Chapter 2 Review of literature

2. REVIEW OF LITERATUREPlants 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; Vayalil et 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 AGENTSMedicinal 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

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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

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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 rhizome of 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 (Assinewe et 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 (Makare et 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

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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 (Mukherjee et 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 and Mishra, 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 of Chlorophytum

borivilianum (Family: Liliaceae) comprising ~31% inulin-type fructans and ~25% acetylated

mannans was found to stimulate NK cell activity in vitro, however, in vivo evaluation 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

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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 the ethanol (50%) extracts of the seed and root of

Nyctanthes arbortristis L. 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

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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 AGENTSResearch 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 as Endod, 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 of Nyctanthes

arbortristis L. 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

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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

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Chapter 2 Review of literature

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 vitro study aiming to find new drug candidates with antileishmanial activity was

conducted on 13 plant-derived compounds and 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 falciparum and P. yoelii in vitro and in vivo. In vitro, the

alkaloid Tazopsine was found to fully inhibit the development of P. yoelii and P. falciparum in

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).

Atjanasuppat et al. (2009) have screened the methylene chloride and methanol extracts of 32 plant

species for their in vitro anthelminthic activity against nematode Caenorhabditis elegans, the

digeneans Paramphistomum epiclitum and Schistosoma mansoni cercariae. Cytotoxicity of the

extracts was also evaluated against two cancer cell lines, human amelanotic melanoma (C32) and

human cervical carcinoma (HeLa). The Plumbagin, a pure compound from Plumbago indica had

the strongest activity against Caenorhabditis elegans and the methylene chloride extract of Piper

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Chapter 2 Review of literature

chaba fruits had the strongest activity against Schistosoma cercariae. However, the strong

cytotoxicity was shown by the methylene chloride extract of Michelia champaca bark and the

methanol extract of Curcuma longa rhizome against C32 and HeLa, respectively. Extracts from 6

selected plant species, used as medicinal plants by indigenous local communities in Kenya, were

screened for in vitro antiplasmodial and antileishmanial activity using Plasmodium falciparum and

Leishmania major. The best antiplasmodial activity was exhibited by methanol extract of

Suregada zanzibariensis leaves, moderate antiplasmodial activity by methanol extracts of Albizia

coriaria and Aspergillus racemosus followed by mild antiplasmodial activity by Acacia tortilis

and Albizia coriaria methanol extracts and Aloe nyeriensis var kedongensis aqueous extracts. The

remaining extracts did not exhibit any antiplasmodial activity. Although the leishmanicidal

activities of extracts were lower than for pentosam (80%), reasonable activity was observed for

Aloe nyeriensis methanol extract, Albizia coriara aqueous extract, Maytenus putterlickoides

methanol extract, Asparagus racemosus methanol and aqueous extracts, Aloe nyeriensis and

Acacia tortilis aqueous extracts at 1000 µg/ml (Kigondu et al., 2009). Tucaresol, a novel

immunomodulator, was found inactive against Leishmania donovani amastigotes in both

peritoneal and bone marrow macrophages in vitro at concentrations between 100 and 1 µM, with

toxicity to macrophages and parasites at 300 µM. However, the same compound against

L. donovani in BALB/c mice at doses between 80 and 1.25 mg/kg of body weight when

administered once daily by oral route during days 7 to 11 of infection at an optimal dose of

5 mg/kg produced 43.8 to 62.4% suppression in amastigotes numbers in the liver biopsy.

However, the drug had no activity against L. donovani infections in C.B-17 SCID mice when the

same regimen was used (Smith et al., 2000). Tylor et al. (2011) have isolated three antimalarial

compounds, N-Nitrosoxylopine, Roemerolidine and Duguevalline from the bark extract of Annona

squamosa. They reported all the compounds to be moderately active against chloroquine-sensitive

and chloroquine resistant strains of P. falciparum with IC50 values ranging between 7.8 and 34.2

μM/mL.

