induction of apoptosis in B16F-10 melanoma cells The precisely controlled cellular event apoptosis, also known as programmed cell death, is a well-recognized process essential for normal human embryogenesis as well as to maintain tissue homeostasis and protective processes.[26] The methanolic extract of B. sensitivum and amentolavone, a bilavonoid present in this plant, were able to inhibit the production of proinflammatory cytokines such as Interleukins (IL-1β and IL-6), GM-CSF, Tumor necrosis factor (TNF-α), and Nitric oxide (NO) by Tumor-associated macrophages.[27,28] Efect of B. sensitivum on cell-mediated immune response Guruvayoorappan and Kuttan evaluated the effect of B. sensitivum on mitogen-induced proliferative response of lymphocytes and on Natural Killer (NK) cell-mediated, Antibody-dependent cellular cytotoxicity (ADCC), and Antibody-dependent complement-mediated cytotoxicity (ACC) in Ehrlich ascites carcinoma (EAC)-bearing mice. The administration of B. sensitivum stimulates the immune system, leading to enhanced immune cell proliferation of splenocytes, thymocytes, and bone marrow cells by stimulating the mitogenic potential of various mitogens such as Lipopolysaccharide (LPS), Concanavalin A (Con A), Phytohemagglutinin, and Poke Weed Mitogen. NK cell activity, ADCC, and ACC were found to be enhanced in tumor-bearing animals that suggest the immunomodulatory property of B. sensitivum.[29] Immunostimulation and Antitumor Activity Figure 2: Pharmacological dimensions of B. sensitivum The agents that activate the host-defense mechanisms or modulate the immune response by stimulation or Table 1: Ethnopharmacological uses of B. sensitivum Part of the plant Ailment Group / Country Whole plant decoction as a tonic (Oral) Whole plant (Topical) Root (Oral) Leaves (Oral) Stomach ache, asthma, insomnia, convulsions, chest complaints, tumors Inflammations, chronic skin diseases Gonorrhea and Lithiasis Diabetes Powdered leaves and seeds (Topical) Whole plant decoction (Oral) Wound healing Ayurveda, Foot hills of Himalayas, India Ayurveda, India Ayurveda, India “Nagbeli” a folk medicine, Nepal, Foot hills of Himalayas, India Mali, Africa Snake envenomation Both in Ayurveda and Siddha, India Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 References 9 9 9 51,80,81 18 19 85 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum suppression are referred as Immunomodulators. The methanolic extract of B. sensitivum was screened for its immunomodulatory and antitumor activity in BALB/c mice. The administration of methanolic extract of B. sensitivum increase the total WBC count and stimulate the hematopoietic system by increasing the count of bone marrow cells and also enhanced the diferentiation of stem cells by increasing the presence of γ-esterase-positive bone marrow cells. Increase in circulatory antibody titer and antibody-forming cells indicates its stimulatory efect on the humoral arm of the immune system and production of immune cells by increasing weight of spleen and thymus. The antitumor activities of B. sensitivum extract was determined by both in vitro and in vivo methods. Administration of B. sensitivum inhibited the growth of solid tumor induced by DLA cells and ascites tumor induced by EAC cells. It also decreased cellular Glutathione (GSH), Serum Gamma Glutamyl Transpeptidase (GGT), and NO levels.[30] This was congruent to the report that the aqueous extract of B. sensitivum leaves showed signiicant antitumor activity against the transplantable murine tumor.[14] Natural antioxidant defense mechanism Antioxidative action plays a major role in protection of human beings against oxidative damage caused by the highly reactive unpaired electrons referred as free radicals, which atracted a great deal of atention in recent years. The increased lux of free radicals can react with number of biomolecules including DNA, lipids, and proteins and produce toxic efects.[31] Flavonoids can able to scavenge free radicals by means of directly donating the hydrogen atoms and also by various mechanisms.[32] The possible mechanism by which lavonoids can act is through interaction with various antioxidant enzymes.[33] The antioxidant potential of B. sensitivum was evaluated in both by in vitro and in vivo. B. sensitivum was found to inhibit in vitro lipid peroxidation and also scavenge superoxide radicals and NO in vitro. Intraperitoneal administration of B. sensitivum in BALB/c mice inhibited Phorbol-12-myristrate-13-acetate-induced superoxide radical generation in macrophages in vivo. The extract also produced a signiicant increase in catalase activity, Glutathione-S-transferase, GSH, Glutathione reductase and decrease in Glutathione peroxidase.