Chapter 2 Review of literature
2.REVIEW OF LITERATURE
Plants are considered as the key reservoirs of natural entities having tremendous medicinal value. It is estimated that around 2, 50,000 flowering plant species are found on the earth, of which around 1, 25,000 are found in the tropical forests and around 2000 species of higher plants are used for medicinal purposes throughout the world. However, till date only about 1% of tropical species have been exploited for their pharmaceutical potentials. India harbors more than 45,000 plant species, among these thousands have been claimed to possess medicinal properties (Botanical Survey of India, 1983; Srinivasan et al., 2001; Tag et al., 2007; Carneiro et al., 2008; Mahesh and Satish, 2008; Anonymous, 2011). Ayurveda and other traditional literature mention about 1500 plants with medicinal uses and around 800 of these have been used as ethno medicine in the treatment of various human ailments (Chopra, 2000; Vayalilet al., 2002;Rao and Das, 2011). More than 70% of the Indian population still relies on these natural product derived medicines (Paul et al., 2006). Large numbers of tropical plants have not yet been exploited in detail for their pharmacological properties or chemical constituents. Use of plants had been spontaneously recognized for centuries to resist and recover from various ailments without any untoward side effects. Emergence of drug resistance in pathogens against commonly used drugs has warranted an urgent need to develop new and safe drug with an alternate mode of action. Despite the introduction of allopathic medicines plant based medicines are much in demand. The compounds isolated from medicinal plants may serve as lead molecules for development of safe and better drugs against various diseases or may enhance the efficacy of presently available synthetic drugs when used in combination. Some of the important medicinal uses of these plants are discussed below.
2.1.MEDICINAL PLANTS AS IMMUNOMODULATORY AGENTS
Medicinal plants have tremendous potential to yield novel compounds as model for future drugs (Cragg et al., 1997). One of the fast developing area of drug development involves the search for novel immunomodulatory agents having either immune-stimulatory or immune-suppressant activity that could be used for the treatment of various immune dysfunctions or in case of residual cancer (Yamamoto, 1996;Mahiuddin and Shaikh, 2010; Singh et al., 2011). The intralesional application of BCG (Patard et al., 1998) and systemic use of chemical non-specific immunomodulator levamisole (Rahman et al., 1989; Kurman, 1993) have shown promising results, however, break-through in the field of immunomodulators was achieved when a strong immunosuppressant drug, cyclosporine (Walsh et al., 1992) was discovered that prevented the rejection of graft and had use in other auto-immune diseases. Immu-21 a polyherbal immunomodulator has been reported to promote host immune functions by augmenting both arms of the immune system i.e. humoral and cell-mediated immunity (Chatterjee, 1994; Chauhan, 1998) and has been reported to be safe and non-toxic (Das et al., 1997). There are a number of medicinal plants whose crude preparations, formulations or purified compounds have shown strong immunomodulatory activity.
Curcumin, an active ingredient of Curcuma longa (‘Haldi’ or Turmeric, Family: Zingiberaceae) and powdered root extract from W. somnifera (20 mg/dose/animal, i.p) were found to increase total WBC count, increase in circulating antibody titre against Sheep Red Blood Corpuscles (SRBC), splenic plaque forming cells (PFC), bone marrow cellularity, alpha esterase positive cells and an increase in the phagocytic activity of macrophages in BALB/c mice (Antony et al.,1999;Davis and Kuttan, 2000). The petroleum ether extract of the rhizomes of C. longa also showed significant anti-inflammatory activity and was effective in delayed hypersensitivity (Yegnarayan et al., 1976). Piper betle Linn. (Family: Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. P. betle has several landraces. Singh et al. (2009) have described the in vivo immunomodulatory efficacy in the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various doses in murine model. The crude methanol extract and n-hexane fraction were found to potentiate both the humoral (PFC and hemagglutination titre) and cellular (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The splenocytes of treated mice displayed enhanced population of CD4+ and CD8+ T cells as well as CD19+ B cells. The n-hexane fraction induced a biased type 2 cytokine response (increased IL-4, decreased IFNγ) while the chloroform fraction produced a predominant type 1 cytokine response. On the other hand, the crude methanolic extract possessed a mixed TH cell response suggesting a remarkable immunomodulatory property in this plant. Phytochemical investigations of the aerial parts of the plant Nepeta ucrainica L. (used as herbal tea in Kazakhistan) resulted in the isolation of verbascoside. The in vitro immunomodulatory activity of verbascoside was investigated by assessing neutrophil function, chemotaxis and intracellular killing activity. Verbascoside showed an increased chemotactic activity and had a positive effect on respiratory burst of neutrophils. The opposite effect was observed with increasing doses demonstrating a possible suppression of neutrophil intracellular killing activity (Akbay et al., 2002).
