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Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Vol-III, Nov 2013 Special Issue Preliminary phytochemical and in vitro anti-diabetic activity of Ficus
racemosa (L.) stems bark extract
aRajendra Chary Vijayagiri, aEstari Mamidala
aInfectious Diseases & Metabolic Disorders Research Lab, Department of Zoology,
Kakatiya University, Warangal, Andhra Pradesh, India.
Abstract
Ficus racemosa Linn. (Family Moraceae) is traditionally used in the treatment of
metabolic disorders and skin problems. The present study is aim to identify the
phytochemical components in Ficus racemosa plant extract and to test the anti-diabetic
activity by in vitro study. Phytochemical analysis was performed by various qualitative
methods. In vitro anti-diabetic activity was done by measuring the α-amylase and α-
glucosidase enzyme inhibitory activity. The extraction of Ficus racemosa stem bark was
carried out using sequential extracts of solvents with varying polarity; hexane,
chloroform, ethyl acetate, acetone and methanol respectively. The qualitative
phytochemical analysis shows the presence of alkaloids, carbohydrates, glycosides,
saponins, tannins, phenols, flavonoids, quinones, steroids, amino acids and terpenoids in
various extracts. The chloroform extract shows highest α-amylase and α-glucosidase
enzyme inhibitory activity when compared to other solvent extracts. The results obtained
in the present investigation indicated Ficus racemosa stem bark as a rich source of herbal
medicines and having anti-diabetic compounds.
KEYWORDS: Ficus racemosa (L), phytochemicals, TLC, plant extracts, Rf (retention
factor).
Introduction
Diabetes mellitus is an endocrine and metabolic disorder characterized by chronic
hyperglycemia, dyslipidemia, and protein metabolism that result from defects in both
regulations of insulin secretion and/or insulin action (Bloc et al, 2006). There has been a
dramatic increase in the number of diabetic patients worldwide because of changes in
life-style and diet. The major complications associated with diabetes include retinopathy,
neuropathy, nephropathy, atherosclerotic coronary artery disease and peripheral
atherosclerotic vascular disease. Besides hyperglycemia, several other factors like
hyperlipidemia and enhanced oxidative stress play a major role in diabetic pathogenesis
(Kaczmer, 1998). However, the challenge is to optimize glycaemic control with
minimum number of medication while taking into consideration the cost of the therapy,
adverse effect profiles, ease of administration, and the urgency for blood sugar
normalization. Insulin and Insulin sensitizers, as well as enzyme inhibitors such as α-
glucosidase and α-amylase inhibitors contribute as an important therapeutic option in the
treatment of diabetes (Akhtar and Al, 1984).
Many traditional medicinal plants are good sources of unique phytochemical compounds
such as polyphenols and flavonoids. Recent studies have shown that some medicinal
w w w . o i i r j . o r g I S S N 2 2 4 9 - 9 5 9 8 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Vol-III, Nov 2013 Special Issue plants containing high total polyphenolic compounds and flavonoids yield can be linked
to intestinal α -glucosidase and pancreatic α -amylase inhibitory activities in vitro (Koh et
al, 2010). Thus, efforts have been directed at investigating intestinal α-glucosidase and
pancreatic α–amylase inhibitors from medicinal plants that are largely free of major
undesirable side effects.
Ficus racemosa linn is commonly known as Cluster fig and Udumbara (sanskrit) belongs
to the family Moraceae, is an evergreen, moderate sized, deciduous tree and used as
herbal medicine from ancient times. Its leaves are dark green, ovate and having
traditional medicinal importance (Abu Hasant. 2011). This is native to Australia, South
East Asia and the Indian subcontinent. It is unusual in this plant that its figs grow on or
close to the tree trunk (Joseph. 2010). F. racemosa is widely distributed genus in North
East India and abundant in Warangal district, Andhra Pradesh having used by Warangal
peoples to reduce diarrhea and stop bleeding along with some other ethno medicinal
practices (Rajendra Chary Vijayagiri. 2012; Vinatha Naini and Estari Mamidala, 2013).
So, it was the need of time to explore some of the species of this genus specially Ficus
racemosa which is available in southern part of Andhra Pradesh, India, for better
upgradation of knowledge regarding the phytochemicals and its biological activity of this
genus. Therefore, the aim of this work is to carry out a phytochemical screening of stem
bark extracts of Ficus racemosa and in vitro antidiabetic activity testing.
Materials and Methods:

Plant material
Based on ethno botanical investigation and in our previous study (Rajendra chary
Vijayagiri. 2012), the Ficus racemosa plant was selected for study. Ficus racemosa stem
bark was collected from rural areas of Venkatapuram forest, Warangal dist., Andhra
Pradesh, India. The taxonomic identities of these plants were confirmed by Department
of Botany, Kakatiya University, Warangal, India. The collected fresh stem bark of plant
free from diseases was bought to laboratory in sterile polyethylene bags and washed
thoroughly 2-3 times with running tap water and then shade dried for three weeks,
subsequently ground into fine powder using mechanical grinder and motor driven
grinding mill. The powder was used for extraction of crude extracts.
Preparation of plant extracts
Around 2 kg of stem bark of Ficus racemosa was powdered by using of electric grinder
to obtain coarse powdered which is best suited for extraction. The powder was weighed
and plant powder material was extracted successively with maceration method. In this
method using solvents ranging from non polar to polar i.e., hexane, chloroform, ethyl
acetate, acetone and methanol for 24 hrs. The extract was concentrated under vacuum
using rotary vacuum evaporator. The obtained extracts were stored at 4oC till to use for
phytochemical investigation.
Preliminary phytochemical analysis
Preliminary phytochemical screening of the extracts for alkaloids, carbohydrates,
glycosides, saponins, tannins, phenols, flavonoids, quinones, steroids, amino acids and
w w w . o i i r j . o r g I S S N 2 2 4 9 - 9 5 9 8 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Vol-III, Nov 2013 Special Issue terpenoids using standard phytochemical screening methods (Harbone. 2009, Siddiqui.
1997 and Earnsworth. 1974).
Inhibition of α–amylase:
Starch solution (0.5% w/v) was prepared in Tris HCl buffer with 6.7 mM sodium
chloride (pH 6.9) in boiling water for 5 min and preincubated at 37°C for 5 min.
The extracts of F. racemosa were dissolved in DMSO to obtain concentration of 10, 20,
40, 60, 80 and 100 µg/ml. Then 0.2 ml of plant extract was added to the tubes containing
starch solution. A total of 0.1 ml pancreatic amylase solution (PPA-Sigma Aldrich)
prepared in Tris HCl buffer (2 units/ml) was added to the tube containing plant
extract and starch solution. The reaction was carried out at 37°C for10 min. The reaction
was stopped by adding 0.5 ml 50% acetic acid. The reaction mixture was centrifuged at
3000 rpm for 5 min 4°C. The absorbance of supernatant was measured at 595 nm
(Conforti et al, 2005).
Percentage inhibition = Control-Test/Control × 100

Inhibition of α – glucosidase
Enzyme solution prepared in Tris buffer (pH 8) was added to the tubes containing
increasing concentration of extracts of F. racemosa (10, 20, 40, 60, 80 and 100 µg/ml) at
37°C for 60 min. Then the reaction mixture was heated for 2 min in boiling water to stop
reaction. The absorbance was measured at 540 nm. Percentage inhibition was calculated
by using the following equation (Hansawasdi et al, 2000 and Vogel, 2002).
Percentage inhibition = Control-Test/Control × 100
Results:

Yield of Ficus racemosa stem bark extracts
The yield of sequential extracts (g) is shown in Table 1. The 500 grams of the plant
material used for extraction with each solvent and the amount of the hexane extract
obtained from the extraction was (7.3) (1.46 % w/w yield), chloroform extract was 6.6 g
(1.32 % w/w yield), ethyl acetate extract 5.3 g (1.06 % w/w yield), acetone extract 2.9 g
(0.58 % w/w yield) and methanol extract was 47.6 g (9.52 % w/w yield).