[34] This result strongly supports its usefulness as a natural antioxidant to combat the free radical chain propagation efect. B. sensitivum was reportedly rich in lavonoids which includes Isoorientin, two bilavones: Amentolavone and Cupressulavone,[16] and Phenolic compounds; [19] Amentoflavone inhibits the cyclooxygenase-1- and cyclooxygenase-2-catalyzed prostaglandin biosynthesis.[35] A polysaccharide isolated from B. sensitivum was found to be enhancing the complement ixation.[10] These phytocompounds may be responsible for the antioxidant potential of B. sensitivum and further research works need to be initiated to understand the 86 precise mechanism behind the possible role and to ind out which active ingredient is responsible for antioxidant action. Antibacterial property The use of B. sensitivum as an anti-infective agent was demonstrated recently. The leaves extracts of B. sensitivum (methanol, chloroform, acetone, and petroleum ether) was evaluated for its antibacterial activity by them against several human pathogenic bacterial strains (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumonia, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, and Escherichia coli). All the extracts showed various remarkable levels of activity on diferent test organisms and their activity is quite comparable with the standard antibiotics. This discovery indicates a potent remedy from B. sensitivum that will be a great advancement in bacterial infection therapies.[36] Cardioprotection Coronary heart disease is characterized by the presence of high blood cholesterol which is the main risk factor for this major life-threatening disease.[37] The possible hypocholesterolemic efect of B. sensitivum leaf water extract in inbred male albino rabbits was assessed. Simultaneous administration of B. sensitivum produced a beneicial efect by improving all the parameters of lipid proile which include Very low-density lipoprotein and Low-density lipoprotein, except High-density lipoprotein, and also it causes slight lowering of plasma glucose, but without any risk of causing severe hypoglycemia.[38] Antimetastatic quality The eradication or prevention of highly complex metastatic process, the major cause of mortality in various cancer patients, remains current clinical challenge in the investigation of cancer research. This multistep process involves up and down regulations in the expression of speciic genes and various cytophysiological changes. Cancer cells from the primary neoplasm enter into the blood vessels or lymphatics and circulate in the body luids, then form a new colony at a distant site.[39,40] The treatment with B. sensitivum in B16F-10 melanoma-induced experimental lung metastasis in C57BL/6 mice significantly reduced lung collagen hydroxyproline, hexosamine, uronic acid levels, serum sialic acid, and gamma glutamyl transpeptidase. It inhibits the expression level of several proinlammatory cytokines (IL-1, IL-6, GM-CSF, and TNF) and the proteolytic enzymes matrix metalloproteinases. It also inhibits the activation and nuclear translocation of p65, p50, c-Rel subunits of nuclear factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element binding protein in B16F-10 melanoma cells.[41] Targeting angiogenesis Understanding the key molecular pathways and signaling molecules involved in the formation of new capillaries from preexisting vasculature by the tumor cells has been a highly Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum active area in the investigation of cancer therapy over the past two decades.[42] Angiogenesis is one of the essential hallmarks required for the transformation of normal cell to a cancer cell. Targeting of angiogenesis has become a promising strategy for cancer treatment by developing angiogenesis inhibitors. Inhibition of angiogenesis suppresses the growth of tumor by switching of the supply of oxygen and nutrients.