Investigation of anthelmintic, anti inflammatory activity of leaves extract of Acacia catechu Willd

Journal of Pharmacy Research Vol.5 Issue 5.May 2012
Gulzar Alam et al. / Journal of Pharmacy Research 2012,5(5),2587-2589
2587-2589
Research Article
ISSN: 0974-6943
Available online through
www.jpronline.info
*Corresponding author.
Gulzar Alam
Kailash Institute of Pharmacy & Management,
Gorakhpur, Pin-273209,
Uttar Pradesh (India)
Investigation of anthelmintic, anti-inflammatory
activity of leaves extract of Acacia catechu Willd
*Gulzar Alam1
, Manjul Pratap Singh1
, Anita Singh1
, Roshan Patel 2
1
Kailash Institute of Pharmacy & Management,Gorakhpur, Uttar Pradesh,India
2
Shree Leuva Patel Trust Pharmacy Mahila College, Amreli, Gujarat,India
Received on:16-02-2012; Revised on: 18-03-2012; Accepted on:22-04-2012
ABSTRACT
From ancient timeAcaciacatechu Willd. (AC) is used as a potent medicinal. Preliminary phytochemical tests were conducted with the crude extract obtained
from the leaves of AC reveals the presence of carbohydrates, steroids, alkaloids, glycosides, tannins, saponins, flavones, and phenolic. Anthelmintic activity
of petroleum ether, ethanolic and ethanol:water (1:1) extracts of leaves of AC was evaluated by adult Indian earthworm, (Pheretima posthuma). The
anthelmintic activity of extract was compared with standard albendazole. Ethanol:water (1:1) extract was found to possess potent anthelmintic activity and
petroleum ether extract was found to be least active, while ethanol:water (1:1) extract playing an intermediate role. The anti-inflammatory activity of leaves
extracts at the doses of 300mg/kg body weight was investigated in albino rats of wiester strain using carrageenan induced rat paw oedema. The ethanol extract
of leaves showed significant anti- inflammatory activity (45%) when compared with the standard (54%). Further studies are suggested to isolate the active
principles which are responsible for the anthelmintic and anti-inflammatory activity.
Keywords: Acacia catechu Willd., Leaves extracts, Phytochemical tests, Anthelmintic & Anti-inflammatory activity,
INTRODUCTION
Herbs are staging a comeback and herbal ‘renaissance’ is happening all over
the globe. The herbal products today symbolize safety in contrast to the
synthetics that are regarded as unsafe to human and environment and been
priced for their medicinal, flavouring and aromatic qualities for centuries.
Acacia catechu Willd. (Family: Fabaceae and Sub-family: Mimosoideae)
known as Black Khair and is widely distributed in different part of India. AC
grows slowly and matures to a height of about 12-15 meters. The leaves of
AC are compound. The rachis branching from the mid-rib has 4 to 5 round
prickles. The rachis is nearly 10 to 20 cm long and bears 20 to 60 pinnae each
about 3 to 4 cm long. The fruit of is pod shaped. It is 5 to 7 cm long and 1 to
1.5 cm wide and shining brown in color. The sap wood of is large and yellow-
ish white and heart wood is small and red in colour (1-4).
Deshpande et al (1981) reported the flavonoids like kaempferol,
dihydrokaempferol, taxifolin, isorhamnetin, (+)-afzelchin, and a dimeric
procyanidin AC along with quercetin and (-)-epicatechin in heartwood of
Acacia catechu Willd(5)
. Pradeep Sharma et al (1997), isolate the chemical
constituents of the leaves of Acacia catechu Wliid. and characterized as
poriferasterol, quercetin, poriferasterol-3-ß-D-glucoside, quercetin-3-O-
arabinofuranoside, quercetin-3-O-rhamnoside, and 3-O-galactose(6)
.
