arjunolic acid: a novel phytomedicine with multifunctional therapeutic...

Indian Journal of Experimental Biology

Vol. 48, March 2010, pp. 238-247

Review Article

Arjunolic acid: A novel phytomedicine with multifunctional

therapeutic applications

Thiagarajan Hemalatha1, Sivasami Pulavendran

1, Chidambaram Balachandran

2, Bhakthavatsalam Murali Manohar

3 &

Rengarajulu Puvanakrishnan1*

1Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai 600 020, India, 2Department of Veterinary Pathology, Madras Veterinary College, Vepery, Chennai 600 007 India,

3Centre for Animal Health Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai 600 051 India.

Herbal plants with antioxidant activities are widely used in Ayurvedic medicine for cardiac and other problems.

Arjunolic acid is one such novel phytomedicine with multifunctional therapeutic applications. It is a triterpenoid saponin,

isolated earlier from Terminalia arjuna and later from Combretum nelsonii, Leandra chaeton etc. Arjunolic acid is a potent

antioxidant and free radical scavenger. The scientific basis for the use of arjunolic acid as cardiotonic in Ayurvedic medicine

is proven by its vibrant functions such as prevention of myocardial necrosis, platelet aggregation and coagulation and

lowering of blood pressure, heart rate and cholesterol levels. Its antioxidant property combined with metal chelating

property protects organs from metal and drug induced toxicity. It also plays an effective role in exerting protection against

both type I and type II diabetes and also ameliorates diabetic renal dysfunctions. Its therapeutic multifunctionality is shown

by its wound healing, antimutagenic and antimicrobial activity. The mechanism of cytoprotection conferred by arjunolic

acid can be explained by its property to reduce the oxidative stress by enhancing the antioxidant levels. Apart from its

pathophysiological functions, it possesses dynamic insecticidal property and it is used as a structural molecular framework

in supramolecular chemistry and nanoscience. Esters of arjunolic acid function as gelators of a wide variety of organic

liquids. Experimental studies demonstrate the versatile effects of arjunolic acid, but still, further investigations are necessary

to identify the functional groups responsible for its multivarious effects and to study the molecular mechanisms as well as

the probable side effects/toxicity owing to its long-term use. Though the beneficial role of this triterpenoid has been assessed

from various angles, a comprehensive review of its effects on biochemistry and organ pathophysiology is lacking and this

forms the rationale of this review.

Keywords: Antioxidant, Arjunolic acid, Cardioprotectant, Free radical scavenger, Terminalia arjuna

Ancient Indian medical knowledge known as

Ayurveda goes back millennia. The Vedas and

Puranas refer various materials of medical importance

including herbs, plants and trees, etc. Medicinal plants

are being investigated in the past to unravel the

scientific principles behind their pharmacological

properties. A scientific analysis of Ayurvedic herbal

armamentarium will lead to the identification of

potent drug molecules.

Terminalia arjuna (TA), a deciduous tree of the

Combretaceae family, has been widely used in Indian

system of medicine for cardiac ailments1. Its

cardioprotective property has been mentioned in

ancient Indian medical literature including Charaka

Samhita and Astang Hridayam2. It is also believed to

have the ability to cure hepatic, urogenital, venereal

and viral diseases3. It also possesses antilipidemic,

antioxidant4, antiinflammatory, antinociceptive and

immunomodulatory activities5. The various

constituents present in the bark of TA include tannins,

arjunic acid, arjunolic acid, arjungenin,

arjunglycosides, flavonoids, ellagic acid, gallic acid,

oligomeric proanthocyanidins (OPCs), phytosterols,

calcium, magnesium, zinc and copper6.

Arjunolic acid (AA) AA is used as a cardiac tonic in Ayurvedic

medicine for centuries and it has been first isolated

from TA7. Later, it has been isolated from

Cochlospermum tinctorium8, Cornus capitata

9,

Leandra chaetodon10

, Combretum leprosum11

,

Campsis grandiflora12

, Syzygium guineense13

,

Combretum nelsonii14

etc. Ratsimamanga and

Boiteau15

have filed a patent based on the hormonal,

wound healing and bactericidal properties of AA.

______________________

*Correspondent author

Telephone: +91 9444054875.

Fax: +91 44 491 1589.

E-mail: [email protected]

HEMALATHA et al.: THERAPEUTIC APPLICATIONS OF ARJUNOLIC ACID

239

AA, a triterpenoid saponin, is a major component

of the extracts of the bark of TA (Fig.1).

Triterpenoids, are an important class of plant secondary

metabolites derived from C30 precursors16

and they

possess a wide range of biological acitivities17

. This

chiral triterpenic acid has a rigid pentacyclic backbone

with two equatorial hydroxyl groups and one equatorial

hydroxymethyl group attached to the “A” ring. The

carboxyl group is attached at the ring junction of the

cis-fused “D” and “E” rings18

.

Research on AA aims to untie its multifunctional

therapeutic applications. Being known for its

cardioprotective effect over centuries, experimental

studies have proved functions such as prevention of

myocardial necrosis, platelet aggregation and

coagulation, and lowering of blood pressure, heart rate

and cholesterol levels, which lend support to the claim

for its traditional usage. Apart from its cardioprotective

effects, AA protects the cells from metal induced

toxicity and it also possesses antiinflammatory,

antidiabetic, antitumor, antimicrobial activity etc. AA

is a potent antioxidant and a free radical scavenger. The

free radical scavenging activity could be determined

using 2,2-diphenyl-1-picryl hydrazyl (DPPH) and its in

vivo antioxidant potential could be assayed by ferric

reducing/antioxidant power (FRAP) assay19

.