[43] B. sensitivum displayed antiangiogenic activity in both in vitro and in vivo models. Intraperitoneal administration of methanol extract of B. sensitivum at a concentration of 50 mg/kg inhibited the tumor-directed capillary formation induced by B16-F10 melanoma cells and increased the level of proinlammatory cytokines such as IL-1β, IL-6, TNF-α, GM-CSF, and VGEF (Vascular Endothelial Growth Factor), the direct endothelial cell-proliferating agent. It also increased production of IL-2 and tissue inhibitor of metalloprotease-1 in B16-F10-injected and treated C57BL/6 animal models. The extract at concentrations of 1 μg/ml, 5 μg/ml, and 10 μg/ml inhibited the VGEF-induced vessel sprouting in rat aortic ring assay and inhibited the VGEFinduced proliferation, cell migration, and capillary-like tube formation of primary cultured human endothelial cells and VGEF mRNA levels in B16-F10 melanoma cells. Antiangiogenic activity of B. sensitivum was supported by its signiicant modulation of inlammatory cytokines and inhibition of VGEF expression levels.[44] Chemoprotective ability The potential and powerful approach of modern chemotherapy uses natural, synthetic, or biological chemical agents to reverse, control, or prevent the multistep process of carcinogenesis by altering its molecular and cellular events.[45,46] The ethanolic extract of B. sensitivum was studied against cyclophosphamide (CTX)-induced toxicity in mice. Intraperitoneal administration of the extract with CTX signiicantly increased the total WBC count, bone marrow cellularity, alpha-esterase-positive cells, and weight of lymphoid organs. Reduction of GSH in liver and in intestinal mucosa of CTX-treated controls was signiicantly reversed by B. sensitivum administration with amelioration of changes in serum and liver Alkaline Phosphatse, Glutamic pyruvate transaminase, , and lipid peroxidation. The level of the proinlammatory cytokine, TNF-alpha, which was elevated during CTX administration, was signiicantly reduced by the administration of B. sensitivum extract. The lowered levels of cytokines IFN-gamma, IL-2, and GM-CSF after CTX treatment were also found to be increased by B. sensitivum extract administration. Even though it did not compromise the antineoplastic activity of CTX, there is a synergistic action of CTX and B. sensitivum in reducing the solid tumors in mice was found.[47] Antidiabetic potential The inherited or acquired deficiency in production of insulin leads to hyperglycemic condition or Diabetes mellitus, as a result of high blood sugar which in turn damage many of body systems. The prevalence of diabetes mellitus is alarmingly increasing worldwide from 2.8% in 2000 to 4.4% in 2030.[48] The administration of B. sensitivum reduced the activities of gluconeogenic enzymes present in glucose homeostasis and increased the level of plasma insulin which enhances the glycogenesis in streptozotocin and nicotinamide-induced diabetic rats have recently been reported.[49] The insulinotropic efect was investigated in alloxan-induced diabetic rabbits and the results showed signiicant hypoglycemic efect possibly due to stimulating action of synthesis of insulin from beta cells of islets of Langerhans of pancreas.[50,51] Anti-inlammatory response Inflammation is the important cause known to be responsible for association with several diseases like Cancer, Diabetes, Arthritis, Parkinson’s disease, Alzheimer’s disease, and Ulcerative colitis.[52-56] The antiinlammatory action of B. sensitivum was investigated in aqueous and methanol extracts of the aerial parts and roots in the carrageenin-induced rat paw edema. The inhibition of edema was found to be maximum in aqueous extract of aerial parts and roots and methanol fraction of roots than the methanol fraction of aerial parts.[57] B. sensitivum inhibits the production of NO, proinlammatory cytokines like IL-1β, IL-6, and TNF-α, the important targets for treatment of inlammatory disorders in LPS or Con A-stimulated primary macrophages. The gene expression proile showed that B. sensitivum downregulate the expression of Inducible NO synthase and COX-2 and acts as potential inhibitor in inlammatory conditions.[58] Amentolavone—A potent bilavonoid in B. sensitivum Amentoflavone is a potent biflavonoid present in B. sensitivum and other numerous plant species which include Putranjiva roxburghii Wall. (family: Euphorbiaceae), Mangifera indica Linn. (family: Anacardiaceae), Ginkgo biloba Linn. (family: Ginkgoaceae), Selaginella sinensis, Selaginella willdenowii, Selaginella tamariscina (family: Slaginellaceae) [59,66,71] Antidesma laciniatum (family: Euphorbiaceae),[60] Tratinickia rhoifolia (family: Burseraceae),[61] Thuja orientalis (family: Cupressaceae), [62] Nandina domestica (family: Berberidaceae).