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Research Article

ISSN: 0974-6943

Rajaram K et al. / Journal of Pharmacy Research 2011,4(3),891-893

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In-vitro antioxidant and antidiabetic activity of Tephrosia tinctoria PERS.: an endemic medicinal
plant of South India
Rajaram K1 and Suresh Kumar P 1*
1
Department of Biotechnology, Anna University of Technology, Tiruchirappalli-620024, Tamil Nadu, India

Received on: 05-10-2010; Revised on: 14-12-2010; Accepted on:09-02-2011


ABSTRACT
The present study was carried out to explore the in vitro antioxidant and antidiabetic activity of the different parts (Leaf, Stem & Root) of Tephrosia tinctoria extracted with various solvents
from non polar to polar basis (Hexane, Chloroform, Ethyl acetate & Ethanol). Among the various fractions tested, the Ethyl acetate fraction of stem of T. tinctoria exhibited maximum antioxidant
(DPPH with IC 50 value 33.54 4.27g/ml) and antidiabetic (Glucosidase inhibition IC 50 value 94.33 3.65g/ml) activity. Further the ethyl acetate fraction of stem extract was assayed for
antioxidative assays such as reducing power, lipidperoxidation inhibition activity, total phenols and total flavonoids. The ethyl acetate stem extract fraction of T.tinctoria was subjected to
cytotoxicity studies by performing MTT assay on L6 muscle cell line, which showed nil or low toxicity effect at higher doses.

Key words: Antioxidant, DPPH, -Glucosidase, Tephrosia tinctoria


INTRODUCTION
Diabetes mellitus is one of the worlds major public health burdens. In 2000, there were around
171 million diabetes causes and it is estimated that the number will double by 2030[1].
Numerous studies have been demonstrated that oxidative stress, mediated mainly by hyperglycemia induced generation of free radicals, contributes to the development and progression
of diabetes and its complications [2-4]. Abnormally high levels of free radicals which cause
membrane damage due to peroxidation of membrane lipids and protein glycation and simultaneous decline of antioxidant defense mechanisms leads to cell and tissue damage [5]. The
increase of ROS leads to damage of -cells through the induction of apoptosis and suppression
of insulin biosynthesis [6-7]. As a new strategy for alleviating the oxidative damage in diabetes,
interest has shown in the usage of natural antioxidants. It has been postulated that many of the
negative effect of oxidative stress are diminished upon supplementation with certain dietary
antioxidants such as Vitamin E, Vitamin C and other non-nutrient antioxidants such as
flavonoids[8].
The genus Tephrosia (Fabaceae) with about 400 species is distributed chiefly in Asia, Africa,
Australia and America [9-10]. About twenty-four species of Tephrosia were recorded in India[1112]. Most of the Tephrosias are herbs to undershrub and are grown as weeds. The genus is well
known for its richness in prenylated flavonoids and is considered to possess insect repellent,
larvicidal, pesticidal, antimicrobial and anticancer properties [13-17]. Tephrosia tinctoria (TT)
is widely distributed in Andhra Pradesh, South India [18]. A new prenylated isoflavone 7-OGeranylbiochanin together with three known compound, 7-O-methylglabranin, Flemichapparin
and dehydrodeguelin was isolated from root [19]. The root methanolic extract of TT possess
antimicrobial activity [20-21]. The objective of the present investigation to explore the in vitro
antioxidant and antidiabetic activity of bioactive guided fraction of TT herb. To our knowledge, this is the first report of antioxidant and antidiabetic activity in TT.
MATERIALS AND METHODS
Plant material
The herb TT was collected from Kolli hills of Namakkal (Dt), Tamil Nadu during the month
of November 2009 and authenticated by the Botanical Survey of India (BSI) (Southern Circle),
Coimbatore, Tamil Nadu. A voucher specimen (BSI/SRC/5/23/09-10/Tech-1569) was deposited in the Rappinart Herbarium, St.Josephs College, Tiruchirappalli, Tamil Nadu, India.
The shade dried plant parts of TT (leaf, stem and root) were coarsely powdered with mechanical grinder and stored in dry place.
Chemicals
1,1 diphenyl-2-picryl-hydrazl(DPHH), butylated hydroxyl toluene (BHT) were purchased
from Hi media , Mumbai. All other chemicals and reagents were of analytical grade.
Preparation of plant extracts (Activity guided extraction)
100gm of powder (leaf, stem and root) was extracted with various organic solvents (Petroleum
ether, Chloroform, Ethyl acetate and methanol) based on their polarity. All fractions were
evaporated to dryness at 40C in the rotary evaporator and stored. 100mg of each extract of
different plant parts (Leaf, Stem & Root) of TT was dissolved in 1 ml of DMSO (Dimethyl
sulfoxide) which was used for the following assays.
DPPH radical scavenging activity
The free radical scavenging activities of all extracts were measured by DPPH assay [22]. 0.9 ml
of 1.5 x 10-4 M DPPH radical solution in methanol was prepared, and then mixed with 0.1 ml
of the sample dissolved in DMSO and kept in the dark for 30 min. The quantity of DPPH
remaining in the mixed solution was measured at 517nm. The reduction in the absorbance of