Though a vast repertoire of literature describes the

therapeutic use of AA in experimental animal models

and in vitro studies, a comprehensive review on the

beneficial role of this triterpenoid saponin in organ

pathophysiology is lacking and hence, this review deals

with the multifunctional role of AA, a plant product.

Extraction of arjunolic acid

AA is extracted from the bark of TA7. To state

briefly, the bark (2 kg) of TA is sliced and ground

into powder and extracted with methyl alcohol at

room temperature. To the extract, lead acetate (100 g)

is added and the precipitate material formed is filtered

off. Water (10 l) is added to the filtrate and the

greenish precipitate formed is separated by filtration.

The mother liquor is concentrated under reduced

pressure to have greenish yellow precipitate. The

combined greenish yellow precipitate (Total 3.2 g) is

subjected to separation on a silica gel column. Elution

with chloroform:methanol yields 210 mg of arjunolic

acid (mp: 296°–297°C) and the eluted fractions are

examined by thin layer chromatography. The purity of

the compound is checked by using standard tools such

as nuclear magnetic resonance (1H,

13C), infrared,

mass spectroscopy and optical rotation studies.

Cardioprotective effects

Protection against myocardial necrosisSumitra

et al.20

have clearly portrayed the cardioprotective

effects of AA by showing its effect on platelet

aggregation, coagulation and myocardial necrosis. AA

at an effective dose of 15 mg/kg body wt (pre- and

post-treatment), when administered intraperitoneally

to rats afflicted with isoproterenol (ISO) induced

myocardial necrosis, effects a decrease in serum

enzyme levels such as creatine kinase, creatine

kinase–MB and lactate dehydrogenase and the

electrocardiographic changes get restored towards

normalcy. AA treatment also prevents the decrease in

the levels of superoxide dismutase, catalase,

glutathione peroxidase, ceruloplasmin and α-

tocopherol and also reduces glutathione, ascorbic

acid, lipid peroxide and myeloperoxidase.

Histopathological studies on the heart tissue reveal an

engorgement of coronary vessels due to the

administration of ISO (Fig. 2a) while in liver,

hepatocytes around the portal triad showed intense

cytoplasmic staining (Fig. 2b). Normal architecture of

heart (Fig. 3a) and liver (Fig. 3b) has been observed

after AA pre- and post-treatment20

. This study also

proves the antiplatelet and anticoagulant activity of

AA. The cardioprotective effect of AA (pre- and post-

treatment) has been possibly attributed to the

protective effect against the damage caused by

myocardial necrosis.

Prevention of arsenic induced myocardial

injuryArsenic toxicity is one of the risk

factors associated with cardiovascular diseases. Arsenic

exposure is associated with direct myocardial injury,

cardiac arrhythmias and cardiomyopathy21

. Manna et

al.19

have revealed the protective effects of AA against

Fig. 1Chemical structure of arjunolic acid (2:3:23-trihydroxy

olean-12-en-28-oic acid; Mol wt: 488.88)

INDIAN J EXP BIOL, MARCH 2010

240

arsenic-induced cardiac oxidative damage. Oral

administration of sodium arsenite (NaAsO2), at a dose

of 10 mg/kg body wt for two days, causes a significant

accumulation of arsenic in cardiac tissues of the

experimental mice in addition to reduction in cardiac

antioxidant enzyme activities, viz. superoxide

dismutase, catalase, glutathione-S-transferase,

glutathione reductase and glutathione peroxidase.

Arsenic intoxication also decreases the level of cardiac

glutathione and total thiol contents and increases the

levels of oxidized glutathione, lipid peroxidation end

products and protein carbonyl content.

Treatment with AA at a dose of 20 mg/kg body wt

for four days prior to sodium arsenite intoxication

Fig. 2(a) Marked congestion of vessels and extravasation of erythrocytes (EE), hyalinisation of a few muscle fibers (MF) and

mononuclear cell infiltration (→) in heart tissue in ISO administered rats (H & E; ×320); and (b) Hepatocytes (HP) around the portal triad

showing intense cytoplasmic staining; diffuse degenerative changes in the hepatocytes; hypertrophy of Kupffer cells and focal

mononuclear cell infiltration (MC) in the liver tissue in ISO administered rats (H & E; ×320).

Fig. 3(a) Heart tissue showing normal architecture (MF) in ISO administered group, pre- and post- treated with 15 mg of arjunolic acid

(H & E; ×320); and (b) Liver tissue showing normal architecture (HP) in ISO administered group, pre- and post- treated with 15 mg of

arjunolic acid (H & E; ×320).


241

protects the cardiac tissue from arsenic-induced

oxidative impairment by restoring the levels of

antioxidants. In addition to oxidative stress, arsenic

administration increases total cholesterol level and it

reduces high density lipoprotein (HDL) cholesterol

level in the sera of the experimental mice. AA

pretreatment, however, can prevent this

hyperlipidemia. While sodium arsenite administration

causes the disorganization of normal radiating pattern

of cell plates in the heart, treatment with AA before

sodium arsenite intoxication reduces such changes

and helps to maintain the normal architecture of the

heart. Treatment with AA prior to arsenic intoxication

reduces the extent of arsenic concentration as well as

DNA fragmentation in the heart tissue and also it

prevents the toxin induced reduction in the heart

weight to body weight ratio. Results suggest that AA

can prevent the reduction in the intracellular

antioxidant potential and protect the myocardium

from arsenic exposure by chelating the free arsenic in

the system.