[63] Cycas rumphii (family: Cycadaceae), and Trifolium alexandrinum (family: Fabaceae).[64] Amentoflavone, the active ingredient of B. sensitivum and other plants, has been shown to exhibit various pharmacological activities such as antiviral,[65,66] antiinflammatory, [67] antidepressant, [68] antioxidant, [69] analgesic activities,[70] Inhibitor of phospholipase Cγ1,[71] irreversible inhibitor of lymphocyte proliferation, [72] inhibitor of NO synthase in macrophages, [73] inhibitor of cAMP phosphodiesterase in rat adipose tissues,[74] inhibits nonenzymatic lipid peroxidation, [75] a potent cafeine-like Ca2+ releaser in skeletal muscle sarcoplasmic Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 87 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum reticulum,[58] inhibits the papain super family member of cysteine proteases—Cathepsin B,[76] a potent scavenger of superoxide,[77] and inhibitory efect on the degradation of IkBα and NF-kB translocation into the nucleus.[78] It has potential to activate the proliferation of lymphoid cells and efector cell functions by increasing the production of IL-2 and IFN-γ and could impede the level of markers of proliferating tumor cells, serum sialic acid and serum γ-GGT.[79] It also has the ability to inhibit the production of proinlammatory cytokines such as IL-1β, IL-6, GM-CSF, and TNF-α in B16F-10 cells and peritoneal macrophages;[80] antiangiogenic qualities by distracting the integrity of endothelial cells and by modulating the endogenous factors required for the neovascularization process.[81] In view of the above interpretations, Amentolavone become the potent bioactive principle from B. sensitivum and other several plants; it is evident to accept its worthwhile contribution to healthcare. Other active ingredients Isoorientin is one of the main biologically active phytochemical components of B. sensitivum. It is also reported to be present in different plants species such as Gentiana olivieri (family: Gentianaceae), [82] Cucumis sativus, Cucumis metuliferus, Cucumis myriocarpus (family: Cucurbitaceae),[83] Cymbopogon citrates (family: Poaceae),[84] Centaurea gigantean (family: Asteraceae),[85] Clematis rehderiana (family: Ranunculaceae),[86] Commelina communis (family: Commelinacae), [87] Sasa borealis (family: Poaceae),[88] Daphne gnidium,[89] Passilora edulis (family: Passiloraceae),[90] Caraipa densifolia,[91] Cecropia pachystachya (family: Cecropiaceae),[92] Patrinia villosa [93], Arum palaestinum (family: Araceae), [94] and Zea Mays (family: Poaceae).[95] Isoorientin can also synthesized from commercially available Phloroacetophenone.[96] Literatures on biological activities of isoorientin shows Hypoglycemic and antihyperlipidemic efects,[82] Antinociceptive, antiinlammatory and gastroprotective activities,[97] antioxidant potential, [98] and myolytic activity on uterine smooth muscle of rats and guinea pigs.[94] Another component present in B. sensitivum is luteolin 7-methyl ether. It is also present in Blumea balsamifera (family: Asteraceae) and exhibited strong cytotoxicity against human lung cancer cell lines (NCI-H187).[99] As documented by several workers, further work on clinical investigations of these biologically active phytocomponents seems to be highly essential. the plant B. sensitivum reported in the last decade was highlighted. The bioactivity of the various extracts of B. sensitivum corresponding to its traditional application can validate the traditional folk medicinal usage of the plant. Furthermore, the theories, beliefs, and experimental studies will hopefully broaden our knowledge base needed for designing beneicial multi-target therapeutic agents from the B. sensitivum in near future directions. Studies in the recent past indicate its potential efectiveness of pure chemical compounds atributed to it and its diverse medicinal activities. From this, it is confirmed that B. sensitivum with its active ingredients can be implicated in the maintenance of health as well as in prevention, treatment, or improvement of several disease areas. However, further exploration for development of new drug molecules and to elucidate the mechanism responsible for its therapeutic action is of paramount importance. Further research works need to be initiated to look for the possible role of this plant extract and its chemical constituents to variety of diseases in human models. ACKNOWLEDGMENT The authors acknowledge funding from the DST, New Delhi, India (Grant No: SR/FT/LS-30/2009). The valuable guidance of Dr. V. M. Berlin Grace, Head, Department of Biotechnology and Dr. M. Patrick Gomez, Director, School of Biotechnology and Health Sciences, Karunya University, is gratefully acknowledged. REFERENCES 1. Ameenah Gurib-Fakim. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol Aspects Med 2006;27:1-93. 2. Balunas Mj, Kinghorn AD. Drug discovery from medicinal plants. Life Sci 2005;78:431-41. 3. Barboza GE, Cantero JJ, Nunez C, Paccioroni A, Ariza-Espinar L. Medcinal plants - A general review and a phytochemical and ethnopharmacological screening of the native Argentina lora. Tomo 2009;34:7-365. 4. Silano M, De Vincenzi M, De Vincenzi A, Silano V. The new European legislation on Traditional Herbal Medicines: Main features and perspectives. Fitoterapia 2004;75:107-16. 5. Nahrstedt A, Butterweck VL. Lessons learned from herbal medicinal plants: The example of St.John’s Wort. J Nad Prod 2010;73:1015-21. 6. Steenkamp V. Phytomedicines for the prostate. Fitoterapia 2003;74:545-52. 7. Dev S. Impact of natural products in modern drug development. Indian J Ex biol 2010;48:191-8. CONCLUSION 8. Sahoo N, Manchikanti P, Dey S. Herbal drugs: Standards and regulation. Fitoterapia 2010;81:462-71. Medicinal plants still remains as thriving source of lifesaving drugs for the large majority of people treating health problems. During the past two decades, remarkable progress in medicinal plants research such as chemical characterization, biological, pharmacological, and toxicological activity of the plants has been witnessed. In the present review, the proven scientiic validation for 9. Jirovez L, Buchbauer G, Wobus A, Shai MP, Jose B. Medicinal used plants from India: Analysis of the essential oil of air- dried Biophytum sensitivum (L.) DC. Sci Pharm 2004;72:87-96. 88 10. Inngjerdingen KT, Colibaly A, Diallo D, Michaelsen TE, Paulsen BS. A Complement ixing polysaccharide from Biophytum petersianum Klozch, a medicinal plant from Mali, West Africa. Biomacromolecules 2006;7:48-53. 11. Varghese KJ, Anila J, Nagalekshmi R, Resiya S, Dasapushpam JS. Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum The traditional uses and the therapeutic potential of ten sacred plants of Kerala state in India. International journal of pharmaceutical sciences and research 2010; : 50-59. . 12. Shivanna MB, Vasanthakumari MM, Manala MC. Regeneration of B.sensitivum (Linn), DC through organogenesis and somatic embryogenesis. Indian J Biotechnol 2009;8:127-31. 13. Tian Z, Si J, Chang Q, Zhou L, Chen S, Xiao P, Wu E. Antitumor activity and mechanisms of action of total glycosides from aerial part of Cimicifuga dahurica targeted against hepatoma. BMC Cancer 2007;7:237. 14. Bhaskar VH, Rajalakshmi V. Anti tumour activity of aqueous extract of Biophytum sensitivum Linn. Ann. Biol Res 2010;3:76-80. 15. Kirtikar KR, Basu BD. Indian medicinal plants. Dehradun, India: International book distributors; 2005. p. 3. 16. Lin YL, Wang WY. Chemical constituents of Biophytum sensitivum. Chin Pharm Jr 2003;55:71-5. cancer. Biochem J 1996;313:17-29. 32. Prochazkova D, Bousova I, Wilhelmova N. Antioxidant and prooxidant properties. Fitoterapia 2011;82:513-23. 33. Nijveldt RJ, Van nood E, Van Hoom DE, Boelens PG, Van Norren K, Van Leeuwen PA. Flavonoids: A review of probable mechanisms of action and potential applications. Am J Clin Nutr 2001;74: 418-25. 34. Guruvayoorappan C, Aira AH, Kutan G. Antioxidant potential of B.sensitivum extract in vitro and in vivo. J Basic Clinl Physiol Pharmacol 2006;17:255-67. 35. Bucar F, Jachak SM, Noreen Y, Kartnig T, Perera P, Bohlin L, et al. Amentolavone from B.sensitivum and its efect COX-1/COX-2 catalysed prostaglandin synthesis. Planta Med 1998;64:373-4. 36. Natarajan D, Shivakumar MS, Srinivasan R. Antibacterial activity of leaf extract of Biophytum sensitivum (L.) DC. J Pharm Sci Res 2010;2:717-20. 17. Jachak SM, Bucar F, Kartnig TH, Shubert-Zsilavecz M. C-Glycosyllavones from B. sensitivum leaves. Prague, Czech Republic: 44th Annual Congress of the society for Medicinal Plant Research; 1996: p.188. 37. Zaloga GP, Harvey KA, Stillwell W, Siddiqui R. Trans faty acids and coronary heart disease. Nutr Clin Pract 2006;21:505-12. 18. Bucar FS, Jachak M, Kartnig TH, Noreen Y, Bohlin L, SchubertZsilavecz M. Phenolic compounds of Biophytum sensitivum and their activities on COX catalyzed prostaglandin biosynthesis, International Symposium of Bioassay methods in Natural Product Research and Drug Development, Uppsala University, Uppsala, Sweden: Swedish Academy of Pharmaceutical Sciences; 1997: p.49. 