2.3. MEDICINAL PLANTS AS IMMUNOPROPHYLACTIC AGENTSOne of the most promising methods of controlling diseases is strengthening the defense

mechanisms of the body through prophylactic administration of immunostimulants (Jeba et al.,

2011). Immunostimulants also have ability to increase resistance to viral, bacterial and fungal

infection (Maqsood et al., 2011). Prophylactic treatment with the 50% ethanol extracts of the seed 18

Chapter 2 Review of literature

and root of the Nyctanthes arbortristis L. (p.o. once daily) provided significant protection of Swiss

mice against systemic infection of Candida albicans. Arbortristoside A and C (iridoid glucosides)

isolated from seed at 5 mg/kg (p.o. once daily) enhanced protection. Arbortristoside C was,

however, more protective (77.7%) and curative than arbortristoside A based on the increased

survival time and reduced CFUs of C. albicans from kidney homogenates of treated mice as

compared to control. The prophylactic (-7 to -1 day) and therapeutic (+1 to +7 days post-infection)

regimens (5 mg/kg) of arbortristoside A enhanced protection (71.4%), whereas, arbortristoside C

was detrimental to mice. The protective effect of these extracts or fractions may possibly be

because of the strong stimulatory activity of arbortristoside A and C elicited by significant

increase in humoral and DTH response to SRBC and macrophage migration index (MMI) in

BALB/c mouse. The combined therapy of arbortristoside A or C with ketoconazole did not

improve protection of mice (Khan et al., 1995).

Significant restriction of growth of Ehrlich's carcinoma was observed following prophylactic

treatment on Swiss albino mice with Neem leaf preparation (NLP-1 unit) once weekly for four

weeks. NLP doses were observed to stimulate hematological systems as evidenced by the increase

in total count of RBC, WBC and platelets and hemoglobin percentage. Increased antibody

production against B16 melanoma antigen was detected in mice immunized with 0.5 units and 1

unit of NLP. Number of splenic T lymphocytes (CD4+ and CD8+) and NK cells were also

observed to be increased in mice injected with 0.5 units and 1 unit of NLP. Tumor growth

restriction was observed only when mice were injected with immunostimulatory doses of NLP

(0.5 units and 1 unit) (Haque et al., 2006). The leaves of Adansonia digitata Linn. have been used

prophylactically against fevers while Chrysanthemum vulgare (L.) Bernh. (Family: Asteraceae) is

known for its significant prophylactic treatment of migraine (Khare, 2007). A pilot study explored

the antioxidative properties of oil of Linum usitatissimum (also known as Flax or linseed, belong

to Family: Lineaceae) for its prophylactic action against oxidative stress induced by a

radiomimetic drug, cyclophosphamide. Oral administration of linseed oil (0.1 mL/kg of body

weight/day) for 20 days prior to an acute dose of cyclophosphamide (75 mg/kg) significantly

inhibited the augmented level of malondieldehide, conjugated dienes, and hydroperoxides in the

mouse brain. The cyclophosphamide-induced decline in the levels of reduced glutathione,

glutathione peroxidase, and alkaline phosphatase was also significantly prevented by linseed oil in

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Chapter 2 Review of literature

mouse blood. Similarly, increased activity of acid phosphatase and oxidized glutathione was

significantly inhibited by linseed oil (Bhatia et al., 2006).

Dwivedi et al. (2008) evaluated the potential of combination therapy comprising

immunomodulator Picroliv (a standardized fraction of the ethanolic extract from the root and

rhizome of Picrorhiza kurroa, Family: Scrophulariaceae) and anti-malarial drug Chloroquine

against multi drug resistant (MDR) Plasmodium yoelii infection in BALB/c mice. The study

revealed that picroliv helped in evoking strong immune potentiating response against model

antigen in the immunized mice by increasing T-cell proliferation and antibody production. Further,

the co-administration of picroliv enhanced the efficacy of chloroquine against experimental

murine Malaria which supports that the activation of host immune system can increase the efficacy

of chloroquine for suppression of drug resistant malaria infection in BALB/c mice. Another study

with first of its kind to show the significant anti-ulcer effect of AS was performed on cold

restraint, pyloric ligation, aspirin, alcohol induced gastric ulcer and histamine induced duodenal

ulcer model and further confirmed through in vitro assay of H+ K+-ATPase activity and plasma

gastrin level. Pre-treatment of animals with ethanol extract of AS and its chloroform and hexane

fractions were found to attenuate ulcer formation and imparted gastro-protection via inhibition of

H+ K+- ATPase (proton pump) activity and simultaneous strengthening of mucosal defense

mechanism (Yadav et al., 2011).