The cardioprotective effects of AA can be

attributed to its powerful antioxidant, free radical

scavenging and metal chelating properties.

Protection against arsenic induced toxicity Arsenic, one of the ubiquitous environmental

pollutants, induces tissue damage, which is a major

concern to human population. Its exposure occurs

from inhalation, absorption through the skin and

primarily, by ingestion of contaminated food and

drinking water. An impaired antioxidant defense

mechanism followed by oxidative stress is the major

cause of arsenic-induced toxicity. AA has been shown

to protect arsenic induced cytotoxicity probably due

to its free radical scavenging as well as metal–

chelating properties and thereby, diminishing the

arsenic burden in the cells19

.

Hepatic protection (in vivo)Manna et al.22

have

investigated the hepatoprotective role of AA against

arsenic-induced oxidative damage in murine livers.

Oral administration of sodium arsenite at a dose of 10

mg/kg body wt for two days significantly reduced the

activities of enzymatic and nonenzymatic antioxidants

and in addition, it has also been shown to increase the

activities of serum marker enzymes viz., alanine

transaminase and alkaline phosphatase, DNA

fragmentation, protein carbonyl content, lipid

peroxidation end-products and the level of oxidized

glutathione. Treatment with AA at a dose of 20 mg/kg

body wt for 4 days prior to arsenic administration

prevents the alterations in the activities of all

antioxidant indices and levels of the other parameters

studied. Histological studies reveal less centrilobular

necrosis in the liver treated with AA prior to arsenic

intoxication when compared to the liver treated with

the toxin alone. The ability to attenuate arsenic-

induced oxidative stress in murine liver can probably

be via the antioxidant mechanism of AA.

Hepatic protection (in vitro)Manna et al23

have

further reported that the incubation of isolated murine

hepatocytes with sodium arsenite (1mM) for 2 h caused

reduction in the cell viability as well as activities of the

intracellular enzymatic as well as nonenzymatic

antioxidants. Treatment with sodium arsenite enhancs

lipid peroxidation and also increases the activities of

alanine transferase and alkaline phosphatase.

Administration of AA (100 µg/ml) before and also

along with the toxin almost normalizes the altered

activities of antioxidant indices. This study clearly

reveals the ability of AA to maintain the integrity of

cell membrane. AA is demonstrated to possess free

radical scavenging activity and it can enhance the

cellular antioxidant capability against sodium arsenite-

induced cytotoxicity.

Protection against oxidative insult in brainSinha

et al.24

have demonstrated the ability of AA to

ameliorate arsenic-induced oxidative insult in murine

brain. Dose of arsenic for disease induction as well as

AA concentration for treatment are the same as

reported earlier22

. Oral administration of arsenic in the

form of sodium arsenite significantly decreases the

levels of antioxidant enzymes, cellular metabolites,

reduces glutathione and total thiols and increases the

level of oxidized glutathione. In addition, it enhances

the levels of lipid peroxidation end products and

protein carbonyl content. Pretreatment with AA

almost normalizes the above indices. Histological

findings reveal that arsenic-treated brain tissue shows

more frequent nuclear pyknosis. Treatment with AA

prior to the arsenic intoxication reduces nuclear

pyknosis and shows almost normal architecture

similar to that of the control. The above effects can be

attributed to the antioxidant activity of AA.

Protection against nephrotoxicitySinha et al.25

have reported the efficacy of AA against arsenic-

induced nephrotoxicity in mouse model. Oral

administration of sodium arsenite at a dose of

10 mg/kg body wt for 2 days causes a significant

accumulation of arsenic in renal tissues and it alters

the activities of serum markers, antioxidant enzymes


242

and the levels of lipid peroxidation end products.

Treatment with AA at a dose of 20 mg/kg body wt for

4 days almost normalizes above indices. Histological

studies also indicate preventive role of AA against

sodium arsenite induced nephrotoxicity.

Testicular protectionManna et al.26

have

reported the preventive role of AA against arsenic-

induced testicular damage in mice. Dose of arsenic for

induction of disease and AA concentration for

treatment are the same as reported earlier22

.

Administration of arsenic (in the form of sodium

arsenite) significantly decreases the intracellular

antioxidant activity, the activities of the antioxidant

enzymes and the levels of cellular metabolites. In

addition, arsenic intoxication enhances testicular

arsenic content, lipid peroxidation, protein

carbonylation and the level of glutathione disulfide.

Exposure to arsenic also causes significant

degeneration of the seminiferous tubules with

necrosis and defoliation of spermatocytes.

Pretreatment with AA prevents the arsenic-induced

testicular oxidative stress and injury to the

histological structures of the testes which can be due

to its intrinsic antioxidant property.

Mechanism of action It can be speculated that the preventive role of AA

against arsenic-induced cardiac oxidative stress may

be due to formation of five-membered chelate

complex between arsenic and two equatorial hydroxyl

groups of AA. This chelate formation probably

removes the free toxin from the system and thereby

inhibits it from causing any further oxidative damage

to the tissue. In addition, the presence of one

carboxylic hydrogen atom may be responsible for its

free radical scavenging activity19

. Therefore, AA acts

as a good chelator against arsenic-induced toxicity.

Manna et al.23

have demonstrated that AA exhibits

preventive role similar to that of vitamin C. So, it can

be expected that AA can also be oxidized by reactive

oxygen species to the corresponding keto-derivative

like vitamin C, since it has one primary as well as two

secondary hydroxyl groups. Besides, AA also contains

one carboxylic hydrogen atom which can easily be

abstracted by any free radical. This property of AA

may explain its DPPH radical scavenging activity.