Tinospora cordifolia (also known as Guduchi belongs to family Menispermaceae) has been reported to contain a polysaccharide as an immunomodulatory constituent in its crude stem extract (Desai et al., 2002) and was found to impart its effect as an adjuvant to augment process of ulcer healing, bacterial eradication and polymorphonuclear phagocytosis (Purandare and Supe, 2007). Another polysaccharide isolated from the rhizomeof Alpinia galanga showed a marked stimulant effect on the reticulo-endothelial system (RES) and increased the cellularity in the peritoneal cavity as well as in spleen after treatment of mice (Bendjeddou et al., 2003). "Ginseng", a well known herbal remedy, developed mainly from the roots of Korean or Asian ginseng (Panax ginseng), Siberian ginseng (Eleutherococcus senticosu), or American ginseng (Panax quinquefolius), belong to family Araliaceae was reported to impart its immunomodulatory functions by increasing antibody concentration and natural killer (NK) cell activity in the treated individuals. It has also been reported to confer a lower incidence of influenza and colds. The main active agent in Panax ginseng is identified as ginsenoside, a triterpene saponin (Assineweet al., 2002; Tan and Vanitha, 2004; Qi et al., 2011). Like ginseng, Hydrastis canadensis (Golden seal, Family: Ranunculaceae) was also found fruitful in the treatment of cold and flu. Another well known plant Mangifera indica L. (Mango, Family: Anacardiaceae) possesses a number of therapeutic properties and an alcoholic extract prepared from its stem bark contains mangiferin which is responsible for in vivo immunostimulatory activity (Makareet al., 2001).
An independent study with ethanolic extracts of Mollugo verticillata L. (Family: Molluginaceae) has confirmed stimulation of peritoneal macrophages and increased production of nitric oxide (NO), however, if used along with BCG or Mycobacterium tuberculosis antigen, it suppressed the activity of these cells. An aqueous extract prepared from the fruits of the plant Emblica officinalis (Phyllanthus emblica; Family: Euphorbiacae) has been shown to possess antioxidant properties and protected mice from the chromosome-damaging effects of the well known carcinogen 3, 4-benzo (a) pyrene (Nandi et al., 1997) thereby increasing the life span of tumor bearing mice (Suresh and Vasudeven, 1994). Immunomodulatory effects of the aqueous extract of the tea, Camellia sinensis, was also reported in vitro in the form of enhanced production of neopterin, a sensitive marker of cell-mediated immunity (CMI) in un-stimulated human peripheral mononuclear cells, however, a reduction of neopterin formation was observed when cells were stimulated with mitogens (Zvetkova et al., 2001). Another important traditional medicinal plant, Centella asiatica (Family: Apiaceae) commonly known as “Mandukaparani” in India, is used in folk medicine as rasayana (Mukherjeeet al., 2011). The alcoholic extract of C. asiatica has been reported to have reticulo-endothelial stimulating activity and increases the antibody titer and delayed type hypersensitivity (DTH) response when fed at 100 mg/ kg body weight. The active constituent of C. asiatica was identified as Asiaticosides (Jayathirtha andMishra, 2004). The Indian Vedic plant Neem (Azadirachta indica, Family:Meliaceae) is also a powerful immunostimulatory plant reported to be non-toxic and hematostimulatory (Haque et al., 2006). The methanolic extract of Andographis paniculata (Family: Acanthaceae), also known ‘Kaal Megh, has been reported to contain andrographolide, 14-deoxyandrographolide and 14-deoxy-11, 12-didehydroandrographolide as active constituents which are attributed with the strong immunostimulatory properties. These compounds enhanced the cellular proliferation and interleukin-2 (IL-2) production in human peripheral blood lymphocytes (Kumar et al., 2004) and reversed the cyclophosphamide induced immunosuppression (Naik and Hule, 2009).