2.4. MEDICINAL PLANTS AS CHEMOTHERAPEUTIC AGENTS OR AS

CHEMOTHERAPY ADJUNCTSThe total alkaloid fraction from the roots of AS was reported to have antihypertensive,

antispasmodic, antihistaminic and bronchodilatory properties while its leaves contain a cardiotonic

alkaloid, quinoline. The alkaloids squamone and bullatacinone were selectively cytotoxic to

human breast carcinoma, further adds the medicinal importance of this plant (Khare, 2007). Oral

administration of AS leaves’ aqueous extract (300 mg/kg) to streptozotocin (STZ) –induced

diabetic rats for 30 days was found to significantly reduce the blood glucose, urea, uric acid and

creatinine and increase the activities of insulin, C-peptide, albumin, albumin/globulin ratio and

restored all marker enzymes to near control levels (Kaleem et al., 2008).

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Chapter 2 Review of literature

A review by Joy et al. (Joy et al., 2008) described a number of plants including Alchornea

cordifolia, Cymbopogon densiflorus, Bridelia ferruginea, Ceiba pentandra, Morinda lucida, and

Vigna unguiculata to possess anti-drepanocytary (anti-sickle cell anaemia) properties. Studies on

animals with combination of artemisinin derivative and curcumin have been reported to show a

synergistic interaction in killing Plasmodium falciparum (Nandakumar et al., 2006). Among many

plant products, Saponins, the natural glycosides of steroid or triterpene exhibit many

pharmacological activities. The most widely used saponin based adjuvants are Quil A and its

derivatives QS-21, isolated from the bark of Quillaja saponaria Molina, which stimulate the Th1

immune response and the production of cytotoxic T-lymphocytes (CTLs) against exogenous

antigens. They have been used as adjuvant in vaccine formulations against HIV in guinea pig and

human, cancer in human, malaria in Aotus monkeys and mice, respiratory syncytial virus,

cytomegalovirus, and visceral leishmaniasis in mice. Likewise, ginsenosides (Ginseng saponins)

obtained from the roots of Panax ginseng was reported to increase the blood polymorphonuclear

leukocyte phagocytosis, intracellular killing, lymphocyte proliferation, and IFNγ and TNF

production. Ginseng extract could also enhance specific antibody response against Diphtheric

toxoids in mice and reported to potentiate the antibody response to porcine parvovirus in guinea

pigs. Several other plants including Astragalus membranaceus, Chenopodium quinoa and Quillaja

brasiliensis synthesizing saponins have been described with anti-infective properties (Suna et al.,

2009). Similarly alkaloids vinblatine and vincristine obtained from the plant Catharanthus roseus,

taxol obtained from Taxus and several derivatives of camptothein have proved their potential in

cancer therapy. Another alkaloid, papaverine, has been shown to have potent inhibitory effect on

the replication of several viruses including cytomegalovirus, measles and HIV (Aiyegoro et al.,

2009).

A clinical study demonstrated synergistic effect of the Chinese herbal formulations used to treat

Eczema (Williamson, 2001). A synergistic effect between plants Salvia chamelaeagnea and

Leonotis leonurus, when combined together were also reported against influenza virus and a

number of pathogens including Bacillus cereus, Escherichia coli and Klebsiella pneumoniae

(Kamatou et al., 2006). The inhibitory effect of the two antibiotics penicillin and tetracycline in

combination with various ethanolic extracts of plant Salvadora persica was observed against

Staphylococcus aureus with highest zone of inhibition (31.5 mm) in presence of a combination of

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Chapter 2 Review of literature

tetracycline and Salvadora stem extract followed by another combination with tetracycline and

leaf extract showing 30.0 mm inhibition (Ahmed et al., 2010).

Mohamed et al. (2008) have reported the hepatoprotective activity in the alcoholic and water

extracts of Annona squamosa Linn. leaves against isoniazid and rifampicin induced liver injury in

Wister rats. A number of serum parameters including alanine transaminase (ALT), aspartate

transaminase (AST), serum glutamyl oxaloacetate transaminase (SGOT), alkaline phosphatase

(ALKP) and total bilirubin were found to be decreased in treatment group as compared to the

hepatotoxic group. Although the extracts could not completely revert the liver injury caused by

isoniazid + rifampicin, they could limit the effect of these drugs in liver. Similarly, Raj et al.