Protection against sodium fluoride induced toxicity Fluoride is utilized in a number of industrial

practices and is an ubiquitous ingredient of drinking

water, foodstuffs, and dental products27

. Excess intake

of fluoride, however, causes fluorosis, a slow

progressive degenerative disorder. Along with other

toxic effects, fluoride accumulation induces oxidative

stress. Hence, Ghosh et al.28

have conducted a study

to investigate the effect of AA against sodium

fluoride (NaF)-induced cytotoxicity and necrotic cell

death in murine hepatocytes. Dose-dependent studies

suggest that incubation of hepatocytes with sodium

fluoride (100 mM) for 1 h significantly decreases the

cell viability as well as intracellular antioxidant

potential. Incubation with AA (100 µg/ml) both prior

to and in combination with sodium fluoride almost

normalizes the altered activities of antioxidant

indices. The increased cell viability and reduced

cellular reactive oxygen species (ROS) in AA treated

hepatocytes prove the anticytotoxic effect of AA. AA

treatment enhances the cellular antioxidant capability

and protects hepatocytes against sodium fluoride-

induced cytotoxicity and necrotic death.

Protection against acetaminophen (APAP) induced

toxicity

Renal protectionAcetaminophen (APAP) is a

widely used analgesic and antipyretic drug and it is

safe at therapeutic doses. But accidental or intentional

overdose causes acute liver and kidney failure. Rats

exposed to a nephrotoxic dose of APAP exhibit

alterations in the levels of a number of biomarkers

(blood urea nitrogen, serum creatinine levels, etc.)

related to renal oxidative stress, a decrease in

antioxidant activity and elevation in renal TNF-α and

nitric oxide levels. AA treatment both pre- and post to

APAP exposure protects the alteration of these

biomarkers, compensated deficits in the antioxidant

defense mechanism and suppressed lipid peroxidation

in renal tissue. Experimental evidence suggests that

APAP-induced nephro-toxicity is a caspase-

dependent process that involves the activation of

caspase-9 and caspase-3 in the absence of cytosolic

cytochrome C release. These results provide evidence

that inhibition of nitric oxide overproduction and

maintenance of intracellular antioxidant status may

play a pivotal role in the protective effects of AA

against APAP-induced renal damage. AA represents a

potential therapeutic option to protect renal tissue

from the detrimental effects of acute acetaminophen

overdose29

.

Hepatic protectionAnother study30

has been

undertaken to explore the protective role of AA

against APAP induced acute hepatotoxicity. Exposure


243

of rats with a hepatotoxic dose of APAP (700 mg/kg

body wt, ip) altered a number of biomarkers and

induced necrotic cell death. Pre-treatment (80 mg/kg

body wt, orally) with AA affords significant

protection in liver injury. AA also prevents

acetaminophen-induced hepatic glutathione depletion

and APAP-metabolites formation. Results of this

study suggests that this preventive action of AA can

be due to the metabolic inhibition of the specific

forms of cytochrome P450 that activates

acetaminophen to NAPQI. In addition, administration

of AA 4 h after APAP intoxication, reduces

acetaminophen-induced JNK (Jun N-terminal Kinase

pathway) and downstream Bcl-2 and Bcl-xL

phosphorylation and thus, prevents mitochondrial

permeabilization, loss in mitochondrial membrane

potential and cytochrome C release. AA affords

protection against acetaminophen-induced

hepatotoxicity through inhibition of P450-mediated

APAP bioactivation and inhibition of JNK-mediated

activation of mitochondrial permeabilization30

.

Wound healing activity

Use of Arjuna bark in wound healing has been

mentioned in Sushruta Samhita31

. Effect of topical

application of phytoconstituents (fraction I, II and III)

fractionated from a hydroalcohol extract of the bark

of TA, has been assessed for the healing of rat dermal

wounds32

. Fraction III mainly consists of saponin

from the bark (AA is a major triterpenoid saponin in

the bark of TA). Wounds created on the back of rats

under anesthesia have been treated with various

fractions applied topically as simple ointment. Results

prove that Fraction III prepared as 1% simple

ointment shows complete epithelialization on day 20,

whereas Fraction I shows complete epithelialization

on day 9, which essentially consists of tannins32

.

Antiinflammatory activity

Facundo et al.11

have extracted AA from

Combretum leprosum and demonstrated that when

AA administered at 100 mg/kg body wt is able to

inhibit significantly the carrageenan-induced rat paw

edema by 80.8%, but at this dose, AA has been found

to be gastrotoxic. At a lower dose of 10 mg/kg,

toxicity has not been observed and AA significantly

inhibits the edema by 37.6%. AA (10 mg/kg body wt)

inhibited the acetic acid-induced constrictions by

30.3% in mice. Arachidonic acid and 12-O-

tetradecanophorbol-13-acetate (TPA) induced ear

edemas are widely used to evaluate the

antiinflammatory activity of cyclooxygenase (COX)

and lipoxygenase (LOX) inhibitors. AA shows a

similar profile as that of indomethacin and it inhibits

the arachidonic acid-induced ear edema by 55.5%

without interfering with the TPA-induced one. The

overall results suggest that AA affects the arachidonic

acid metabolism by cyclooxygenase, thus exerting its

antiinflammatory and antinociceptive activities.

Anticholinesterase activity Inhibition of acetyl and butyryl cholinesterase

enzymes by AA has been proved by modified

Ellman’s method using thin layer chromatography11

.