Pradeep Sharma etal (1999), isolate the chemical constituents of the bark and
root of AC and characterized aslupenone, lupeol, poriferasterol, octacosanoic
acid, poriferasterol acyglucoside, hexacosnoic acid ester, ursolic acid,
poriferasterol-3-ß-D-glucoside, 3-methylquercetin, quercetin, dihydrokaemp-
ferol, dihydroquercetin, (-)-epicatechin and catechin(7)
. Azad et al (2001)
reported (+)-catechin and a new poly-phenolic compound with a (+)-homo-
iso-catechin(8)
. Yadava etal (2002) isolate a A new flavone glycoside: 5,7,3',4'-
tetrahydroxy -3-methoxy flavone-7-O-beta-D-galactopyrano syl- (1,4)-O-
beta-D-gluco-pyranoside from the ethyl acetate soluble fraction of the
ethanolic extract of the stems of AC(9)
. Jain et al (2007) reported a flavonoid
“quercetin” from AC(10)
.
The extracts of different Part of AC have been reported to have various
pharmacological effects. AC is useful in stomach problems like diarrhea,
dysentery, colitis and gastric cancer. AC is used as mouthwash for mouth,
gum and throat diseases like gingivitis, stomatitis. Cutch and katha is cooling,
digestive and a very valuable astringent, especially in chronic diarrhea and
dysentery, bleeding piles, uterine haemorrhages, leucorrhoea etc. The decoc-
tion of bark mixed with milk is taken to cure cold and cough(11)
.
Figure 1: Whole tree of Acacia catechu Willd. from where leaves were col-
lected.
The leaves were used traditionally for the treatment of helminthes and as
anti-inflammatory but not yet proved scientifically. Hence, the present study
was designed to evaluate the anthelmintic and anti-inflammatory activity of
petroleum ether, ethanol and aqueous alcoholic extracts obtained from leaves
of the Acacia catechu Willd.
animal models(12)
. Rajni Singh et al (2005) reported antimicrobial activity of
some natural dyes in which AC was effective against all the microbes tested
except Pseudomonas aeruginosa(13)
. Azad Chowdhury etal (1984) reported
anti-fertility activity of a traditional contraceptive pill comprising AC(14)
.
Some activities are also reported by various scientist viz. anti-inflammatory,
hypoglycaemic, hepatoprotective, antioxidant, antimicrobial etc(15-20)
.
hepato-protective effects of the ethyl acetate extract of AC in experimental
Ray et al (2006) reported antipyretic, anti-diarrhoeal, hypo-glycaemic and

2587-2589
MATERIALS AND METHODS
Plant material
The fresh leaves of Acacia catechu Willd was collected from Village Bageha,
District Chitrakoot (U.P., India), was authenticated by the plant taxonomist,
Dept. of Botany, M.M.P.G. College, Modinagar (U.P., India). A voucher
specimen (MMPG/0115) was deposited at the Dept. of Botany, M.M.P.G.
College, Modinagar (U.P., India) for the future reference. After authentica-
tion, fresh plant material (Leaves) was collected in bulk, washed under run-
ning tap water to remove adhering material, dried under shade and pulverized
in a mechanical grinder. The coarse powder was pass through sieve no. 40,
stored in air tight container and used for further extraction.
Preparation of plant Extracts
The extraction of 1 kg dried coarse powder of the leaves of Acacia catechu
Willd was carried out by cold maceration process using with petroleum ether
(60-80o
C), ethanol and ethanol: water (1:1) respectively for 7 days each. The
petroleum ether (60-80o
C), ethanol and ethanol: water (1:1, aqueous alco-
holic) extracts were then filtered through muslin cloth and Whatmann filter
paper. The filtrates were concentrated under reduced pressure to obtain
residues.
Phytochemical Analysis
Preliminary phytochemical screening of the leaves extracts ofAcaciacatechu
Willd. was performed as per standard procedure(21,22)
which revealed that the
presence of carbohydrates, steroids, alkaloids, glycosides, tannins, saponins,
flavones, and phenolic compounds.
Table 1: Phytochemical analysis of leaves extracts of Acacia catechu
Willd.
(+) Indicates positive test result & (-) Indicates negative test result
Preparation of test sample
Samples for in-vitro study were prepared by dissolving and suspending 2.5g
of each extract Petroleum ether (60-80o
C) extract, Ethanol extract and
Ethanol:Water (1:1) extract in 25 ml of distilled water to obtain a stock
solution of 100 mg/ml. From this stock solution, different working dilutions
were prepared to get concentration range of 25, 50, 75 and 100 mg/ml.