(2009) have reported the hepatoprotective activity in the ethanolic extract of Annona squamosa

leaves against diethylnitrosamine (DEN) induced liver carcinoma/ injury in Swiss mice and

reported decrease in SGOT, ALKP and bilirubin in DEN pre-treated mice when fed with the above

extract over those which received only DEN.

Betoni et al. (2006) evaluated the in vitro synergistic effects among eight plant extracts viz. Guaco

(Mikania glomerata), Guava (Psidium guajava), Clove (Syzygium aromaticum), Garlic (Allium

sativum), Lemongrass (Cymbopogon citratus), Ginger (Zingiber officinale), Carqueja (Baccharis

trimera) and Mint (Mentha piperata) in addition to their combinations with thirteen of the

antimicrobial drugs against Staphylococcus aureus strains. Amongst these combinations, Clove,

Guava and Lemongrass produced highest synergistic effects with antimicrobial drugs and

inhibited S. aureus growth, while ginger and garlic showed limited synergistic activity. Another

study aimed to evaluate the interaction between water extracts of Psidium guajava, Rosmarinus

officinalis, Salvia fruticosa, Majorana syriaca, Ocimum basilucum, Syzygium aromaticum, Laurus

nobilis and Rosa damascena alone as also their synergistic activities with known antimicrobial

agents viz. Oxytetracycline HCl, Gentamicin sulfate, Penicillin G, Sulfadimethoxine and

Enrofloxacin using five S. aureus isolates. These interactions demonstrated additive effects of

these plant extracts and antimicrobial agents against five strains of S. aureus with significant

reduction in the MICs of the test antibiotics (Adwan and Mhanna, 2008).

Many compounds of plant origin such as alkaloids, phenolics, polyphenolics, flavonoids,

coumarins, saponins, lectins, tannins and triterpenes have been identified to inhibit different stages

of virus i.e. virus adsorption, cell fusion, reverse transcription, integration, translation, proteolytic

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Chapter 2 Review of literature

cleavage and glycosylation, or assembly/ release in the replication cycle of HIV and are helpful in

cancer chemotherapy (Vlietinck et al., 1998). The purified chloroform fractions identified as

vernodaline, vernolide and vernomygdine from the plant Vernonia amygdalina (Family:

Compositae) possessed cytotoxic effects in human carcinoma of nasopharynx cells. Vernodaline

and vernolide were also reported to elicit antitumoral activities in leukemia cells P-388 and C-

1210. The leaves of this plant have been reported to contain luteolin, luteolin 7-0, β-glucuronoside

and luteolin 7-O-β-glucoside flavonoid compounds which have antioxidant activities. Recently

peptides (edotides) isolated from the aqueous extract of V. amygdalina were shown to be the

potent inhibitors of mitogen-activated proteins kinases (MAPKs) which are crucial for breast

tumor growth and also represent a key regulatory point for tumor growth. Furthermore, extracts

from V. amygdalina have also been suggested to have cell growth inhibitory effects in prostate

cancer cell line (PC-3) without any effect on normal human peripheral blood mononuclear cells

(PBMC) (Farombi, 2003).

Another plant Hibiscus sabdariffa L (Family: Malvaceae) was reported to contain gossypetin,

glucoside, bibiscin, hibiscus anthocyanin and hibiscus protocatechuic acid (PCA, a simple

phenolic compound) in its flowers which may have diuretic and choleretic effects, decreasing the

viscosity of the blood, reducing blood pressure, and stimulating intestinal peristalsis. The crude

extract derived from the dried flowers of this plant is reported to have strong antioxidant potential

as they inhibited xanthine oxidase activity, formation of malondialdehyde and scavenged 1, 1-

dipheny 1-2-picrylhydrazide (DPPH) free radicals most effectively. The extract inhibited the

unscheduled DNA repair, cytotoxicity and genotoxicity induced by tertbutylhydroperoxide (t-

