7

Chapter 2

REVIEW OF LITERATURE

Aegle marmelos (L.) Correa belonging to family Rutaceae is a medium sized

deciduous tree indigenous to the Indian subcontinent. It is also found in

Myanmar, Pakistan, Bangladesh, Burma, Nepal, Vietnam, Laos and

Cambodia. It grows mainly within the sub-Himalayan region (Kala, 2006).

The tree has religious importance as well and is known as “Shivadume”

(Jagetia et al., 2004). It has been studied for various medicinal properties

including diabetes mellitus (Ponnachan et al., 1993; Kamalakkannan and

Prince, 2003; Kar et al., 2003), analgesic, anti-inflammatory and antipyretic

activities (Arul et al., 2005), antifungal (Chakthong et al., 2012), antibacterial

(Gautam et al., 2013) and antiproliferative (Lampronti et al., 2008).

Auraptene present in the extracts of Aegle marmelos has been proposed to

inhibit the deleterious effects of ischemia in isolated cardiomyocytes by

decreasing calcium overload (Kakiuchi et al., 1991). The plant has also been

proposed to inhibit free radical mediated oxidative injury by altering the

biotransformation enzyme systems in rats (Singh et al., 2000). The plant is

also documented to have antifertility effect in male rats (Chauhan and

Agarwal, 2008).The young leaves of Aegle are used in ethno-medicine as

astringent, laxative, febrifuge and expectorant (Nandkarni, 1976).

2.1 TAXONOMIC CLASSIFICATION OF AEGLE MARMELOS

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Subclass : Rosidae

Order : Sapindales

Family : Rutaceae

Genus : Aegle

Species : marmelos

Fig. 1 Aegle marmelos tree and leaves

8

9

Synonyms: Bel, belbaum, bela, bael, sripal, bilwa, wood apple, stone apple,

golden apple, Bengal quince (Nandkarni, 1976)

The bael tree is the only species in the genus Aegle. It is a medium sized

deciduous plant which grows to a height of about 18 m indigenous to India

and Southeast Asia. In India, it is distributed in the Himalayas and South

Indian plateau. The tree is grown mainly for its fruit. It bears thorns and has

fragrant flowers. It bears fruits which are globose with smooth, hard, aromatic

rind and is about 5-15 cm in diameter. The fruit bears numerous seeds which

are covered with dense fibrous hairs embedded in a thick aromatic pulp. The

leaves are trifoliate with a terminal leaflet (Kesari et al., 2006). The

monographs of the fruits and roots are included in Ayurvedic pharmacopoeia

of India (1999). The plant is mostly used in multicomponent formulations in

Ayurvedic medicines. All the parts of the plant have been reported to have

medicinal properties and more than 100 bioactive principles have been

isolated from the plant (Maity et al., 2009). These include aegeline,

imperatorin, skimmianine, psoralen, auraptene, lupeol, eugenol, marmin etc.

which are reported to have diverse pharmacological actions such as anticancer,

gastroprotective, antidiarrhoeal, cardioprotective, antidiabetic etc. (Maity et

al., 2009).

2.2 CHEMICAL CONSTITUENTS OF AEGLE MARMELOS

More than 30 compounds from the leaves of Aegle marmelos (AM) have been

identified. The leaves of the plant have been reported to contain aegeline (1)

and few alkaloids (2-4) of aegeline type (Manandhar et al., 1978;

Govindachari et al., 1983).The major constituents of the root bark of the plant

have been identified to be coumarins, alkaloids, sterols, decursinol (5) and

haplopine (6). The essential oil has been reported to contain p-cymene,

caryophyllene, cineole, citral, citronellal, cinnamaldehyde, d-limonene and

eugenol (Kaur et al., 2006). Other constituents include alkaloids, triterpenoids,

flavonoids and coumarins (Basu and Sen, 1974). Sterols and triterpenoids such

as lupeol and β-sitosterol, α and β-amyrin, flavonoids such as rutin and

coumarins have been isolated from the root and stem-bark of the plant

(Chatterjee et al., 1978). Purified exudate gum from the plant has been

reported to contain D-galactose, L-rhamnose, L-arabinose and D-glucuronic

acid (Mandal and Mukherjee, 1980). Other phytoconstituents include

marmenol (7), praealtin D (8), rutaretin (9), montanine (10), transcinnamic

acid (11), valencic acid (12), N-p-cis-coumaroyltyramine (13) and N-p-trans-

coumaroyltyramine (14) (Ali and Pervez, 2004). Valencic acid (12) from

Aegle marmelos has been shown to possess neuroprotective effect (Epifano et

al., 2008).

10

11

Phuwapraisirisan et al. (2008) have isolated a series of phenylethyl

cinnamides from the leaves of the plant with α- glucosidase inhibitory activity

(15-17).

