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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; 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 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
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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, nbutanol 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
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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, 4benzo (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
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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
hexane and aqueous extracts/ fractions of Carica papaya seeds significantly enhanced the
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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 antihyperglycemic 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 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 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
Leishmania amazonensis. The study concluded a strong antileishmanial activity conferred by both
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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
chaba fruits had the strongest activity against Schistosoma cercariae. However, the strong
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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 AGENTS
One 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
and root of the Nyctanthes arbortristis L. (p.o. once daily) provided significant protection of Swiss
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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
mouse blood. Similarly, increased activity of acid phosphatase and oxidized glutathione was
significantly inhibited by linseed oil (Bhatia et al., 2006).
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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 ADJUNCTS
The 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).
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
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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
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
21
Chapter 2
Review of literature
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
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 C1210. The leaves of this plant have been reported to contain luteolin, luteolin 7-0, β-glucuronoside
22
Chapter 2
Review of literature
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, 1dipheny 1-2-picrylhydrazide (DPPH) free radicals most effectively. The extract inhibited the
unscheduled DNA repair, cytotoxicity and genotoxicity induced by tertbutylhydroperoxide (tBHP) 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
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
23
Chapter 2
Review of literature
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 antileishmanial 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 AGENTS
Helminth 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
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
24
Chapter 2
Review of literature
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
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)
25
Chapter 2
Review of literature
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.
(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,
26
Chapter 2
Review of literature
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 acetatesoluble 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
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
27
Chapter 2
Review of literature
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