BHP) in the rat hepatocyte cultures thereby protect rat hepatocytes. This may be due to the

presence of PCA, which significantly decrease the leakage of lactate dehydrogenase (LDH) and

alanine transaminase (ALT) and the formation of malondialdehyde induced by t-BHP in rat

primary hepatocytes (Tseng et al., 1996 and 1997; Farombi, 2003). Guggulsterone [4, 17 (20)-

pregnadiene-3, 16-dione] is a plant sterol derived from the gum resin (guggulu) of the tree

Commiphora mukul and has been reported to possess antiarthritic and anti-inflammatory activities

by inhibiting inducible nitric oxide synthetase (iNOS) induced by lipopolysaccharide (LPS) in

macrophages. Another compound Curcumin (diferuloylmethane) isolated from Curcuma longa

(Turmeric) has been shown to suppress carcinogenesis of the various organs, lower down blood

cholesterol level, promote wound healing and skin wrinkling, suppress rheumatoid arthritis and

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Chapter 2 Review of literature

inhibit inflammation and HIV replication. Similarly, Zerumbone, a terpenoid isolated from an

essential oil of the rhizomes of a wild ginger, Zingiber zerumbet has been found to suppress the

proliferation of colon cancer, breast cancer and dextran sodium sulphate-induced colitis in mice

and the activation of phorbol ester-induced Epstein-Barr virus. Experimental studies have shown

WS to possess anti-inflammatory, antitumour, cardioprotective and antioxidant properties. It bears

a compound Withaferin A which is a reported as a potent inhibitor of angiogenesis and exerts a

positive influence on the endocrine, urogenital and central nervous systems (Aggarwal et al.,

2006).

The extract from the leaves of Vitex negundo Linn. (Family: Verbenaceae) revealed

hepatoprotective effect on d-galactosamine (Yang et al., 1987) and carbon tetrachloride (CCl4)

(Tasduq et al., 2008; Raj et al., 2008) induced liver damage. An alcoholic extract of the whole

plant of Boerhavia diffusa also exhibited hepatoprotective activity against CCl4 induced

hepatotoxicity in rats and mice, when fed orally (Goyal et al., 2010). Another study showed that

trip chloride from Tripterygium wilfordii is useful in the treatment of rheumatoid arthritis due to

its inhibitory action on production of PGE2 by synovial cells (Singh, 1999).

A study was designed to investigate the immunomodulatory effect of Picroliv in combination with

paromomycin and miltefosine using Leishmania donovani hamster model. Picroliv significantly

enhanced the antileishmanial efficacy and lymphocyte proliferation when given in combination

with paromomycin and miltefosine. Increased production of toxic oxygen metabolites and

phagocytosis were also observed thus establishing the possible use of picroliv as adjunct to anti-

leishmanial chemotherapy (Sane et al., 2010). In a clinical trial, the therapeutic efficacy of WC

Dunal seeds and Trigonella foenum-graecum Linn (Family: Papilionaceae) powder was studied on

patients suffering from type 2 diabetes mellitus. The study reported a significant reduction in

blood sugar level in test group as compared to control group (Alam et al., 2009).

2.5. MEDICINAL PLANTS AS ANTIFILARIAL AGENTSHelminth infections are among the most common infections in humans, affecting a large

proportion of population all over the world and pose a large threat to public health. Lymphatic

filariasis (LF), a mosquito-borne disease, is one of the most prevalent helminth infections in

tropical and subtropical countries and is accompanied by a number of pathological conditions. Due

to the lack of an adulticidal drug or vaccine against the adult filarial parasite, the disease has got 24

Chapter 2 Review of literature

an uncontrolled prevalence in tropical countries (Singh et al., 2010a). Hence it is essential to

develop an effective antifilarial drug which could either kill or permanently sterilize the adult

filarial parasites and also produce less toxicity or side effects. WHO has recognized the potential

of plant derived products as natural medicine (WHO, 2002). The Government of India also

showed interest in the traditional system of treatment of the diseases and constituted a separate

department named as Department of AYUSH (Ayurveda, Yoga, Unani, Siddha and

Homoeopathy) in an effort to integrate this legacy (AYUSH, 2007).