12

Narender et al. (2007) have reported the antihyperglycemic and

antidyslipidemic agent from the alcoholic leaf extract of A.marmelos to be

aegeline (1) and proposed it to possess β-adrenergic receptor agonist using

predictive pharmacophoric hypothesis and 3D-QSAR model. Skimmianine,

lupeol and eugenol have also ben documented to be present in the plant (Maity

et al., 2009).

Skimmianine (18) Lupeol (19) Eugenol (20)

Maity et al. (2009) has reviewed the various phytoconstituents present in the

leaves of Aegle marmelos. Sharma et al. (1980) have reviewed the

phytoconstituents present in the fruits, Singh and Malik (2000) have reviewed

the phytochemical constituents in the seeds and Shoeb et al. (1973) have

reported the chemical constituents reported in the roots of the plant (Table 1).

Maity et al. (2009) have reviewed the pharmacological activities for these

phytoconstituents as presented in the Fig. 2-5. Skimmianine has been proposed

to have antimalarial, antipyretic, analgesic, anticancer and anticonvulsant

activities (Maity et al., 2009; Das and Das, 1995). Aegeline is reported to have

antihyperglycemic, antihyperlipidemic and cardioprotective actions (Narender

et al., 2007). Lupeol has been reported to be cardioactive and eugenol to have

antioxidative, antiulcer and antibacterial activities (Maity et al., 2009).

13

14

Table 1 Phytoconstituents reported in various parts of Aegle marmelos

Plant Part Phytoconstituent Reference

Leaf Tannins Maity et al., 2009

Limonene Maity et al., 2009

Aegelin Maity et al., 2009

O-(3,3-dimethylallyl)-halfordinol Manandhar et al., 1978

p- Cymene Maity et al., 2009

Phellandrene Maity et al., 2009

Cineole Maity et al., 2009

Marmelosin Nandkarni, 1976

Marmesinin Sharma et al., 1980

Rutin Sharma et al., 1980

Skimmianine Maity et al., 2009

Umbelliferone Arul et al., 2004

β- Sitosterol-D-glucoside Sharma et al., 1980

Fruit Alloimperatorin Sharma et al., 1980

Auraptene Kakiuchi et al., 1991

Calcium compounds Maity et al., 2009

Imperatorin Sharma et al., 1981

Glutamic acid Barthakur and Arnold, 1989

Glycine Barthakur and Arnold, 1989

Linoleic acid Maity et al., 2009

Lysine Barthakur and Arnold, 1989

Magnesium compounds Barthakur and Arnold, 1989

Marmelosine Badam et al., 2002

Marmeline Sharma et al., 1980

15

Phenylalanine Barthakur and Arnold, 1989

Proline Barthakur and Arnold, 1989

Psoralen Chakthong et al., 2012

Scoparone Sharma et al., 1980

Scopoletin Sharma et al., 1980

Skimmin Sharma et al., 1980

Umbelliferone Sharma et al., 1980

Xanthotoxol Sharma et al., 1980

Stem Bark Fagarine Chatterjee and Mitra, 1949

Marmin Chatterjee and Mitra, 1949

Seed Anthraquinones Mishra et al., 2010 b

Linoleic acid Singh and Malik, 2000

Linolenic acid Singh and Malik, 2000

Palmitic acid Singh and Malik, 2000

Stearic acid Singh and Malik, 2000

Root Α- Methyl scopoletin Shoeb et al., 1973

Psoralen Basu and Sen, 1974

Skimmin Shoeb et al., 1973

Scopoletin Shoeb et al., 1973

Timbamine Shoeb et al., 1973

Umbelliferone Basu and Sen, 1974

Xanthotoxin Basu and Sen, 1974

16

Skimmianine

Fig. 2 Pharmacological activities of Skimmianine

Antipyretic Anticancer Antimalarial

Analgesic Anticonvulsant

Aegeline

Antidyslipidemic Hypoglycemic

Cardioactive

Fig. 3 Pharmacological activities of Aegeline.

Antiinflammatory Cardioactive

Lupeol

Fig. 4 Pharmacological activities of Lupeol.

17

Eugenol

Antioxidative Antiulcer

Antiulcer

Fig. 5 Pharmacological activities of Eugenol.

18

2.3 PHARMACOLOGICAL ACTIVITIES OF AEGLE MARMELOS

Aegle marmelos has been studied for various pharmacological activities

including antiproliferative, antimicrobial, antidiabetic etc. which are discussed

in detail as under.

2.3.1 ANTICANCER AND ANTIPROLIFERATIVE ACTIVITY

Studies have revealed extracts of AM to have significant antiproliferative

activity (Lambole et al., 2010). Intraperitoneal administration of ethanol

extract of AM leaves has been found to have a strong inhibitory effect on

Dalton’s lymphoma ascites bearing mice (Chockalingam et al., 2012).