*Corresponding author.
P.Suresh Kumar
Department of Biotechnology
Anna University of Technology
Tiruchirappalli - 620024, Tamil Nadu, India
Tel.: + 91-9786075353
E-mail: sureshbiotech2003@yahoo.co.in

the DPPH solution indicated the free radical scavenging activities of the test extracts. DMSO
without the sample was used as a control.
The DPPH radical scavenging activity was calculated according to the following formula: (%)
inhibition ratio [(AbsControl Abssample)/Abscontrol] x 100.
a-Glucosidase inhibitory activity
The inhibitory effect was measured using the method slightly modified from Dahlqvist [23].
After fasting for 20h, the small intestine between the part immediately below duodenum and
the part immediately above the cecum was cut, rinsed with ice cold saline and homogenized
with 12ml of maleate buffer (100mM, pH 6.0). The homogenate was used as a-glucosidase
solution. The assay mixture consisted of 100mM maleate buffer (pH 6.0), 2% (w/v) each
maltose substrate solution (100l), and the extracts (50-500g/ml). It was pre-incubated for
5min at 37C. The glucose released in the reaction mixture was determined with the kit (GODPOD Method). The rate of carbohydrate decomposition was calculated as percentage ratio to
the amount of glucose obtained when the carbohydrate was completely digested. The rate of
prevention was calculated by the following formula:
Inhibition rate (%) = [(A0-A1)-B/A0] x 100
A0 Amount of glucose produced by the positive control
A1 Amount of glucose produced by the addition of Extracts
B Glucose production value in blank
Measurement of total phenol and flavonoid contents
Determination of total phenolic content was made using Folin Ciocalteaus phenol reagent [24].
Test sample (1ml), 0.5ml of Folin-Ciocalteaus phenol reagent (2N), and 2ml of Na2CO3 (5%)
were mixed and the reaction mixture was allowed to proceed for 5 min at room temperature,
before dilution with 5ml of deionized water. Each sample was mixed thoroughly and placed in
dark for 1 h and the absorbance was measured at 725nm with a UV-Vis spectrophotometer.
Gallic acid equivalent (mg/g) was determined from a standard concentration curve.
Flavonoid content was determined according to the aluminum chloride colorimetric method
[25] with some modifications. Quercetin was used as a standard to make the calibration curve.
The sample solution (0.5ml) was mixed with 1.5ml of 95% ethanol, 0.1ml of 10% aluminum
chloride hexahydrate, 0.1ml of 1M potassium acetate, and 2.8ml of distilled water. After
incubation at room temperature for 40 min the absorbance of the reaction mixture was measured
at 415 nm. The same amount (0.1ml) of distilled water substituted for the amount of 10%
aluminum chloride as the blank and a seven point standard curve (0-500g/ml) was obtained.
All tests were performed in independent triplicates (n=3) and the datas were expressed as mean
SD.
Reducing power assay
Various concentrations of Ethyl acetate extract of stem (1ml) were mixed with 2.5 ml of 1%
sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. Then the mixture
was incubated at 50C for 30 min. After 2.5 ml of 10% TCA were added to the mixture was
centrifuged at 3,000 rpm for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml
deionized water with 0.5 ml of 0.1% of ferric chloride and the absorbance was measured at 700
nm. The assays were carried out in triplicate and the results were expressed as mean values
SD. Ascorbic acid was used as a standard [26].
Lipid peroxidation inhibition assay
This assay was carried out using rat liver homogenate as substrate. Male rat of Wister strain
was sacrificed by cervical dislocation and liver was immediately excised, and a homogenate
(5:1 w/v) was prepared using phosphate buffered saline (PBS) in cold condition. It was
centrifuged at 200 g for 10 min. The supernatant was collected and finally suspended in PBS
so as to contain 10 mg protein in 1 ml suspension to perform in vitro experiment. Protein
content was estimated by using diagnostic kit (Span Diagnostics, India). Various concentrations of the ethyl acetate stem extracts dissolved in 1 ml of PBS were mixed with 3 ml of
homogenate. Lipid peroxidation was initiated by adding 100 l of H2O2 (10 mM), gently

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Rajaram K et al. / Journal of Pharmacy Research 2011,4(3),891-893


mixed and incubated at 37C for 30 min. After 30 min the thiobarbituric acid-reactive
substances (TBARS) were estimated by standard method [27] and reduced glutathione was
estimated according to the standard method [28]. A control is run without the addition of extract
and the percent inhibition of lipid peroxidation was calculated by the following formula.
Abs control Abs sample
Percentage of inhibition (%) =x 100
Abs control
Abs control - absorbance of the control reaction (Containing all reagents except the test
compound),Abs sample- absorbance of the test compound. BHT was used as positive controls
and all tests were carried out in triplicates.