A polysaccharide fraction derived from hot water extraction ofChlorophytum borivilianum(Family: Liliaceae) comprising ~31% inulin-type fructans and~25% acetylated mannans was found to stimulate NK cell activity in vitro, however,in vivoevaluation of the aqueous extract was found to strengthen the humoral immune functions of Wistar strain of albino rats. A study conducted by Dhuley (1997) with plants Asparagus racemosus, Tinospora cordifolia, Withania somnifera and Picrorhiza kurroa revealed significant attenuation of ochratoxin carcinogen induced suppression of chemotactic activity as well as IL-1 and TNFα production by macrophages of treated mice. These plants also produced leucocytosis with predominant neutrophilia and prevented, to varying degrees, the leucopenia induced by cyclophosphamide. The plant A. racemosus induced excessive production of TNFα while W. somnifera potentiated macrophage chemotaxis, NO production over that of controls (Iuvone et al., 2003). Immunostimulatory effects have also been reported from the bark aqueous extract of pule (Alstonia scholaris, Family: Apocynaceae) in BALB/c mice. The extract stimulated non specific immune response, restored the prednisolone induced reduction of phagocytic activity and protected the body from opportunistic infection caused by Escherichia coli (Iwo et al., 2000). The hexane and aqueous extracts/ fractions of Carica papaya seeds significantly enhanced the proliferation of lymphocytes towards phytohemagglutinin (PHA) sensitization and inhibited the classical complement-mediated hemolytic pathway (Mojica-Henshaw et al., 2003).
Among the numerous plants known for their traditional medicinal use, Pterospermum acerifolium commonly known as Kanak Champa was also reported to have hepatoprotective activity. Preliminary phytochemical analysis of the leaf extract of this plant revealed the presence of several compounds like steroids, flavonoids, alkaloids and glycosides. We have reported a contrasting immunomodulatory activity in different vegetative parts of this plant i.e. a strong immunosuppressive activity in the seed part (Pathak et al., 2010) and an excellent immunostimulatory activity in the floral ethanolic extract derived from this plant (Soni et al., 2011) demonstrating the diverse pharmacological activity in different parts of the same plant. Withania coagulans has been ascribed to possess various biological activities including anti-hyperglycemic activity (Maurya et al., 2008). This plant is known to contain withanolides (withaferin-A), phenolic tannin, flavonoids and flavonols (Prasad et al., 2010). Shah and Juvekar (2008) have reported the macrophage activating effects in the form of up-regulated production of nitric oxide and augmentation in the antibody levels of animals treated with an aqueous extract from the leaves of Murraya koenigii, commonly known as curry leaf tree (Family: Rutaceae). M. koenigii extract has also been reported to have anti-hyperglycemic and cholesterol lowering effects (Shah et al., 2008). Bafna and Mishra (2010) described the in vitro antioxidant and immunomodulatory activity in mice treated with the alkaloidal fraction from the roots of Cissampelos pareira Linn in a dose dependent manner. The alkaloid fraction was found to have significant immunosuppressive activity at lower doses (25 and 50 mg/kg) while no activity was observed at higher doses (75 and 100 mg/kg) revealing the immunosuppressive and antioxidant activities in the alkaloidal fraction of C. pareira roots. Khan et al. (1995) for the first time have reported immunomodulatory activity in theethanol (50%) extracts of the seed and root of Nyctanthes arbortristisL.against systemic candidiasis in mice. Sharififar et al. (2009) demonstrated immunomodulatory activity of the aqueous extract from the fruits of Heracleum persicum Desf. (Apiaceae) at 50, 100 and 200 mg/kg in female Swiss mice. The extract was found to stimulate both humoral and cellular arms of the immune system. The alkaloids, tetrandrine found in the roots of Cyclea arnotii Miers (Family: Menispermaceae) and rohitukine present in the stem bark of Dysoxylum binectariferum Hook. f. (Family: Meliaceae) also possessed immunomodulatory properties and plant has been indicated in the treatment of various immune disorders (Khare, 2007).