Secondary metabolites produced by plants constitute a rich source of bioactive substances and

have recently gained attention of the scientific community for the search of new drugs from plant

origin. Till date, majority of the drugs available are in fact derived from the medicinal plants or

prepared as analogs to the active entities (leads). The extracts derived from Piper betle L. leaves

demonstrated in vitro antifilarial as well as antileishmanial activity against B. malayi and L.

donovani (Tripathi et al., 2006). Crude extract prepared from the stem portion of Lantana camara

revealed significant antifilarial activity against B. malayi and sterilized surviving female worms in

vivo in animal models (Misra et al., 2007). The n-hexane fraction of P. betle induced biased type 2

cytokine response characterized by an increased IL-4 and decreased IFNγ production while the

chloroform fraction triggered a predominant type 1 cytokine response in BALB/c mice and

exhibited antifilarial activity against human lymphatic filariid B. malayi (Singh et al., 2009). The

extracts from three plant species i.e Xylocarpus granatum (Family: Meliaceae), Tinospora crispa

(Family: Menispermaceae), Andrographis paniculata (Family: Acanthaceae) showed in vitro

activity against adult worms of B. malayi (Zaridah et al., 2001). The bark of Streblus asper shrub

was effective against chronic stages of filarial infection (Singh and Ram, 1988). The extracts from

parts of Carapa procera, Polyalthia suaveolens and Pachypodanthium staudtii also exhibited

microfilaricidal activity on Onchocerca volvulus (Titanji et al., 1990).

Mathew et al. (2007) have screened the methanolic extracts of 20 medicinal plants at 1–10 mg/ml

for in vitro macrofilaricidal activity by worm motility assay against adult cattle filarial worm,

Setaria digitata. Four plant extracts showed macrofilaricidal activity by worm motility at

concentrations below 4 mg/ml and an incubation period of 100 min. Complete inhibition of worm

motility and subsequent mortality was observed at 3, 2, 1 and 1 mg/ml concentrations of the

extracts prepared from Centratherum anthelminticum, Cedrus deodara, Sphaeranthus indicus and

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Chapter 2 Review of literature

Ricinus communis respectively. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide

(MTT) reduction assay was carried out at 1 mg/ml over a 4 hour exposure, and the results showed

that C. deodara, R. communis, S. indicus and C. anthelminticum exhibited 86.56, 72.39, 61.20 and

43.15% inhibition respectively in MTT reduction (formazan formation) as compared to that of

control. Mathew et al. (2008) also identified a lead molecule against the adult bovine filarial worm

Setaria digitata from the methanolic extract of fruits of Trachyspermum ammi (Family: Apiaceae)

in vitro. The crude extract and the active fraction showed significant activity against the adult S.

digitata by both a worm motility and MTT reduction assays. The isolated active principle was

chemically characterized by IR, 1H-NMR and MS analysis and identified as a phenolic

monoterpene. It was further screened in vivo against B. malayi in a rodent model Mastomys

coucha and showed profound macrofilaricidal activity apart from female worm sterility.

Singh et al. (2009) have reported antifilarial activity in the plant Piper betle Linn. (Family:

Piperaceae). Based on the immunostimulatory findings, the antifilarial activity of crude methanol

extract, chloroform and n-hexane fractions of P. betle was evaluated against B. malayi in

mastomys. The crude MeOH extract was reported to suppress microfilaraemia, caused significant

female worm sterilization and had moderate action on adult worms (noticeably at 100 mg/kg) as

compared to the untreated control groups. However, both the fractions led to suppressed

microfilaraemia at 30 mg/kg dose on day 90. Further, oral administration of the P. betle extract

and both the fractions (n-hexane and chloroform) at 100 mg/kg for 5 consecutive days caused

significant increase in NO production correlating well with the reduced blood microfilarial

density. The extract and fractions also led to increased T- and B-cell proliferation in presence of

mitogens as well as filaria-specific antigen and increased IgG antibody production. The effect of

dried husks (HX), dried seeds (SX) and dried leaves (LX) of Xylocarpus granatum, dried stem

(ST) of Tinospora crispa and dried leaves (LA) of Andrographis paniculata were tested in vitro

on adult B. malayi parasites for antifilarial activity in which SX extract of X. granatum

demonstrated the best activity, followed by the LA, ST, HX and LX extract (Zaridah et al., 2001).