Administration of AM extract has been found to inhibit micronuclei

frequencies in the bone marrow cells of cyclophosphamide treated mice in a

dose dependent manner (Gupta et al., 2011). Extracts from AM have been

documented to have antiproliferative activity against MCF-7 and MDA-MB-

231 breast cancer cell lines at higher doses (Lambertini et al., 2004). Studies

have revealed the plant extract to inhibit the proliferation of Ehrlich Ascites

carcinoma transplanted into mice (Jagetia et al., 2005).

The plant is documented to be used in Bangladeshi folk medicine for the

treatment of cancer (Costa-Lotufo et al., 2005). Bark extract of AM has been

postulated to have an inhibitory effect on proliferation of various human

tumour cell lines including leukemia, lymphoma, colon and breast cancer cell

lines in vitro and phytochemicals such as butyl-p-tolyl sufide, 6-methyl-4-

chromanon and butylated hydroxyl anisole have been identified in these

extracts (Lampronti et al., 2006; Khan et al., 2002). Imperatorin, a linear

furanocoumarin isolated from the fruit of AM has been documented to inhibit

the proliferation of human leukemia cell lines (Pae et al., 2002).

2.3.2 ANTIHYPERGLYCEMIC ACTIVITY

Diabetes mellitus is a global epidemic. The number of people afflicted with

diabetes has increased manifolds. The progress of the ailment is marked by a

high rate of morbidity due to the complications including diabetic retinopathy,

neuropathy, nephropathy and cardiomyopathy. Plant extracts have emerged as

useful sources of nutraceuticals due to the rich diversity of the

19

phytoconstituents present in these extracts. Extract of green leaves of the plant

has been shown to have hypoglycaemic activity in diabetic animals

(Chakarbarty et al., 1960; Rao et al., 1995). Studies have revealed that a 75 %

methanol extract of AM decreases the blood glucose levels in alloxan induced

diabetic rats when administered at a dose of 100 mg Kg-1 (Sabu and Kuttan,

2004). AM extract has also been found to increase the levels of reduced

glutathione in the erythrocytes and decreases the levels of malondialdehyde in

alloxan induced diabetic rats (Upadhya et al., 2004). Studies have revealed

significant improvement in the glucose tolerance in rats administered the

aqueous decoctions of the plant (Karunanayaka et al., 1984). Administration

of ethanol extract of the plant for a period of two weeks has been reported to

have a hypoglycemic effect in diabetic rats (Kar et al., 2003). Sachdewa et al.

(2001) have reported glucose lowering effect of AM on one week

administration of extract in diabetic rats. The AM extract has been

documented to have maximal antihyperglycemic effect at the end of second

week of administration in a four week study (Upadhya et al., 2004). AM

leaves have been investigated to have hypoglycemic effect in normoglycemic

rats as well (Sharma et al., 1996).

Numerous mechanisms have been reported for the hypoglycemic effect of the

plant in diabetic conditions. These include regeneration of the damaged

pancreatic beta cells (Das et al., 1995), increase in the concentration of insulin

secreted by the beta cells (Kamalakkannan and Prince, 2003; Nammi et al.,

2001; Rao et al., 1995; Sachdewa et al., 2001; Sharma et al., 1996), inhibition

of glucose absorption from the gastrointestinal tract (Rao et al., 1995),

increase in sensitivity of the peripheral tissues to glucose (Sachdewa et al.,

2001) and restoration of the liver and renal vasculature damaged due to

diabetes (Das et al., 1996). Treatment with AM extract is also postulated to

reverse the muscarinic M1 receptor gene expression which is decreased in

diabetic rats and subsequently increase the vagal nerve stimulation and insulin

secretion thereby having a regulatory effect on glucose homeostasis in

diabetes (Gireesh et al., 2008). Studies have revealed AM to have stimulatory

action on PPAR-γ in vitro (Anandharajan et al., 2006) and in vivo (Sharma et

al., 2011).

20

2.3.3 CARDIOPROTECTIVE ACTIVITY

Hyperlipidemia characterized by an increase in blood cholesterol, low density

lipoproteins cholesterol and triglycerides and decreased high density

lipoprotein cholesterol leads to a number of chronic cardiac ailments. The

incidence and prevalence of cardiovascular disorders has increased

tremendously over the last few decades and plants have emerged as attractive

targets for finding pharmacologically active molecules with improved

therapeutic profile. The aqueous and alcohol extract of the leaves of AM has

been shown to reduce the pulse rate and increase the amplitude and tone of

contractions in isolated frog heart (Haravey, 1968). The plant extract has also

been found to attenuate the deleterious effects of calcium overload in frogs

(Haravey, 1968). The methanol extract of the root bark of the plant is reported

to contain auraptene which decreases the calcium paradox induced ischemic

injury and spontaneous beating in isolated myocardial cells (Kakiuchi et al.,

1991).