Evaluation of Anthelmintic activity
Prepared extracts were evaluated for anthelmintic activity separately as per
the prescribed method(23,24)
. The assay was performed on adult Indian earth-
worm, Pheretima posthuma due to its anatomical and physiological resem-
blance with the intestinal roundworm parasite of human beings(25)
. Because
of easy availability, earthworms have been used widely for the initial evalu-
ation of anthelmintic compounds in-vitro(26)
. Indian adult earthworms
(Pheretima posthuma) collected from moist soil and washed with normal
saline to remove all faecal matter. The earthworms of 3-5 cm in length and
0.1-0.2 cm in width were used for all the experimental protocol. The earth
worms were divided into six groups of six earth worms in each. Albendazole
was diluted with 5% DMF (Dimethyl formamide) in normal saline solution
to obtain 10, 25, 50 and 100 mg per ml served as standard and poured into
Petri dishes. The extracts were dissolved in 5% DMF in normal saline solu-
tion and diluted to prepare four concentrations such as 10, 25, 50 and 100 mg
per ml and poured into petri dishes. 5% DMF in normal saline solution was
taken as control. Earth worms were placed in petri dishes containing differ-
ent concentrations of standard and extracts at room temperature. The mean
paralysis time and mean death time for each sample was calculated (all the
readings were taken in triplicate). Paralysis was said to occur when the
worms do not revive even in normal saline. Death was concluded when the
worms lost their motility, followed with fading away of their body colour.
Table 2: Anthelmintic activity leaves extracts of Acacia catechu Willd.
Test substance Concentration Time taken for Time taken
(mg/ml) paralysis (min.) for death (min.)
Control (5% DMF in normal saline) - - -
Albendazole 25 5:15 ± 0.01 6:30 ± 0.01
50 3:28 ± 0.01 4:37 ± 0.01
75 2.38 ± 0.02 3.20 ± 0.05
100 1.11 ± 0.05 2.53 ± 0.02
Petroleum ether (60-80o
C) Extract 25 28.35 ± 0.01 32.15 ± 0.02
50 22.45 ± 0.04 25.25 ± 0.05
75 18.30 ± 0.02 20.52 ± 0.01
100 13.25 ± 0.01 15.32 ± 0.03
Ethanol Extract 25 14.24 ± 0.01 16.32 ± 0.04
50 11.28 ± 0.02 14.35 ± 0.02
75 7.55 ± 0.05 10.12 ± 0.01
100 4.14 ± 0.03 6.25 ± 0.04
Ethanol: Water (1:1) Extract 25 7.18 ± 0.04 9.47 ± 0.02
50 5.35 ± 0.03 7.39 ± 0.03
75 3.22 ± 0.02 5.21 ± 0.01
100 2.43 ± 0.02 4.26 ± 0.02
(All values are expressed as mean ± SEM (n=6) Control worms were alive up
to 24 hrs)
Anti-inflammatory Activity
Healthy inbred Wister albino rats of either sex, (150-200g) were selected and
housed in polypropylene cages at a well-ventilated, temperature-controlled
(30±1o
C) animal room with food and water ad libitum. Animals were periodi-
cally weighed before and after experiments. Animals were divided in five
groups of 6 animals each. The control group receives vehicle orally, while
other groups receives standard drug and test drug respectively. The animals
were treated with drugs by oral route and subsequently one hour after treat-
ment, 0.1ml of 1% suspension of carrageenan in normal saline was injected to
the sub planter region of left hind paw to induce edema. The paw volume was
measured initially at 1, 2, 3 and 5 hours after carrageenan injection using
plathismometer. The difference between the initial and subsequent reading
gave the actual edema volume which was compared with control. The differ-
ence of average values between treated animals and control group is calcu-
lated for each time interval and evaluated statistically. The percent inhibition
is calculated using the formula as follows: {% edema inhibition = [1- (Vt/
Vc)] × 100}. Vt and Vc are edema volume in the drug treated and control
group’s respectively (27-29)
.