The hexane, chloroform and ethanol extracts of Boerhavia diffusa (Commonly known as Punarnava, Family: Nyctaginaceae) were evaluated in vitro for their immunomodulatory activities on human peripheral blood mononuclear cells (PBMCs). The hexane extract was found to stimulate proliferation of PBMCs, LPS induced NO production and, PHA/ LPS induced IL-2 and TNFα production. The pure compound Bd-I (eupalitin-3-O-h-D galactopyranoside) purified from the ethanolic extract inhibited production of PHA stimulated IL-2 at the protein and mRNA transcript level and LPS stimulated TNFα production in human PBMCs at equivalent dose. This compound also blocked the activation of DNA binding of nuclear factor-ќ B and AP-1, the two major transcription factors centrally involved in expression of IL-2 and IL-2R gene, which are necessary for the activation and proliferation of T cells (Goyal et al., 2010).
2.2.MEDICINAL PLANTS AS ANTI-INFECTIVE AGENTS
Research efforts in herbal remedies have contributed in developing outstanding new drugs against many parasitic diseases (Li and Vederas, 2009). There are a number of plants which have been shown to contain anti-infective potential. An extract, commonly known asEndod, from the African plant Phytolacca dodecandra, is used as an effective molluscicide to control schistosomiasis (Lemma, 1991). Khan et al.(1995) have reported the significant (P < 0.05) protective effect of 50% ethanol extract prepared from the seeds and roots ofNyctanthes arbortristisL. against a systemic fungal infection of Candida albicans in Swiss mice. The mechanism behind this protection was assumed to be the strong immunostimulatory activity of arbortristoside A and C alkaloids which significantly (P < 0.001) increased the humoral and DTH response to SRBC and macrophage migration index (MMI) in BALB/c mouse. Alstonia scholaris(Pule), a medicinal plant found in South East Asia is mainly used as antimalarial and antidysentery(Iwo et al., 2000). Pterospermum acaerifolium commonly known as ‘Kanak Champa’ was also reported to have its beneficial effects in various disease conditions such as inflammation, ear pain, blood trouble, small pox, leucorrhoea, leprosy, ulcer, cancer and hemicranias, as laxative, anthelmintic, antimicrobial, hepatoprotective and in maintaining homeostasis. MK leaves mixed with fat separated butter is used for the treatment of amoebiasis, diabetes and hepatitis in Ayurveda. The steam distillate of the leaves is reported to exhibit antifungal and insecticidal activities (Khare, 2007; Pathak et al., 2010).