Oral administration of the aqueous leaf extract of AM has been studied to

prevent isoprenaline induced myocardial infarction in rats (Prince and

Rajadurai, 2005). AM extract treatment for a period of 35 days has been

postulated to reduce the levels of creatine kinase, lactate dehydrogenase,

Na+/K+ ATPase in isoproterinol treated rats and hence confer cardioprotective

effect (Prince and Rajadurai, 2005). Kamalakkannan and Prince (2003) have

proposed AM extract to have a marked hypolipidemic effect in diabetic rats.

Ethanol extract of AM leaves is documented to inhibit the increase in serum

cholesterol and triglycerides and increase high density lipoproteins in triton

and diet induced hyperlipidemic rats (Vijaya et al., 2009).

A polyherbal preparation containing AM and five other plant extracts has been

documented to decrease the rise in serum lipids, cholesterol and triglycerides

significantly in experimental model of hyperlipidemia in rats (Ansarullah et

al., 2012). Periplogenin, a cardenolide obtained from the leaves of AM has

been reported to have a protective effect against doxorubicin induced

cardiotoxicity and lipid peroxidation in rats (Panda and Kar, 2006). Oral

administration of methanol extract of the leaves of AM has been proposed to

21

have significant hypolipidemic effect in streptozotocin induced diabetic rats

(Juvekar and Bandawane, 2009). Lupeol obtained from the leaves of AM has

been studied to have antidyslipidemic activity in streptozotocin diabetic rats

(Papi Reddy et al., 2009). Unripe fruit extract has been documented to be used

in cardiac ailments (Dhankar et al., 2011). AM has been proposed to have

therapeutic potential in cardiovascular disorders due to inhibition of apoptosis

induced by ischemia-reperfusion induced myocardial injury (Ahmad et al.,

2010).

Padma-28, a polyherbal Tibetian preparation containing AM has been reported

of have beneficial effect on patients suffering from peripheral arterial disease

in a meta- analysis study (Melzer and Saller, 2010). Aegeline, an alkaloidal-

amide isolated from the leaves of AM has been found to have antidyslipidemic

effect in streptozocin induced diabetic rats (Narendra et al., 2007).

Pharmacophoric and 3D QSAR studies have suggested that aegline may have

a β3 adrenergic agonistic activity (Narendra et al., 2007). Studies have

suggested fresh juice of AM to be better tolerated and less toxic cardiotonic in

isolated frog hearts as compared to digoxin (Dama et al., 2010).

2.3.4 ANTIMICROBIAL ACTIVITY

Consumer preferences to naturally derived antimicrobial agents have increased

and plant medicines are being explored for their antimicrobial activities.

Several studies have revealed the antibacterial activity of AM (Dey and De,

2012; Gautam et al., 2013; Prasannabalaji et al., 2012). Petroleum ether

extract obtained from callus culture of AM has been documented to have an

inhibitory effect on the growth of Salmonella typhi in in vitro studies

(Thangavel et al., 2008). Shahidine, a highly labile oxazoline obtained from

AM, has been reported to have antibacterial activity against Gram positive

bacteria (Faizi et al., 2009). Aqueous and ethanol extracts of AM have been

postulated to have strong inhibitory effect on the growth of some bacteria

causing common human diseases including Staphylococcus aureus,

Pseudomnas aerugenosa and Escherichia coli (Chattopadhyay et al., 2009).

Maity et al. (2009) have suggested AM to have therapeutic potential for

developing novel antimicrobials. Hot aqueous decoction of unripe fruits of

22

AM has been demonstrated to have cidal activity against Giardia and rota

virus in comparison to limited activity against bacteria such as E. coli (Brijesh

et al., 2009). Studies have hypothesized AM to have a broad spectrum

antibacterial activity that includes both the Gram positive and Gram negative

bacteria, while certain bacterial strains such as Bacillus subtilis are resistant to

the action of the plant extracts (SaradhaJyoti and SubbaRao, 2010).

Plant extracts of AM and Andrographis paniculata have been reported to have

synergistic antibacterial action (Rasi and Daniel, 2011). The bael plant has a

long history of use in ethnomedicine for its antibacterial property (Baliga et

al., 2011). The chloroform extract of the leaves of AM has been reported to

have a higher activity against Proteus mirabilis and Klebsiella pneumoniae

whereas the methanol extract has been studied to have a better action against

Salmonella typhi (Kothari et al., 2011). Aqueous and ethanol extract of the

leaves of AM have shown significant activity against multi drug resistant

strains of uropathogenic bacteria including Acinetobactor baumannii,

Citrobactor freundii, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris

and Pseudomonas aeruginosa (Rath et al., 2012).

Chakthong et al. (2012) have isolated alkaloids and coumarins from the

acetone extract of green fruits of AM and investigated the antibacterial activity

of these compounds. The aqueous and ethanol extracts of AM have been

shown to have significant antimicrobial activity against ten species of multi

drug resistant strains of enteropathogenic bacteria (Rath and Padhy, 2012).