Phytochemical screening of Ficus racemosa stems bark extracts
Phytochemical screening of the sequential extract of Ficus racemosa stem bark revealed
the presence of various bioactive components of which phenolics, saponins, steroids,
alkaloids, flavonoids, terpenoids, tannin, and cardiac glycosides are the most prominent
components and the result of phytochemical test is presented in Table 2. Among these
phytochemical tests, methanol extract was found to contain maximum saponin content
along with plant phenolics such as alkaloids, glycosides and tannins.
Inhibition of α–amylase
All solvent extracts of F. racemosa as a test drug and Acarbose as reference
standard were analysed for α-amylase inhibitory activity at concentration of 10, 20, 40,
w w w . o i i r j . o r g I S S N 2 2 4 9 - 9 5 9 8 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Vol-III, Nov 2013 Special Issue 60, 80 and100 µg/ml. A dose dependent, gradual rise in inhibition of α-amylase was
observed for test and standard drugs as shown in Figure 1. The chloroform extract of F.
racemosa shows highest α-amylase inhibitory activity when compared remaining solvent
extracts. The IC50 value for chloroform extract and and Standard drug were found to be
64.5 µg/ml and 51.8 µg/ml, respectively. The chloroform extract shows 81% α–amylase
inhibitory activity where standard drug acarbose shows 84% at 100 µg/ml concentration.
Inhibition of α–glucosidase:
A gradual rise in inhibitory activity of α-glucosidase was observed for all solvent extracts
of F. racemosa (Test Drug) and acarbose (Standard reference) as shown in Figure 2. The
chloroform and acetone extracts shows highest α-glucosidase inhibitory activity when
compared to methanol, ethyl acetate and hexane extracts. The IC50 value for test drug
chloroform extract and standard drug were found to be 71.2 µg/ml and 52.9 µg/ml,
respectively. The chloroform extract shows 66% α-glucosidase inhibitory activity
compared to acarbose (68%).
Discussion

In the present study, we investigated F. racemosa stem bark extracts (n-hexane,
chloroform, ethyl acetate, acetone and methanol) with anti-diabetic properties for
intestinal α -glucosidase and pancreatic α -amylase inhibitory activities. There is an
abundant medicinal plant throughout the world but only small amounts are investigated
for its biological activity (Awadh Ali. 2001). Previous study of the phytochemical
analysis of Ficus racemosa report as found that most of the biologically active
phytochemicals were present in the ethanolic extract of Ficus racemosa bark
(Poongothai, 2011). The results confirm the presence of constituents which are known to
exhibit medicinal as well as physiological activities (Ismail. 2011). The results obtained
in this study thus suggest that the identified phytochemical compounds may be the
bioactive constituents responsible for the efficacy of the stem bark of the plants studied
(Ogu. 2012). Qualitative tests performed on the stem bark extracts of Ficus racemosa
indicate the presence of alkaloids, carbohydrate, glycosides, saponins, tanins, phenolic
compounds, proteins, flavonoids, terpenoids, quinines, steroids in different extracts.
The basic fundamental lying behind hyperglycemia involved overproduction and
decreased utilization of glucose (Koh et al, 2010). Alloxan, destroys β cells of islet of
langerhens of pancreas resulting in decrease in the insulin secretion and leads to
decreased use of glucose by tissues (Conforti et al, 2005). α- amylase is main enzyme
present in pancreas responsible for the digestion of starch and absorption of glucose. Its
inhibitors such as acarbose inhibit the release of glucose in the blood and thereby
achieving the anti-diabetic effect. Our finding revealed that the chloroform extract of
F. racemosa efficiently inhibited the enzyme [Figure 1]. α-glucosidase is responsible
for the digestion of carbohydrates to simpler carbohydrates and its absorption in
small intestine (Abu hasant, 2011) Chloroform and ethyl acetate extracts of F.
racemosa significantly inhibit the enzyme and thus attributed for anti-diabetic activity.
Further purification, identification and characterization of the active compounds would
be our priority in the future studies.
w w w . o i i r j . o r g I S S N 2 2 4 9 - 9 5 9 8 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Vol-III, Nov 2013 Special Issue Acknowledgement;
The authors wish to acknowledge the Department of Botany, Kakatiya University,
Warangal for identification and preservation of the plant and also thank to traditional
healers of Warangal district, Andhra Pradesh.
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Figure 1: α-amylase inhibitory activity

itio
ib 60
Concentration of extracts (µg/ml)
Figure-2. α-glucosidase inhibitory activity
sid
co
lu 30
Concentration of extracts (µg/ml)
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