The ether and ethyl acetate extracts of Alpinia galanga (Family: Zingiberaceae) were screened for their antibacterial activity in vitro against different multi-resistant gram positive and gram negative bacteria isolated from hospitalized patients. Both the extracts had significant effects on Staphylococcus aureus and Klebsiella pneumonia(Thomas et al., 1996). The walnut tree contains naphthoquinone compounds (Daglish, 1950) with antihelminthic properties (Papageorgiou et al., 1979). A recent in vitro study also demonstrated its specific antimicrobial activity against Helicobacter pylori, a bacterium associated with chronic gastric ulcers. When the extract was incubated with human colonic muscosal cells and H. pylori, it resulted in the accumulation of prostaglandin E and prostacycline; the prostaglandins are known to be protective for gastric mucosa (Goel et al., 2003). The berries of WC contain fatty oil, an essential oil which is reported to be active against Micrococcus pyogenes var. aureus and Vibro cholerae and also showed anthelminthic activity. The roots of Cyclea arnotii Miers (Family: Menispermaceae) were effective against smallpox, malaria, jaundice and stomach ache. The root bears tetrandrine as a major alkaloid which was found effective in the treatment of chloroquine resistant malaria at a concentration of 0.1 g/day. A bitter principle, tamarindienal, isolated from the fruit pulp of Tamarindus indica Linn. (Family: Caesalpiniaceae), showed fungicidal and bactericidal activity against Aspergillus nigar, Candida albicans, Bacillus subtilis, Staphylococcus aureus, E. coli and Pseudomonas aeruginosa. The fruits of Adansonia digitata Linn. (also known as Gorakshi, Gorakh Imli, Gorakh Chinchaa in India, Family: Bombacaceae), have antidysenteric, antiseptic and antihistaminic properties. Oral administration of agglutinins isolated from the seeds of Abrus precatorius Linn. (Family: Fabaceae) is reported to be useful in the treatment of hepatitis and AIDS while its seed extract exhibited antischistosomal activity in male hamsters (Khare, 2007).
The root extract of Boerhavia diffusa contains basal proteins which induce strong systemic resistance in susceptible host plants against plant viruses and also induce resistance against TMV infection. The methanol extract from the leaves of the same plant, however, had significant in vitro antimicrobial activity (Goyal et al., 2010). A study with chloroform extract of aerial parts from the plants Portulaca werdermannii and P. hirsutissima, native to the semi-arid area of northeastern Brazil was conducted with an objective to evaluate its antiparasitic activities on axenic culture of Leishmania amazonensis. The study concluded a strong antileishmanial activity conferred by both the plant extracts by inhibiting the growth of L. amazonensis and thus supports these plants as potential source of new active molecules for treatment of leishmaniasis (Costa et al., 2007). B. Patwardhan (2000) described several plants or their products useful against various infections viz. Holarrhena alkaloids for amoebiasis, Mucuna pruriens for Parkinson’s disease, phyllanthins as antivirals and curcumines for inflammation. Quinine, an alkaloid isolated from the stem bark of Cinchona was reported useful in the treatment of malaria and nocturnal leg cramps (Iwu et al., 1999). An in vitrostudy aiming to find new drug candidates with antileishmanial activity was conducted on 13 plant-derived compoundsand reported two acridones, 5-hydroxynoracronycine and yukocitrine, two flavaglines, aglafoline and rocaglamide and the sulfur-containing amide methyldambullin to possess promising antileishmanial activities (Astelbauer et al., 2011). Similarly an alkaloid, tazopsine isolated from the hot aqueous stem bark extract of Strychnopsis thouarsii, a plant traditionally used against malaria in Madagascar and a few semisynthetic derivatives of this compound were investigated for inhibitory activity against liver stages of malaria protozoan parasite Plasmodium falciparumandP. yoelii in vitroand in vivo. In vitro, the alkaloid Tazopsine was found to fully inhibit the development ofP. yoeliiandP. falciparumin cultured primary hepatocytes (Carraz et al., 2006). The sesquiterpene and steroidal constituents isolated from Vernonia amygdalina (Family: Compositae) were also effective against P. falciparum in vitro (Farombi, 2003). The acetone, chloroform, hexane, petroleum ether and ethanol extracts of Annona squamosa leaves have been studied against the early fourth instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. The larval mortality was observed after 24 h exposure. All extracts showed moderate larvicidal effects, however, the highest larval mortality was found in the petroleum ether extract. The bioassay guided fractionation of Annona squamosa led to the separation and identification of saponin as a potential mosquito larvicidal compound (Kumar et al., 2011).