Gaur et al. (2007) evaluated the antifilarial activity of Caesalpinia bonducella seed kernel crude

extract and its various fractions against rodent filarial parasite Litomosoides carinii in cotton rats

and against human filarial parasite B. malayi in mastomys. The crude extract and fractions showed

microfilaricidal, macrofilaricidal and female-sterilizing efficacy against L. sigmodontis and

microfilaricidal as well as female-sterilizing efficacy against B. malayi in animals. Sahare et al.

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Chapter 2 Review of literature

(2008) have reported significant inhibition in motility of microfilariae of B. malayi after in vitro

incubation for 48 h with aqueous/ methanol extracts (in the concentration range of 20 to 100

ng/ml) prepared from the roots of Vitex negundo L. (Family: Verbenaceae), roots and leaves of

Butea monosperma L. (Family: Fabaceae) and leaves of Aegle marmelos Corr. (Family:

Rutaceae). Methanolic extracts from Centratherum anthelminticum, Cedrus deodara,

Sphaeranthus indicus and Ricinus communis had macrofilaricidal activities and completely

inhibited worm motility in vitro (3-1 mg/ml) when exposed for 100 min (Mathew et al., 2007).

Misra et al. (2011) have reported the in vitro and in vivo antifilarial activity in the fruit extract of

Xylocarpus granatum, a mangrove plant from Andaman, India. The various extracts/ fractions and

pure molecule/s of X. granatum fruits were evaluated in vitro on adult worms and microfilariae of

B. malayi and the active samples were further evaluated in vivo in the primary and secondary in

vivo screens. The crude aqueous ethanolic extract was active in vitro and demonstrated 52.8% and

62.7% adulticidal and embryostatic effect on B. malayi, respectively, in mastomys at a dose of

5×50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-

soluble fraction which revealed IC50 of 8.5 and 6.9 μg/ml in case of adult and Mf, respectively.

This fraction possessed moderate adulticidal and embryostatic action in vivo in mastomys. Out of

eight pure molecules isolated from the active fraction, two compounds gedunin and photogedunin

at 5×100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the

death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in

addition to noticeable microfilaricidal action on the day of autopsy.

The above review of literature demonstrates the importance of natural products especially plant

derived test substances in treatment of various diseased conditions. In spite of rich biodiversity in

India and the traditional knowledge available through Ayurveda, Unani and Homeopathy, the

pharmacological exploitation of these medicinal plants has still been limited. Though some of the

plants have been extensively investigated, others await thorough investigation. Not only this, the

scientific validation of the traditional value of the medicinal plants need in depth study. Numerous

studies on the pharmacological activity of WS have been conducted in the past and the plant has

been used in many formulations available in the market. However, it becomes important to

identify the active constituents present in the crude extract which is responsible for biological

activity. Since it is well known that the active constituents also vary in the plant during different

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Chapter 2 Review of literature

seasons and geographical variation also has impact on the compound composition. Thus it

becomes worth investigating the pharmacological profile of chemotypes of the most popular plant

such as Withania somnifera which is a well known immunomodulatory plant. The

immunostimulatory plant products also find use as anti-infectives as well as

immunoprophylactants and can be used in combination with chemotherapeutic agents to enhance

their anti-infective activity. The withanolides from WS reported in the present study are well

known and even their diverse bioactivity Including anti-cancer, anti-inflammatory, anti-bacterial,

anti-oxidant, health adjuvant and for treating skin disease, diabetes, arthritis, and epilepsy have

also been explored by other groups (Priyandoko et al., 2011; Fong et al., 2012; Grin et al., 2012;

Grover et al., 2012; Soman et al., 2012) however, no one has ever looked in to the anti-filarial

activity of any withanolide. Limited data is available on other important useful plants such as

Annona squamosa, Murraya koenijii or Withania coagulans which need detailed investigation as

immumodulators and anti-infective agents. We have selected one of the important helminth

parasites i.e. lymphatic filaria, B. malayi and the plants exhibiting immunostimulatory activity in

naïve BALB/c mice were later investigated as immunoprophylactant and chemotherapy adjunct

using B. malayi infection in the susceptible rodent host, Mastomys coucha.

28