Inhibition of Acyl-CoA:Cholesterol acyltransferase

(ACAT), which catalyses acylation of cholesterol to

cholesteryl esters with long chain fatty acids is a very

attractive target for the treatment of

hypercholesterolemia and atherosclerosis33

. ACAT-1

and ACAT-2 are the two isoforms of ACAT in

mammals34

. AA isolated from Campsis grandiflora12

,

exhibits relatively high hACAT-1 inhibitory activity

of 60.8% at a concentration of 100 µg/ml, while it is

not shown to inhibit hACAT-2. Microsomal fractions

of Hi5 cells containing baculovirally expressed

hACAT-1 or hACAT-2 and rat liver microsomes are

used as the source of enzyme. Presence of three

hydroxyl groups at C-2, C-3 and C-23 in the A ring of

AA is attributed to its high hACAT-1 inhibitory

action.

Antidiabetic activity Manna et al.

35 have investigated the prophylactic

role of AA against streptozotocin (STZ) induced

diabetes in the pancreatic tissue of Swiss albino rats.

STZ administration (at a dose of 65 mg/kg body wt,

injected into the tail vein) causes an increase in the

production of both ROS and reactive nitrogen species

(RNS) in the pancreas of experimental animals.

Formation of these reactive intermediates decreases

the intracellular antioxidant defense, increases the

levels of lipid peroxidation, protein carbonylation,

serum glucose and TNF-α. Treatment of animals with

AA (at a dose of 20 mg/kg body wt, orally) both pre-

and post- to STZ administration effectively reduces

these adverse effects by inhibiting the excessive ROS

and RNS formation as well as by down-regulating the

activation of phospho-ERK1/2, phospho-p38, NF-κB

and mitochondrial dependent signal transduction

pathways leading to apoptotic cell death, thus

showing the beneficial role of AA against STZ-

induced diabetes.


244

Manna et al.36

have reported the efficacy of AA

against STZ induced diabetic nephropathy in rats.

Diabetic renal injury is associated with increased

kidney weight to body weight ratio, glomerular area

and volume, blood glucose (hyperglycemia), urea

nitrogen and serum creatinine. This nephro

pathophysiology increases the production of ROS and

RNS, enhances lipid peroxidation and protein

carbonylation and decreases intracellular antioxidant

defense in the kidney tissue. Treatment of AA (20

mg/kg body wt) effectively ameliorates diabetic renal

dysfunctions by reducing oxidative as well as

nitrosative stress and deactivated the polyol pathways.

Histological studies reveal multiple foci of

hemorrhagic necrosis and cloudy swelling of tubules

in the kidney of toxin control and this is reduced after

treatment with AA, suggesting that AA may act as a

ameliorating agent against the renal dysfunctions

developed in STZ-induced diabetes.

α-Glucosidase and α-amylase inhibitors are widely

used in the treatment of patients with type II diabetes37

.

AA isolated from the leaves of Lagerstroemia speciosa

inhibits α-glucosidase to some extent, while it shows

weak inhibitory activity against α-amylase38

.

Antiasthmatic activity Mast cells release mediators such as histamine,

acetylcholine etc. Mast cell disruption is induced in

rat by compound 48/80 (a condensation product of p-

methoxy-N-methyl phenyl amine). Rats treated with

AA (50 and 100 mg/ kg body wt) and alcoholic

extract of TA (250 and 500 mg/kg body wt) shows

significant protection against mast cell disruption.

While the control group demonstrates 62%

degranulation, the AA treated groups (50 and 100

mg/kg body wt) show 42 and 33% degranulation,

respectively. Animals treated with 250 and 500 mg/kg

body wt of alcoholic extract of TA exhibit 51 and

39% degranulation, respectively, while the standard

drug disodium chromoglycate (50 mg/kg body wt)

produces 22% degranulation. The results reveal that

AA and alcoholic extract of TA have significant mast

cell stabilization activity and specifically, AA exhibits

comparatively better stabilization activity than

alcoholic extract of TA39

.

TA and AA also protect the guinea pig against

histamine as well as acetylcholine induced

bronchospasm. Histamine is the most implicated

mediator in bronchoconstriction that accompanies

asthma40

. Acetylcholine can cause bronchoconstriction

by activating efferent cholinergic fibers, secondary to

the stimulation of histamine41

. AA at a dose of 50 and

100 mg/kg body wt, shows 49 and 64% protection,

respectively against histamine challenge and 40 and

51% protection, respectively against acetylcholine

challenge. The alcoholic extract of TA at 250 and 500

mg/kg body wt, exhibited 28 and 40% protection,

respectively against histamine challenge and 25 and

32% protection, respectively against acetylcholine

challenge. The standard drug chlorpheniramine

maleate (2 mg/kg body wt) produced 81% protection

against histamine challenge and atropine sulphate (2

mg/kg body wt) produced 72% protection against

acetylcholine challenge. The above results clearly

reveal that both AA and alcoholic extract of TA show

greater percentage of protection against histamine

challenge than against acetylcholine challenge. This

antiasthmatic and antianaphylactic activity of TA and

AA may be due to the mast cell stabilizing potential

and inhibition of antigen induced histamine and

acetylcholine release39

.

Antitumour activity

AA, isolated from the rhizome of Cochlospermum

tinctorium and its triacetate derivative and their

methyl esters have been tested using the short term in

vitro assay on Epstein Barr virus-EA activation in

Raji cells induced by 12-O-tetradecanoyl-phorbol-13-

acetate (TPA). Their inhibitory effects on skin tumor

promotors are greater than those of previously studied

natural products8.