Volatile oil obtained from the leaves of plant has been documented to have

potent antifungal activity against various strains of fungi (Rana et al., 1997).

2.3.5 HEPATOPROTECTIVE ACTIVITY

Traditional systems of medicines have been widely practiced through

generations to cure various hepatic ailments. The chloroform, alcohol and

aqueous extracts of AM leaf have been documented to decrease the blood

levels of serum glutamate pyruvate, serum glutamate oxaloacetate

transaminase, alkaline phosphatase and bilirubin in ethanol induced

hepatotoxicity in rats (Modi et al., 2012). The aqueous fruit extract of the plant

23

has also been documented to have a protective action in paracetamol induced

hepatotoxicity in rats (Sastry et al., 2011). Administration of the dried leaf

powder of AM for a period of 14 days has been reported to have a

hepatoprotective effect in carbon tetrachloride induced hepatotoxicity in rats

(Jayachandra and Sivaraman, 2011). Furthermore, the hepatoprotective effect

of the dried leaf powder of the plant was comparable to that of the standard

drug Liv 52 (Jayachandra and Sivaraman, 2011).

2.3.6 ACTIVITY IN ULCERATIVE COLITIS

Ulcerative colitis is a highly debilitating ailment and therapeutic interventions

available are only a few. Plants have emerged as useful sources of drugs to

mitigate a number of chronic ailments such as ulcerative colitis. AM has been

reported to be used for gastroprotective effect in ethnomedicine (Romano et

al., 2012). Oral administration of the extract of unripe fruit of AM at different

concentrations, once daily, has been reported to have a significant protective

effect on acetic acid and indomethacin induced ulcerative colitis in rats

(Behera et al., 2012). The extract was found to decrease mast cell

degranulation, disease activity index, macroscopic and microscopic scores of

disease severity in both the models significantly (Behera et al., 2012). AM

treatment has also been postulated to have protective effect on 2,4-

dinitrobenzene sulfonic acid induced colitis in rats (Gandhi et al., 2009).

2.3.7 ANTIFERTILITY EFFECT

Aegle marmelos has been used in ethno medicine for antifertlity effect (Jain et

al., 2004). Studies have revealed that the various parts of AM including the

stems, fruits, seeds and leave have antifertility effect in male animals

(Agrawal et al., 2012; Gnanasam et al., 2002). The bark extract of AM

contains marmin and fagarine, which are postulated to reduce male fertility in

rats (Agrawal et al., 2012). Treatment of male rats with methanol extract of

AM caused dose dependent decrease in sperm density, motility, viability and

acrosomal integrity by decreasing the serum testosterone levels and the weight

of the reproductive organs in male animals. These observations suggest that it

may be used as a male contraceptive (Agrawal et al., 2012).

24

Administration of the aqueous leaf extract of AM has been associated with

reversible loss of fertility without affecting the vital parameters in rats

(Chauhan et al., 2009). Alkaloids, phenolics and triterpenoids present in the

aqueous extract of the leaves of AM have been reported to decrease the

vitality of human sperms in in vitro studies (Mohanraj et al., 2009). A dose of

300 mgKg-1 of 50% ethanol extract of AM is documented to produce a

complete inhibition of fertility in rats (Chauhan and Agarwal, 2007, 2008).

2.3.8 ANTIDIARRHEAL ACTIVITY

Aegle marmelos is used by the tribal populations in West Bengal and Assam

for its antidiarrheal action (Dry and De, 2012; Sharma et al., 2012). Methanol

extract of the unripe fruit of AM is documented to have protective effect on

castor oil induced diarrhea (Maity et al, 2009; Shoba and Thomas, 2001). AM

is documented to have a protective action against gastric mucosal damage and

diarrhea induced by various agents including hypothermia restraint, absolute

ethanol, indomethacin and castor oil and study reported AM to decrease the

intestinal fluid accumulation and gastric mucosal damage significantly (Shoba

and Thomas, 2001). Mebarid, an Ayurvedic polyherbal formulation containing

AM as one of the components, has been documented to have potent

antidiarrheal, antimotility and antiulcer activities in rats (Bafna and

Bodhankar, 2003). Studies have revealed that intracaecal administration of

methanol extract of AM is more effective than oral dose in protection against

experimentally induced diarrhea (Shoba and Thomas, 2003).

It has been postulated that free radical scavenging activity of AM plays a

significant role in protection against experimentally induced ulcers and

diarrhea in rats (Rao et al., 2003). The chloroform extract of the root of AM

has also been suggested to have in vitro and in vivo antidiarrheal activity

(Mazumdar et al., 2006). Hot water decoction of the dried unripe fruit of AM

is documented to prevent infective diarrhea caused by micro-organisms such

as Giardia and rota virus both of which are highly virulent (Brijesh et al.,

2009).