Free radical scavenging activity (DPPH Assay)


The DPPH free radical is a stable free radical, which has been widely accepted as a tool for
estimating free radical-scavenging activities of antioxidants [31-32]. DPPH is violet colour in
methanol has got a strong UV absorbance at 517nm.The presence of reducing agent in the test
solution pairs the odd electrons of DPPH radical and further the solution losses colour and also
absorbance of the solution decreases at 517nm [33]. The ethyl acetate fraction of stem extracts
of TT has strong antioxidant activity (IC 50 value 33.544.27 g/ml) compared to other
extracts (leaf & root) shown in Table 1 & Fig. 1. The percentage inhibition of Tephrosia
purpurea showed IC 50 value of 523.8 g/ml [34].Our result shows that the Ethyl acetate stem
extract of TT has significant potential to scavenge free radicals at minimum dosage which was
comparable to the positive control a-tocopherol (IC 50 value 31.793.81 g/ml).

MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) Assay


Muscle cell line (L6) were grown in DMEM supplemented with 10% fetal calf serum and
antibiotics (100 units/ml penicillin, 100 mg/ml streptomycin, and 5 mg/ml amphotericin B)
at 37C in a humidified atmosphere composed of 5% CO2 in a 24 well plate with 3.5x105 L6
myotubes/well. The medium was replaced at every third day. All experiments were performed
in plastic tissue culture flasks, dishes, or in microplates. For differentiation, the L6 myotubes
were transferred to DMEM having 2% FBS for 46 days post-confluence. The extent of
differentiation was established by observing the formation of elongated and multinucleate
myotubes. These differentiated cells were used for further studies.
Cell viability was evaluated by measuring the mitochondrial dependent reduction of colourless
MTT to a coloured blue formazan. The cells were seeded at 5x104 cells/ml density and
incubated plant extracts (50-1000 g/ml) for 24-h and 48-h. Then the medium was changed
and the cells were incubated with MTT (0.5 mg/ml in PBS) for 4h at 37C. The formazan was
dissolved in HCl in isopropanol and the absorbance at 595 nm was measured with spectrophotometer. The number of viable cells is directly proportional to the production of formazan [29].
Percentage Growth inhibition (%) =100 - [Mean of individual test group/ Mean OD of the
control group X 100]
Statistical Analysis
All the experiments were repeated at least three times. Results were reported as mean SE.
RESULTS AND DISCUSSION
Owing to the presence of complicated phytoingredient diversities in plants, the in vitro
activity guided extraction has been effectively applied to screen the biological activities. These
activities may contribute important indications for investigating the characteristics of active
component [30]. In this context different plant parts (Leaf, Stem & Root) of TT were subjected
to activity guided extraction based on polarity to identify the potent bioactive fraction for
antioxidant (DPPH assay) and antidiabetic activity (a-Glucosidase inhibitory activity). Later
the total phenol and flavanoid content were quantified. The reducing power, lipidperoxidation
inhibition assay and its toxicology study in L6 muscle cell line using MTT Assay were also
investigated.

Table .1 a-Glucosidase inhibitory activity of TT extracts


T.tinctoria
Extract Fractions

a-Glycosidase inhibitory
activity IC50 Value(g/ml)

DPPH Assay IC50


Value (g/ml)

Pet-Leaf
Pet-Stem
Pet-Root
C-Leaf
C-Stem
C-Root
EA-Leaf
EA-Stem
EA-Root
E-Leaf
E-Stem
E-Root
Acarbose
a-Tocopherol

1506.022.69
1650.165.95
621.892.94
94.333.65
1143.364.79
545.372.84
1160.633.59
2446.184.61
38.923.52
-

555.554.52
477.823.85
596.233.42
508.854.31
499.54.71
566.122.72
693.095.54
33.544.27
472.054.37
452.732.41
455.042.84
542.061.75
31.793.81

*Pet-Petroleum ether; C-Chloroform; EA-Ethyl acetate; E-Ethanol;Results are expressed as