39. Fidler IJ, Hart IR. Biological diversity in metastatic neoplasm, origin and implications. Science 1982;217:998-1001. 38. Puri D. Hypocholestrolemic efect of Biophytum sensitivum leaf water extract. Pharm Biol 2003;41:253-8. 40. Weiss L. Metastatic ineiciency. Adv Cancer Res 1990;54:159-211. 19. Bucar FS, Jachak M, Kartnig TH, Schubert-Zsilavecz M. Phenolic compounds from Biophytum sensitivum Pharmazie 1998;53: 651-653. 41. Guruvayoorappan C, Kutan G. Antimetastatic efect of B.sensitivum is exerted through its cytokine and immunomodulatory activity and its regulatory efect on the activation and nuclear translocation of transcription factors in B16F-10 melanoma cells. J Exp Ther Oncol 2007;7:49-63. 20. Puri D, Baral N, Upadhyaya BP. Indigenous plant remedies in Nepal used in heart diseases. J Nepal Med Assoc 1997:36:334-7. 42. Tortora G, Melisi D, Ciardiello F. Angiogenesis: A Target for Cancer Therapy. Curr Pharm Des 2004;10:11-26. 21. Pant PC, Joshi MC. Studies on some controversial indigenous herbal drugs based on ethnobotanical research: A review. J Res Edu in Indian Med 1993;12:19-29. 43. Cook KM, Figg WD. Angiogenesis Inhibitors: Current Stratergies and Future Prospects. Cancer J Clin 2010;60:222-43. 22. The Wealth of India: First Supplement Series, NISC, CSIR, India, 1 A-Ci: 2000. p.140. 23. Gomes A, Das R, Sarkhel S, Mishra R, Mukherjee S, Bhatacharya S, et al. Herbs and Herbal constituents active against Snake bite. Indian J Ex boil 2010;48:865-78. 24. Paul P, Unnikrishnan MK, Nagappa AN. Phytochemicals as radioprotective agents A review. IJNPR 2011;2:137-50. 25. Guruvayoorappan C, Kutan G. Protective efect of B.sensitivum (L.) DC on Radiation- Induced Damage. Immunopharmacol Immunotoxicol 2008;30:815-35. 26. Woodle ES, Kulkarni S. Programmed cell death. Transplantation 1998;66:681-91. 27. Guruvayoorappan C, Kutan G. Apoptotic efect of Biophytum sensitivum on B16F-10 cells and its regulatory efects on nitric oxide and cytokine production on tumor- associated macrophages. Integr Cancer Ther 2007;6:373-80. 28. Guruvayoorappan C, Kuttan G. Amentoflavone stimulates apoptosis in B16F-10 melanoma cells by regulating bcl-2, p53 as well as caspase-3 genes and regulates the nitric oxide as well as proinlammatory cytokine production in B16F-10 melanoma cells, tumor associated macrophages and peritoneal macrophages. J Exp Ther Oncol 2008;7:207-18. 29. Guruvayoorappan C, Kutan G. Efect of Biophytum sensitivum on Cell-Mediated Immune response in Mice. Immunopharmacol Immunotoxicol 2007;29:337-50. 30. Guruvayoorappan C, Kutan G. Immunomodulatory and Anti tumour activity of Biophytum sensitivum Extract. Asian Pac J Cancer Prev 2007;8:27-32. 31. Wiseman H, Haliwell B. Damage to DNA by reactive oxygen and nitrogen species: Role of inlammatory disease and progression to 44. Guruvayoorappan C, Kuttan G. Anti-angiogenic effect of Biophytum sensitivum is exerted through its cytokine modulation activity and inhibitory activity against VEGF mRNA expression and endothelial cell migration and capillary tube formation. J Exp Ther Oncol 2007;6:241-50. 45. Tsao AS, Kim ES, Hong WK. Chemoprevention of Cancer. Cancer J Clin 2004;54:150-80. 46. Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-80. 47. Guruvayoorappan C, Kutan G. Evaluation of chemoprotective effect of Biophytum sensitivum (L.) DC extract against cyclophosphamide induced toxicity in Swiss albino mice. Drug metabol Drug Interact 2007;22:131-50. 48. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the 2000 and projections for 2030. Diabetes Care 2004;27:1047-53. 49. Prabu KA, Kumarappan CT, Christudas S, Kalaichelvan VK. Efect of B.sensitivum on streptozotocin and nicotinamide induced diabetic rats. Asian Pac J Trop Med 2012; 2: 31-35. 50. Puri D, Baral N. Hypoglycemic efect of B.sensitivum in the alloxan diabetic rabbits. Indian J Physiol Pharmacol 1998;42:401-6. 51. Puri D. The insulinotropic activity of a Nepalese medicinal plant Biophytum sensitivum preliminary experimental study. J Ethnopharmacol 2001;78:89-93. 52. Virani SS, Polsani VR, Nambi V. Novel markers of inlammation in artherosclerosis. Curr Atheroscler Rep 2008;10:164-70. 53. Maiti R, Agarwal NK. Artherosclerosis in Diabetes Mellitus: Role of inlammation. Indian J Med Sci 2007;5:292-306. 54. Bartsch H. Chronic infections and inlammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994;2:253-64. Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 89 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum 55. Stevens RJ, Douglas KM, Sarazis AN, Kitas GD. Inlammation and artherosclerosis in rheumatoid arthritis. Expert Rev Mol Med 2005;7:1-24. 56. da Silva MS, Sánchez-Fidalgo S, Talero E, Cárdeno A, da Silva MA, Villegas W, et al. Anti-inlammatory intestinal activity of Abarema cochliacarpos (Gomes) Barneby, Grimes in TNBS colitis model. J Ethnopharmacol 2010;128: 467-75. 57. Jachak SM, Bucar F, Kartnig Th. Anti-inlammatory activity of extracts of B. sensitivum in Carrageenin-induced Rat Paw Oedema. Phytother Res 1999;13:73-4. 58. Guruvayoorappan C, Kutan G. Methanol extract of B.sensitivum alters the cytokine profiles and inhibits iNOS and COX-2 expression in LPS/Con A stimulated macrophages. Drug Chem toxicol 2008;31:175-88. 59. Ravishankara MN, Pillai DA, Padh H, Rajani M. A Sensitive HPTLC Method for estimation of Amentolavone, a Bioactive Principle from Biophytum sensitivum (Linn.) DC. and Putranjiva roxburghii Wall. J Planar Chromat 2003;16:3-6. 60. Alembert T, Tchinda, Teshome A, Dagne E, Arnold N, Ludger A. Wessjohann. Squalene and Amentoflavone from Antidesma Laciniatum. B Chem Soc Ethiopia 2006;20:325-8. 61. Ubaldo J SS and Porcor CR.Chemical constituents of the leaves from Tratinickia rhoifolia. Avances en Quimica 2010;5:63-5. 62. Xu GH, Ryoo IJ, Kim YH, Choo SJ, Yoo ID. Free Radical Scavenging and Anti elastase activities of Flavonoids from the Fruits of Thuja orientalis. Arch Pharmacal Res 2009;32:275-82. 63. Suzuki A, Matsunaga K, Mimaki Y, Sashida Y, Ohizumi Y. Properties of Amentolavone, a potent cafeine like Ca 2+ releaser in skeletal muscle sarcoplasmic reticulum. Eur J Pharmacol 1999;372:97-102. 64. Uddin Q, Malik A, Azam S, Hadi N, Azmi AS, Parveen N, et al. The Bilavonoid, Amentolavone degrades DNA in the presence of copper ions. Toxicol in vitro 2004;18:435-40. 65. Lin YM, Flavin MT, Schure R, Chen FC, Sidewell R. Antiviral activities of biolavonoids. Planta Med 1999;65:120-5. 66. Ma SC, But PP, Ooi VE, He YH, Lee SH. Antiviral Amentolavone from Selaginella sinensis. Biol Pharm Bull 2001;24:311-2. 67. Kim HK, Son KH, Chang HW, Kang SS, Kim HO. Amentolavone, a plant biolavone: a new potential anti-inlammatory agent. Arch Pharm Res 1998;21:406-10. 68. Baureithel KH, Buter KB, Engesser A, Bukard W, Schafner W. Inhibition of enzodiazepine binding in vitro by Amentolavone, a constituent of various species of Hypericum. Pharm Acta Helv 1997;72:153-7. 69. Cholbi MR, Paya M, Alcaraz MJ. Inhibitory efects of phenolic compounds on CCl4 – induced microsomal lipid peroxidation. Experientia 1991;72:153-7. 70. Da Silva KL, Dos Santos AR, Matos PE, Yunus RA, Delle Monache F, Cechine-Filho V. Chemical composition and analgesic acivity of Calophyllum brasiliense leaves. Therapie 2001;56:431-4. 71. Lee H, Oh W, Kim B, Ahn S, Kang D, Shin D, et al. Inhibition of phospholipase Cγ1 activity by Amentolavone isolated from Selaginella tamariscina. Planta Med 1996;62:293. 72. Lee SJ, Choi JH, Son KH, Chang HW, Kang S, Kim HP. Suppression of mouse lymphocyte proliferation in vitro by naturally occuring biolavonoids. Life Sci 1995;57:551-8. 73. Woo ER, Lee JY, Cho IJ, Kim SG, Kang KW. Amentoflavone inhibits the induction of nitric oxide synthase by inhibiting NF-kB activation in macrophages. Pharmacol Res 2005;51:539-46. 74. Saponara R, Bosisio E. Inhibition of rat adipocyte cAMP phosphodiesterase by biolavones of Ginkgo biloba L. J Nat Prod 1998;61:1386-7. 75. Mora A, Paya M, Rios JL, Alcaraz MJ. Structure activity relationships of polymethoxylavones ad other lavonoids as inhibitors of non 90 enzymic lipid peroxidation. Biochem Pharmacol 1990;40:793-7. 76. Pan X, Tan N, Zeng G, Zhang Y, Jia R. Amentolavone and its derivatives as novel natural inhibitors of human Cathepsin B. Bioorg Med Chem 2005; 13:5819-25. 77. Huguet AI, Manez S, Alcaraz MJ. Superoxide scavenging properties of lavonoids in a non enzymic system. Z Naturforsch C 1990;45:19-24. 78. Banerjee T, Valacchi G, Ziboh VA, van der Vliet A. Inhibition of TNF alpha a induced cyclooxygenase-2 expression by amentolavone through suppression of NK-κ κB activation in A549 cells. Mol Cell Biochem 2002;238:105-10. 79. Guruvayoorappan C, Kutan G. Amentolavone, a biolavonoid from Biophytum sensitivum augments lymphocyte proliferation, natural killer cell and antibody dependent cellular cytotoxicity through enhanced production of IL-2 and IFN-γ and restrains serum sialic acid and gamma glutamyl transpeptidase production in tumor bearing animals. J Exp Ther Oncol 2007;6:285-95. 