25

2.3.9 ANTIVIRAL ACTIVITY

Viral infections are one of the most severe microbial infections and lead to a

high incidence of morbidity and mortality globally. Infections such as HIV,

dengue, hepatitis B and C, and influenza are among the most notorious viral

infections. Due to high toxicity of the existing antiviral drugs, natural products

are being explored for potential antiviral activity. Studies have revealed the

bark, fruit and root of Aegle marmelos to have activity against human

coxsackie viruses and marmelide has been isolated as the antiviral

phytoconstituent (Badam et al., 2002). Marmelide is reported to interfere with

the early stages of viral replication. The fruit extract of AM has been

postulated to have an action similar to interferons (Babbar et al., 1968). The

ethanol extract of bael fruit has been documented to inhibit the Ranikhet

disease virus (Dhar et al., 1968).

2.3.10 MISCELLANEOUS ACTIONS

Arul et al. (2005) have investigated the extracts of leaves of AM in various

solvents including petroleum ether, chloroform and ethanol for analgesic and

anti-inflammatory activities. The results of the study revealed marked

analgesic and anti-inflammatory activities of the extracts. Ethanol extract of

AM has been shown to have most significant analgesic activity on oral

administration (Muruganandan et al., 2000). Methanol extract of AM has been

reported to decrease acetic acid induced writhing and thermal hyperalgesia in

mice (Shankarananth et al., 2007). Ethanol extract of AM leaves has been

postulated to produce relaxant effect in histamine induced contractions in

isolated ileum and tracheal chain from guinea pigs (Arul et al., 2004). AM is

used in indigenous medicine for the treatment of asthma (Prasad et al., 2009).

Studies have revealed the fruit extract of the plant to have immunomodulatory

activity (Patel et al., 2010).

Water extract of AM leaves has been shown to inhibit the levels of thyroid

hormones in rats when administered at a dose of 1 g Kg-1 (Panda and Kar,

2006). The decrease in serum T3 and T4 levels has been postulated to be due

to antiperoxidative action of AM (Kar et al., 2003). PHF, a polyherbal

preparation containing seven herbs including AM has been documented to

have prokinetic effect in mice and rats (Srinivasan et al., 2005).

Aqueous extract of the fruits of AM has been reported to lower the intraocular

pressure in water loading and steroid induced models of raised intraocular

pressure in New Zealand white rabbits when applied topically (Agarwal et al.,

2009). Studies have revealed the ointment containing aqueous and methanol

extracts of bael seeds to increase the percentage wound contraction, tensile

strength and decrease the period of epithelialization of excision and incision

wound models in rats (Sharma et al., 2011). Toxicological studies have

revealed LD50 of AM leaves to be relatively high when given orally and

intraperitoneally in a single dose as well as on chronic administration for 14

days (Veerappan et al., 2007).

The number of papers on Aegle marmelos published in the last ten years and

the journals where these have been published are given in Fig. 6 and Fig. 7

respectively.

0

50

100

150

200

250

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2013

Years

No.

of P

ublic

atio

ns

Fig. 6 Evolution of published work on Aegle marmelos

[Retrieved from SCOPUS database]

26

0 5 10 15 20 25 30 35

Indian Drugs

J. App. Pharma. Sci.

American Eurasian J. Sustainable Agri.

Pharmaceutical Biology

Indian J. Pharmacol.

BMC Complementary Alter. Med.

Int. J. Pharma Bio Sci.

Evidence Based Complementary & Alternat. Med.

Int. J. Pharma. Sci. Review Res.

Asian Pacific J. Tropical Disease

Food Chemical Toxicol.

Pharmacologyonline

Asian Pacific J. Tropical Biomedicine

Int. J. Pharmacy Pharmaceut. Sci.

Journal of Ethnopharmacol.

Fig. 7 Journals publishing research articles on Aegle marmelos from year 2007-2012 [Retrieved from SCOPUS database]

2.4 COUMARINS IN PLANTS

Coumarins are synthesized as secondary metabolites in plants and are

benzopyrone derivatives. Coumarins contain α-pyrone group where as

flavonoids, which are structurally close to coumarins have γ-pyrone moiety.

Linear furanocoumarins are typical to the families Rutaceae, Apiaceae,

Leguminosae and Moraceae (Berenbaum et al., 1991). In their natural form,

coumarins may be linked with sugars and may be present as glycosides. The

furanocoumarins have a five membered furan ring attached to the coumarin

nucleus and are synthesized via shikimic acid pathway in plants (Berenbaum

et al., 1991). In plants coumarins act as phytoalexins which are formed in

response to injury to the plant and are abundantly present in leaves, fruits and

seeds of the plants (Berenbaum et al., 1991).