Hemisynthetic derivatives of AA isolated from

Mitragyna ciliata, have been tested in vivo on a two-

stage carcinogenesis assay in mouse skin. The

activities have been evaluated by rate (%) of

papilloma-bearing mice and average number of

papillomas per mouse. In the group of mice treated

with hemisynthetic derivatives of AA, the occurrence

of papillomas is delayed, compared to the control.

Hence, Diallo et al.42

have suggested that AA

derivatives can be valuable compounds as antitumour-

promoters.

Antimicrobial activity AA isolated from the leaf extracts of Syzygium

guineense13

shows the most significant antibacterial

activity against Escherichia coli (3 µg/spot), Bacillus

subtilis (0.5 µg/spot) and Shigella sonnei (30 µg/spot).

The hydroxyl group present at C-23 position may be

responsible for its expression of antibacterial activity.

AA shows inhibitory activity against Cryptococcus

neoformans with an IC50 of 20 µg/ml10

.


245

Moderate antifungal activity against Candida

krusei, C. albicans and C.parapsilosis has been

exhibited by a mixture of AA [2α, 3β, 23-trihydroxy-

olean-12-en-28-oic acid/2α, 3β, 23-trihydroxy- urs-

12-en-28-oic acid] with minimum inhibitory

concentration (MIC) values in the range of 50 -200

µg/ml43

. The MIC values of a mixture of asiatic acid

and AA against five fungal animal pathogens ranged

from 0.2 to 1.6 µg/ml, viz., Candida albicans (0.9

µg/ml), Cryptococcus neoformans (0.4 µg/ml),

Aspergillus fumigatus (1.6 µg/ml), Microsporum

canis (0.2 µg/ml) and Sporothrix schenckii (0.2

µg/ml)14

.

Insecticidal property

Bhakuni et al.9 have identified another important

property of AA viz. insecticidal property. AA isolated

from the stem of Cornus capitata exhibits significant

inhibitory activity towards fourth instar larvae of

Spilarctia obliqua. A dose-dependent relationship of

activities has been observed. Effective concentration

to reduce feeding and growth of the larvae has been

found to be 617.8 and 666.9 ppm, respectively.

Analogs of AA AA has potential to be used as a structural

framework for the design of molecular receptors and

supramolecular architectures. The design and

synthesis of a chiral arjuna-18-crown-6, with two

additional functional groups at the 23 and 28 positions

and its high association constants make it attractive

for the design of molecular receptors, biomimetic

systems and supramolecular systems capable of

performing specific tasks44

. The nanosized chiral

triterpenoid on derivatization can immobilize varieties

of organic solvents at low concentrations. Arjunolic

acid-derived crown ether shows efficient binding to

monovalent cations, including a primary ammonium

ion paving the way for chiral recognition of amino

acids. Its use in the field of supramolecular chemistry

and nanoscience are also prospective45

.

Though triterpenoids have been recognized

renewables in nanoscience, a major difficulty in their

use is their availability in pure form. The mixture of

the triterpenic acids extractable from TA contains AA

as the major component along with asiatic acid, as a

minor component having a close structural

resemblance7,10

. Hence, Bag et al.46

have reported a

simple method of separation for the two nano-sized

triterpenic acids. Arjuna-bromolactone46

and the nine

esters of AA synthesised with alkyl chains47

were

found to function as excellent gelators of a wide

variety of organic liquids.

Conclusions

The scientific basis for the use of AA in Ayurvedic

medicine has been clearly proved by its versatile

effects viz. prevention of myocardial necrosis, platelet

aggregation, anticoagulant property, free radical

scavenging property, antioxidant property,

metal chelating property, antimicrobial activity etc

(Table 1). Though a large database could be cited to

prove the efficacy of AA in vivo (in experimental

animals) and in vitro, preliminary clinical trials (alone

or combined with conventional drugs) have to be

undertaken to establish its therapeutic effectiveness.

Studies on bioavailability and route of

administration have to be furnished. Also, further

investigations are necessary to identify the functional

group(s) of AA responsible for its cytoprotective role.

More studies have to be undertaken to unravel the

molecular mechanisms by which AA exerts its

preventive role against various cellular stress

conditions. Though it has been observed that the use of

AA has little side effects20

, further work has to be

initiated to evaluate its toxicity/side effects due to its

long term use, as well as to delineate the side effects

due to the use of AA analogs.

Acknowledgement

The authors thank Dr A B Mandal, Director, Central

Leather Research Institute, Chennai, India for his kind

permission to publish this work. The award of CSIR

Table 1Multifunctional activities of AA

S. No. Activity References

1 Antioxidant activity Sumitra et al.20, Manna et

al.19,22,23,26, Sinha et al.24,25

2 Antiplatelet activity Sumitra et al.20

3 Anticoagulant activity Sumitra et al.20

4 Antinecrotic activity Sumitra et al.20, Ghosh et al.28

5 Free radical scavenging

activity

Sumitra et al20, Manna et

al.19,22,23, Sinha et al.24,25

6 Antinephrotoxic activity Ghosh et al.29

7 Antihepatotoxic activity Ghosh et al.29

8 Antiinflammatory activity Facundo et al.11

9 Antinociceptive activity Facundo et al.11

10 Anticholinesterase activity Facundo et al.11, Kim et al.12

11 Antidiabetic activity Manna et al.35,36, Hou et al.38

12 Antiasthmatic activity Prasad et al.39

13 Antitumour activity Diallo et al.8,42

14 Antimicrobial activity Djoukeng et al.13, Masoko et

al.14, Liu et al.43, Zhang et al.10

15 Insecticidal activity Bhakuni et a.l9


246

Research Associateship to T Hemalatha, Senior

Research Fellowship (Extended) to S Pulavendran

and the CSIR Emeritus Scientist Project to

Dr R. Puvanakrishnan are gratefully acknowledged.