Table 3: Effect of Acacia catechu Willd extract and Indomethacin on time course of Carrageenan induced rat paw oedema
Groups 0 Hr 1 Hr 2 Hr 3 Hr 4 Hr 5 Hr
Control 5.41 ± 0.065 4.89 ± 0.03 4.68 ± 0.05 4.54 ± 0.03 4.45 ± 0.07 4.25 ± 0.09
Standard (Indomethacin) (10 mg/kg) 4.21 ± 0.03 3.34 ± 1.01 3.01 ± 0.11 2.85 ± 0.13 2.45 ± 0.05 1.95 ± 0.07
C) Extract (300 mg/kg) 4.58 ± 0.02 3.93 ± 0.15 3.36 ± 1.01 3.11 ± 0.05 2.85 ± 0.07 2.65 ± 0.04
Ethanol Extract (300 mg/kg) 4.61± 0.02 4.01 ± 0.04 3.66 ± 0.07 3.21 ± 1.01 2.91 ± 1.02 2.32 ± 0.05
Ethanol: Water (1:1) Extract (300 mg/kg) 4.68 ± 0.21 4.21 ± 0.11 3.75 ± 0.05 3.31 ± 0.02 3.01 ± 0.10 2.74 ± 0.10
[All values are expressed as mean ± SEM (n = 6)]
Chemical Tests Leaves Extracts
classes Petroleum Ethanol Aqueous
ether extract alcoholic
extract extract
Carbohydrates Molish Test - + +
Fehling’s Test - + +
Protein Biuret Test - - -
Millon’s Test - - -
Amino Acid Ninhydrin Test - - -
Steroids Salkowski Test + - -
Liebermann-Burchard Test + + +
Alkaloids Dragendroff’s Test - + +
Wagner’s Test - + +
Glycosides Legal’s Test - + +
Killer-Kiliani Test - + +
Tannins Ferric chloride Test - + +
Vanillin-HCl Test - + +
Flavonoid NaOH Test - + +
Catechin Test Vanillin-HCl Test - + +
Saponin Ammonia Test - + +

2587-2589
Table 4: Percentage inhibition of the edema in albino rats
Groups % Inhibition in Hr
1 Hr 2 Hr 3 Hr 4 Hr 5 Hr
Control — — — — —
Standard (Indomethacin) 31.69 35.68 37.22 44.94 54.12
(10 mg/kg)
C) 19.63 28.21 31.49 35.96 37.65
Extract (300 mg/kg)
Ethanol Extract (300 mg/kg) 17.99 21.79 29.29 34.61 45.41
Ethanol: Water (1:1) 13.90 19.87 27.09 32.36 35.53
Extract (300 mg/kg)
Figure 2: Anti-inflammatory activity of the leaves extracts of Acacia
catechu Willd.
RESULTS AND DISCUSSION
In the present study the potential anthelmintic activity and anti-inflamma-
tory activity of the leaves extracts of Acacia catechu Willd. were investi-
gated. The Results in the Table 2 depict the time taken for paralysis and
death of worms after treating with the test substances. It was observed that
the Ethanol: Water (1:1) extract of leaves of AC is more potent than the
ethanol and petroleum ether extracts and their activity was comparable with
the standard drug albendazole. It causes paralysis followed by death of the
worms at all tested dose levels.
The anti-inflammatory activity was evaluated and the percentage of inhibi-
tion of the extract was compared with the standard indomethacin. The high-
est percentage inhibition of the ethanol extract was found to be 45%, com-
pared to standard which is 54%. The observations and results are shown in
Table 3 & 4. Figure 1 shows the graphical representation of comparative
anti-inflammatory activity.
There is a need for further studies in order to isolate the active ingredients
that are responsible for its potential anthelmintic activity and anti-inflam-
matory activity of the leaves extracts AC and to elucidate the mechanism of
action of these active ingredients.
ACKNOWLEDGEMENTS
The authors express their deepest gratitude to Dr. Y. Kumar, Director, I.T.S.
Paramedical College, Muradnagar, Ghaziabad (U.P.), for providing me nec-
essary facilities for carrying out research work and also thankful to Asutosh
Sewa Avem Siksha Sansthan, Gorakhpur, for its financial support.
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