These phytoconstituents have been studied for a wide variety of biological

activities including facilitating skin pigmentation in vitiligo and psoriasis

(Dewick, 2009), cytotoxic activity against cancer cell lines (Um et al., 2010),

cardioprotective activity (Vimal and Devaki, 2004). Imperatorin is a linear

furanocoumarin isolated from the fruit extract of AM (Kamalakkanan and

Prince, 2003). Studies have revealed that imperatorin inhibits the action of γ-

27

28

aminobutyric acid (GABA)-transaminase, the enzyme that causes degradation

of the neurotransmitter GABA (Choi et al., 2005). Imperatorin has been

documented to ameliorate electro-shock induced convulsions in rats (Luszczki

et al., 2009).

2.5 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS

Peroxisome Proliferator-Activated Receptors (PPARs) are a family of nuclear

receptors discovered in 1990 (Issemenn and Green, 1990). They are members

of steroid hormone receptor superfamily (Youssef and Badr, 2011). Three

types of PPARs have been identified. These include PPAR-α, PPAR-β/δ and

PPAR-γ, which differ in their tissue distribution, and the ligand specificity

(Berger and Moller, 2002). These receptors form heterodimers with the

retinoid X receptor (RXR) to which the ligands bind (Fig. 8). The complex

then translocates into the nucleus where it binds to specific PPAR response

elements in the DNA in specific promoter regions and brings about a cascade

of complex events involving the influx of co-activators and release of co-

repressors, and finally culminating into transcription (Youssef and Badr,

2011).

PPARs are mainly involved in fatty acid mobilisation and utilization, insulin

sensitivity, lipid storage in liver and lipid mobilization (Jungbauer and

Medjakovic, 2012). PPARs have emerged as interesting and promising targets

for developing new drugs to treat disorders such as cancer, diabetes mellitus,

obesity etc. PPARs have also been postulated to be present in the central

nervous system where they may be involved in various neurological processes

regulating the brain function (Heneka and Landreth, 2007). Studies have

indicated a role of PPARs in H.pylori infection (Lee et al., 2012). The PPARs

are characterized by the presence of six structural regions with four functional

domains. Out of these the A/B region is ligand-independent transactivation

domain, the C region is the region for DNA binding, E/F is the ligand binding

domain that has co-activator/co-repressor binding regions (Jungbauer and

Medjakovic, 2012).

Fig. 8 Mechanism of PPAR signalling.

2.5.1 PPARs IN CANCER

Studies have revealed that the PPARs may be involved in the pathogenesis of

various types of tumors (Panigrahy et al., 2008). It has been reported that

PPAR-γ agonists have an inhibitory effect on the growth of brain tumor cells

(Grommes et al., 2006). Pioglitazone, a PPAR-γ agonist, has been found to

reduce the proliferation of glioblastoma (Papi et al., 2009). Treatment with

L16504, a PPAR-β/δ agonist, has been shown to inhibit lung carcinoma cells

(Fukumoto et al., 2005). It has been postulated that decreased expression of

PPAR-γ is associated with poor prognosis in the patients suffering from lung

cancer (Sasaki et al., 2002). PPAR-γ receptor has been postulated to be

involved in the antiproliferative effect of prostacyclin (Nemenoff et al., 2008).

PPAR-γ has also been linked to decrease in the proliferation of colonocytes

(Matthiessen et al., 2005).

It has been documented that PPAR-γ is expressed in the urinary tract and is

associated with decreased incidence of tumour proliferation (Myloma et al.,

2009). PPAR-γ agonists have also been associated with decreased incidence of

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hematological cancers (Garcia-Bates et al., 2008). The molecular mechanisms

of inhibition of tumours include promoting apoptosis in the tumor cells,

alteration in membrane permeability and, up-regulation of anti-apoptotic

proteins in normal cells (Youssef and Badr, 2011). In clinical studies,

rosiglitazone treatment has been found to have a significant inhibitory effect

on pancreatic cancer (Youssef and Badr, 2011). The synergistic effect of

PPAR ligands with radiation therapy and chemotherapy of cancers has been

shown both in vitro and in vivo (Shimizu and Moriwaki, 2008).

2.5.2 PPARs IN DIABETIC NEPHROPATHY AND CARDIOMYOPATHY

Diabetes mellitus is now prevalent in epidemic proportions world wide. The

availability of numerous options for controlling blood glucose levels has

increased the life expectancy of diabetic patients and this increase in life span

of the patients has led to a higher incidence of the complications due to

diabetes such as diabetic neuropathy, diabetic nephropathy (DN) and diabetic

cardiomyopathy (DCM). These complications are a major cause of concern in

the diabetic patients and cause a very high morbidity, and mortality. It is

estimated that by the year 2030 more than 360 million patients suffering from

type II diabetes mellitus will be at a high risk of DN and DCM

(Kouromichakis et al., 2012). The pathogenesis of these syndromes is

complex and multifactorial. The therapeutic options currently available in the

treatment of these complications are insufficient. Inflammatory process is now

believed to be involved in the pathogenesis of chronic ailments including

diabetes mellitus (Jungbauer and Medjakovic, 2012). PPARs have been

postulated to play a vital role in controlling inflammation (Chinetti et al.,

2000). Various chemical mediators are involved in the inflammatory process,

including, interleukins, tumour necrosis factor, interferons, leukotrienes,

prostanglandins, endoperoxides etc. (Fain, 2006).