References

1 Vaidya A B, Terminalia arjuna in cardiovascular therapy, J

Assoc Physic India, 42 (1994) 281.

2 Nandkarni A K, Terminalia arjuna, in Indian Materia Medica

edited by A K Nandkarni and K M Nandkarni (Popular

Prakashan Private Limited, Mumbai, India) 1976, 1199.

3 Kumar D S & Prabhakar Y S, On the ethnomedical

significance of the arjun tree, Terminalia arjuna (Roxb.)

Wight & Arnot, J Ethnopharmacol, 20 (1987)173.

4 Chander R, Singh K, Khanna A K, Kaul S M, Puri A, Saxena

R, Bhatia G, Rizvi F & Rastogi A K, Antidyslipidemic and

antioxidant activities of different fractions of Terminalia

arjuna stem bark, Indian J Clin Biochem, 19 (2004) 141.

5 Halder S, Bharal N, Mediratta P K, Kaur I & Sharma K K,

Anti-inflammatory, immunomodulatory and antinociceptive

activity of Terminalia arjuna Roxb bark powder in mice and

rats, Indian J Exp Biol, 46 (2009) 577.

6 Anonymous, Monograph: Terminalia arjuna, Altern Med Rev,

4 (1999) 436.

7 King F E, King T J & Ross J M, The constitution of arjunolic

acid, a triterpene from Terminalia arjuna, J Chem Soc, 23

(1954) 3995.

8 Diallo B, Vanhaelen M, Vanhaelen-Fastré R, Konoshima T,

Kozuka M & Tokuda H, Studies on inhibitors of skin-tumor

promotion. Inhibitory effects of triterpenes from

Cochlospermum tinctorium on Epstein-Barr virus activation, J

Nat Prod, 52 (1989) 879.

9 Bhakuni R S, Shukla Y N, Tripathi A K, Prajapati V & Kumar

S, Insect Growth inhibitor activity of arjunolic acid isolated

from Cornus capitata, Phytother Res, 16 (2002) S68.

10 Zhang Q, ElSohly H N, Li X C & Walker L A, A new

triterpene from Leandra chaetodon, Planta Med, 69 (2003)

582.

11 Facundo V A, Rios K A, Medeiros C M, Militao J S L T,

Miranda A L P, Epifanio R A, Carvalho M P, Andrade A T,

Pinto A C & Rezende C M, Arjunolic acid in the ethanolic

extract of Combretum leprosum root and its use as a potential

multi-functional phytomedicine and drug for

neurodegenerative disorders: anti-inflammatory and

anticholinesterasic activities, J Braz Chem Soc, 16 (2005)1309.

12 Kim D H, Han K M, Chung I S, Kim D K, Kim S H, Kwon B

M, Jeong T S, Park M H, Ahn E M & Baek N I, Triterpenoids

from the flower of Campsis grandiflora K. Schum. as human

Acyl-CoA: Cholesterol acyltransferase Inhibitors, Arch Pharm

Res, 28 (2005) 550.

13 Djoukeng J D, Abou-Mansour E, Tabacchi R, Tapondjou A L,

Boud H & Lontsi D, Antibacterial triterpenes from Syzygium

guineense (Myrtaceae), J Ethnopharmacol, 101 (2005) 283.

14 Masoko P, Mdee L K, Mampuru L J & Eloff J N, Biological

activity of two related triterpenes isolated from Combretum

nelsonii (Combretaceae) leaves, Nat Prod Res, 22 (2008)1074.

15 Ratsimamanga A R & Boiteau P, GB Pat, 923414 (CA 59:

P10239a) 1963.

16 Xu R, Fazio G C & Matsuda S P T, On the origins of

triterpenoid skeletal diversity, Phytochem, 65 (2004) 261.

17 Sindambiwe J B, Calomme M, Geerts S, Pieters L, Vlietinck A

J & Van den Berghe D A, Evaluation of biological activities of

triterpenoid saponins from Maesa lanceolata, J Nat Prod, 61

(1998) 585.

18 Ajees A A & Balakrishna K, Arjunolic acid, Acta Crystallogr,

E 58 (2002), 682.

19 Manna P, Sinha M & Sil P C, Arsenic–induced oxidative

myocardial injury: protective role of arjunolic acid, Arch

Toxicol, 82 (2008) 137.

20 Sumitra M, Manikandan P, Kumar D A, Arutselvan N,

Balakrishna K, Murali Manohar B & Puvanakrishnan R,

Experimental myocardial necrosis in rats: role of arjunolic acid

on platelet aggregation, coagulation and antioxidant status.

Mol Cell Biochem, 224 (2001) 135.

21 Benowitz N L, Cardiotoxicity in the workplace, Occup Med, 7

(1992) 465.

22 Manna P, Sinha M & Sil P C, Protection of arsenic-induced

hepatic disorder by arjunolic acid, Basic Clin Pharmacol

Toxicol, 101 (2007) 333.

23 Manna P, Sinha M, Pal P & Sil P C, Arjunolic acid, a

triperpenoid saponin, ameliorates arsenic-induced cytotoxicity

in hepatocytes, Chem Biol Interact, 170 (2007) 187.