80. Guruvayoorappan C, Kuttan G. Amentoflavone stimulates apoptosis in B16F-10 melanoma cells by regulating bcl-2, p53 as well as caspase-3 genes and regulates the nitric oxide as well as proinlammatory cytokine production in B16F-10 melanoma cells, tumor associated macrophages and peritoneal macrophages. J Exp Ther Oncol 2008;7:207-18. 81. Guruvayoorappan C, Kuttan G. Inhibition of tumor specific angiogenesis by Amentoflavone. Biochemistry Moscow 2008;73:209-18. 82. Sezik E, Aslan M, Yesilada E, Ito S. Hypoglycemic activity of Gentiana olivieri and isolation of the active constituent through bioassay - directed fractionation techniques. Life Sci 2005;76:1223-38. 83. Krauze-Baranowska M, Cisowski W. Flavanoids from some species of the genus Cucumis. Biochem Syst Ecol 2005;29:321-4. 84. Orrego R, Leiva E, Cheel J. Inhibitory effect of three C-glycosyllavanoids from Cymbopogon citrates on Human Low Density Lipoprotein oxidation. Molecules 2009;14:3906-13. 85. Shoeb M, Jaspars M, Macmanus SM, Celik S, Nahar L, Thoo lin PK, et al. Anticolon cancer potential of phenolic compounds from aerial parts of Centaurea gigante (Asteraceae). J Nat Med 2007;61:164-9. 86. Du ZZ, Yang XW, Han H, Cai XH, Luo XD. A new lavones C-Glycoside from Clematis rehderiana. Molecules 2010;15:672-9. 87. Shibano M, Kakutani K, Taniguchi M, Yasuda M, Baba K. Antioxidant constituents in the daylower Commelina communis and their alpha glucosidase inhibitory activity. J Nat Med 2008;62:349-53. 88. Park HS, Lim JH, Kim HJ, Choi HJ, Lee IS. Antioxidant lavones Glycosides from the leaves of Sasa borealis. Arch Pharm Res 2007;30:161-6. 89. Deiana M, Rosa A, Casu V, Cotiglia F, Bonsignore L, Dessi MS. Chemical composition and antioxidant activity of extracts from Daphne gnidium L. Journal of American Oil Chemists Society 2003;80:65-70. 90. Li H, Zhou P, Yang Q, Shen Y, Deng J, Li L, et al. Comparitive studies on anxiolytic and favonoid compositions of Passilora edulis ‘edulis’and Passilora edulis ‘lavicarpa’. J Ethnopharmacol 2011;133:1085-90. 91. Silveira Da CV, Trevisan MT, Rios JB, Erben G, Haubner R, Pfundstein B, et al. Secondary plant substances in various extracts of the leaves, fruits stem, bark of Caraipa densifolia Mart. Food Chem Toxi 2010;48:1597-606. 92. Aragao DM, Guarize L, Lanini J, Da Costa JC, Garcia R M, Scio E. Hypoglycemic efects of Cecropia pachystachya in normal and alloxan induce diabetic rats. J Ethnopharmacol 2010;128:628-33. 93. Peng J, Fan G, Hong Z, Chai Y, Wu Y. Preparative separation Journal of Advanced Pharmaceutical Technology & Research | Apr-Jun 2012 | Vol 3 | Issue 2 [Downloaded free from http://www.japtr.org on Sunday, June 17, 2012, IP: 117.230.77.234] || Click here to download free Android application for this journal Sakthivel and Guruvayoorappan: Pharmacological activities of B.sensitivum of isovitexin and isorientin from Patrinia villosa Juss by high spped counter current chromatography. J Chromatogr A 2005;1074:111-5. 94. Aii FU, Khalil E, Abdalla S. Efect of isorientin isolated from Arum palaestinum on uterine smooth muscle of rats and guinea pigs. J Ethnopharmacol 1999;65:173-7. 95. Liu J, Wang C, Wang Z. The antioxidant and free radical scavenging activities of extract and fractions from corn silk (Zea mays L) and related lavone glycosides. Food Chem 2011;126:261-9. 96. Kumazawa T, Minatogawa T, Matsuba S, Sato S, Onodera J. An efective synthesis of isoorientin: The regioselective synthesis of a 6-C-glucosyllavone. Carbohydr Res 2000;329:507-13. 97. Küpeli E, Aslan M, Gürbüz I, Yesilada E. Evaluation of invivo Biological activity proile of Isoorientin. Z Naturforsch C 2004;59:787-90. 98. Lim JH, Park HS, Choi JK, Lee IS, Choi HJ. Isoorientin Induces Nrf2 pathway driven antioxidant response through Phosphatidylinositol 3- kinase signaling. Arch Pharm Res 2007;12:1590-8. 99. Saewan N, Koysomboon S, Chantrapromma K. Anti-tyrosinase and anti-cancer activities of lavonoids from Blumea balsamifera DC. J Med Plants Res 2011;5:1018-25. How to cite this article: Sakthivel KM, Guruvayoorappan C. Biophytum sensitivum : Ancient medicine, modern targets. J Adv Pharm Tech Res 2012;3:83-91. Source of Support: DST, New Delhi, India (Grant No: SR/FT/LS30/2009)., Conlict of Interest: Nil. 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