PPAR ligands have emerged as molecules with pleiotropic actions and are

being explored for their value in treating these ailments (Kouromichakis et al.,

2012). DN is the leading cause of end stage renal disease in clinical settings.

Various factors such as altered lipid balance, proinflammatory mediators,

increased blood pressure, and oxidant stress etc. lead to DN (Levi, 2011).

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Studies have revealed PPAR-α agonists to be of benefit in decreasing the

symptoms of DN in type II diabetic mice (T2DM) (Park et al., 2006). Patients

with mutations in PPAR-γ have been associated with increased severity of

insulin resistance and decrease in the onset time of hypertension, DN and

DCM (Lombard and Cowley, 2012).

Several studies have reported the protective effect of PPAR-γ agonists in DN

(Yoshioka et al., 1993; Levi, 2011). The glitazones which are PPAR-γ

agonists have been found to decrease the symptoms of DN such as

albuminuria, maintain glomerular filteration rate, prevent fibrosis in the

interstitium and glomerulosclerosis (Sarafidis et al., 2006). PPAR γ agonists

have been shown to have synergistic effects with PPAR-α agonists in

decreasing the severity of diabetic symptoms in animal studies (Cha et al.,

2007). In addition to the above effects, PPAR-γ agonists offer other

advantages including antifibrotic, anti-inflammatory and antiproliferative

effects through inhibition of activation of NFkB, free radical species and

infiltration of inflammatory cells like the macrophages (Wu et al., 2004).

Human type I and type II diabetes mellitus is associated with altered lipid

balance and fatty acid, and cholesterol deposition in the renal tissue as well

(Levi, 2011). The PPARs because they help in mobilising the fatty deposits

are receiving special attention in mitigating the renal complications of diabetes

(Yoshioka et al., 1993).

The vascular function has been found to be altered in patients with mutations

in PPAR- γ and impaired relaxant response of the blood vessels to

acetylcholine has been found in these patients (Lombard and Cowley, 2012).

Vascular effects of PPARs include direct effects mediated by PPARs in the

blood vessel wall and indirect effects which are caused by regulation of

glucose, and fatty acid metabolism (Wang et al., 2006). Also, activation of

PPAR-δ has anti-inflammatory effects and decrease in apoptosis in the blood

vessels (Katusic et al., 2012). The PPARs regulate fuel metabolism in the

heart. PPAR-γ is involved primarily with the differentiation of adipose cells

and fat storage. It is expressed in the myocardium as well although the extent

is lower (Madrazo and Kelly, 2008). Studies have revealed beneficial effects

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of thiazolidinediones in the diabetic heart and this protection is due to anti-

inflammatory effect of the PPAR-γ ligands (Abdelrahman et al., 2005). The

PPAR ligands have diverse target sites to mediate the protective effects in

DCM such as anti-inflammatory action, antihypertensive effect, preserving the

endothelial function, suppression of renin-angiotensin system (RAS) etc

(Sugawara et al., 2010).

2.5.3 PPARs IN EPILEPSY

PPARs have been postulated to be present in the brain. PPAR-γ is the most

widely expressed PPAR in the brain (Fajas et al., 1998). Three splice variants

of PPAR-γ are known PPAR-γ1, PPAR-γ2 and PPAR-γ3 (Fajas et al., 1998).

PPAR-γ in brain is involved in many regulatory functions. The inhibition of

neuroinflammation is proposed to be involved in the inhibition of various

neuropsychiatric disorders such as depression, epilepsy etc as well as

neurodegenerative disorders including Alzheimer’s disease, Parkinson’s

disease, Huntington’s disease (Garcia-Bueno et al., 2008). Administration of

thiazolidinediones is documented to decrease the expression of inflammatory

mediators including tumour necrosis factor alpha and inducible nitric oxide

synthase (iNOS) in brain cells (Garcia-Bueno et al., 2008). Rosiglitazone has

been found to produce a dose dependently increase in the dendritic spine

density in rat neurons (Brodbeck et al., 2008). Modulation of bioenergetics in

the brains of stressed animals such as to inhibit excitotoxicity has been

documented with PPAR γ ligands (Garcia- Bueno et al., 2008). The methanol

extract of A. marmelos has been documented to promote glucose uptake by

activation of PPAR-γ in in vitro studies (Anandharajan et al., 2006). Recently,

the fruit extract of Aegle marmelos has been reported to increase the

expression of PPAR-γ diabetic rats (Sharma et al., 2011).