24 Sinha M, Manna P & Sil P C, Protective effect of arjunolic

acid against arsenic-induced oxidative stress in mouse brain, J

Biochem Mol Toxicol, 22 (2008) 15.

25 Sinha M, Manna P & Sil P C, Arjunolic acid attenuates

arsenic-induced nephrotoxicity, Pathophysiology, 15, (2008)

147.

26 Manna P, Sinha M & Sil P C, Protection of arsenic-induced

testicular oxidative stress by arjunolic acid, Redox Rep, 13

(2008) 67.

27 World Health Organization, Environmental Health Criteria,

(227 Fluorides IPS, Geneva, Switzerland) 2002.

28 Ghosh J, Das J, Manna P & Sil P C, Cytoprotective effect of

arjunolic acid in response to sodium fluoride mediated

oxidative stress and cell death via necrotic pathway, Toxicol in

vitro, 22 (2008)1918.

29 Ghosh J, Das J, Manna P & Sil P C, Acetaminophen induced

renal injury via oxidative stress and TNF-alpha production:

therapeutic potential of arjunolic acid, Toxicology, 13 (2009)

(in press).

30 Ghosh J, Das J, Manna P & Sil P C, Arjunolic acid, a

triterpenoid saponin, prevents acetaminophen (APAP) induced

liver and hepatocytes injury via the inhibition of APAP

bioactivation and JNK-mediated mitochondrial protection,

Free Radic Biol Med, (2009) (in press).

31 Ghanekar B G, Ayurveda Rahasyadipika, in Sushruta Samhita

with Hindi commentary-Sutra and Nidansthan (Mehar Chand

Lachhmandas, Lahore) 1936, 213.

32 Chaudhari M & Mengi S, Evaluation of phytoconstituents of

Terminalia arjuna for wound healing activity in rats, Phytother

Res, 20 (2006) 799.

33 Brown M S, Dana S E & Goldstein J L, Cholsterol ester

formation in cultured human fibroblasts, J Biol Chem, 250

(1975) 4025.

34 Coses S, Novak S, Zheng Y, Myers H M, Lear S R, Sande E,

Welch C B, Lusis A J, Spancer T A, Krouse B R, Erickson S

K & Farese R V, ACAT-2, a second mammalian acyl-CoA:

Cholesterol acyltransferase, J Biol Chem, 273 (1998) 26755.

35 Manna P, Sinha M & Sil P C, Protective role of arjunolic acid

in response to streptozotocin-induced type-I diabetes via the


247

mitochondrial dependent and independent pathways,

Toxicology, 257 (2009) 53.

36 Manna P, Sinha M & Sil P C, Prophylactic role of arjunolic acid

in response to streptozotocin mediated diabetic renal injury:

Activation of polyol pathway and oxidative stress responsive

signaling cascades, Chem Biol Interact, 181 (2009) 297.

37 Floris L L, Peter P A, Reinier H V, Eloy E R, Guy & Chris V W,

α-Glucosidase inhibitors for patients with Type 2 diabetes,

Diabet Care, 28 (2005) 154.

38 Hou W, Li Y, Zhang Q, Wei X, Peng A, Chen L & Wei Y,

Triterpene acids isolated from Lagerstroemia speciosa leaves as

alpha-glucosidase inhibitors, Phytother Res, 23 (2009) 614.

39 Prasad M V V, Anbalagan N, Patra A, Veluchamy G &

Balakrishna K, Antiallergic and anti-asthmatic activities of the

alcoholic extract of Terminalia arjuna and arjunolic acid, Nat

Prod Sci,10 (2004) 240.

40 Summers R, Sigler R, Shelhamer J H & Kaliner M, Effects of

infused histamine on asthmatic and normal subjects;

Comparison of skin test responses, J Allergy Clin Immunol, 67

(1981) 456.

41 Akah P A, Ezike A C, Nwafor S V, Ololiad C O & Enwerem N

M, Evaluation of asthmatic property of asystasia gangetic leaf

extracts, J Ethnopharmacol, 89 (2003) 25.

42 Diallo B , Vanhaelen-Fastré R , Vanhaelen M , Konoshima T,

Takasaki M & Tokuda H, In vivo inhibitory effects of arjunolic

acid derivatives on two-stage carcinogenesis in mouse skin,

Phytother Res, 9 (2006) 444.

43 Liu M, Katerere D R, Gray A I & Seidel V, Phytochemical and

antifungal studies on Terminalia mollis and Terminalia

brachystemma, Fitoterapia, 80 (2009) 369.

44 Bag B G, Pramanik S R & Maity G C, Arjunolic acid in

molecular recognition: first synthesis and cation binding

studies of a novel Arjuna-18-crown-6, Supramol Chem, 17

(2005) 297.

45 Bag B G & Dinda S K, Arjunolic acid: A renewable template

in supramolecular chemistry and nanoscience, Pure Appl

Chem, 79 (2007) 2031.

46 Bag B G, Dey PP, Dinda S K, Sheldrick W S & Oppel I M,

Simple route for renewable nano-sized arjunolic and Asiatic

acids and self-assembly of arjuna-bromolactone, Beilstein J

Org Chem, 2 (2008) 24.

47 Bag B G, Dinda S K, Dey P P, Mallia V A & Weiss R G, Self-

assembly of esters of arjunolic acid into fibrous networks and

the properties of their organogels (dagger), Langmuir, (2009)

(in press).

arjunolic acid: a novel phytomedicine with multifunctional therapeutic...

Documents