Mean S.E; n=3

*Pet-Petroleum ether; C-Chloroform; EA-Ethyl acetate; E-Ethanol


Figure. 2 a-Glucosidase inhibitory activity of T.tinctoria extracts compared with Acarbose

a-Glucosidase inhibitory activity


Inhibition of a-Glucosidase delay carbohydrate digestion, causing a reduction in the rate of
glucose absorption and consequently blunting the postprandial plasma glucose rise [34]. aglucosidase inhibition is one of the therapeutic approaches for reducing postprandial hyperglycemia [35]. Screening of plants for enzyme inhibitors like a-glucosidase despite their natural
constituents, have been performed [36]. The a-Glucosidase inhibitory activity was evaluated
using crude enzyme extracts (small intestine of rat). The extracts of different parts (Leaf, Stem
& Root) of TT was tested against this crude enzyme at various concentration (50-1000 g/ml)
among them the EA-stem extract of TT shown significant inhibitory activity (IC 50 94.333.65
g/ml) compared to standard (Acarbose - IC 50 38.923.52 g/ml) as shown in Table 1 & Fig.
2. Ahmad et al (2008) observation in some Iranian medicinal plants of fabaceae family [37]
demonstrated reduced activity when compared to our findings. (EA-Stem extract of TT at100
g/ml).This results encourage us to further probe the active molecule involved in antidiabetic
activity.

*Pet-Petroleum ether; C-Chloroform; EA-Ethyl acetate; E-Ethanol


Figure. 2 a-Glucosidase inhibitory activity of T.tinctoria extracts compared with Acarbose

Total Phenol and Flavonoids


The phenol and flavonoid content of the EA-Stem extract of TT was analysed, since most of
the flavonoids have antioxidant activity and they ascribed to various properties like anticancer,
antidiabetic, antiaging and prevention of cardiovascular diseases [38-40]. The ethyl acetate extract
of TT was found to have maximum antioxidant activity which may be due to the presence of
high amount of flavonoids and moderate amount of phenols. Total phenol and flavonoid
content of the EA-Stem extract of TT was 40 mg/gm (Gallic acid/gm) and 64 mg/gm
(Quercetin/gm) respectively. The high content of flavonoid in the extracts substantiates the
claim for the maximum antioxidant activity.

*Pet-Petroleum ether; C-Chloroform; EA-Ethyl acetate; E-Ethanol;


Figure. 1 Radical scavenging activity of TT compared with a-Tocopherol

Reducing power assay


Reducing power assay is based on the principle that substances, which has reduction potential,
react with potassium ferricyanide (Fe+3) to form potassium ferrocyanide (Fe+2), which then
reacts with ferric chloride to form ferrous complex that has an absorption maximum at 700nm.
The reducing capacity of the compound may serve as a significant indicator of its potential
antioxidant activity [41]. So the hydrogen donating ability of the EA-Stem extract of TT

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Rajaram K et al. / Journal of Pharmacy Research 2011,4(3),891-893


terminates the free radical chain reaction. Fig. 3 shows the EA-Stem extract of TT increase,
when the concentration increases from 0.25 to 2.30 at 50-500g/ml concentration; the
standard drug (BHT) absorbance value range from 0.69 to 3.0 at 50-500g/ml concentration,
which shows the reducing capacity of the extract (EA-Stem extract of TT) comparable to the
BHT.

2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

*EA-Ethyl acetate;

13.

Figure. 3 Reducing power of TT compared with Ascorbic acid

Lipid peroxidation inhibition assay


The peroxidation of membrane lipids initiated by oxygen radicals may lead to cell injury.
Initiation of lipid per oxidation by ferrous sulphate takes place either through ferryl-perferryl
complex or through OH radicals by Fenton reaction [42-43], thereby initiating a cascade of
oxidative reactions. Generation of free radicals eventually causes depletion of antioxidants and
glutathione (GSH) and increases TBARS. Hence the estimation of the reduced glutathione
may serve as a better marker of antioxidant status [44]. Fig.4 shows the lipid peroxidation assay
of EA-Stem & BHT (IC 50 value 64.81.5 g/ml & 38.19 2.14 g/ml respectively). The
results obtained in the present studies may be credited to several reasons viz, the inhibition of
ferryl-perferyyl complex formation; scavenging of OH or superoxide radicals or by changing
the ratio of Fe3+/ Fe2+; reducing the rate of conversions of ferrous to ferric or by chelating of the
iron itself [45].

14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.

*EA-Ethyl acetate; BHT-Butylated hydroxytoluene;

28.

Figure.4 Lipidperoxidation inhibition assay of TT

29.

MTT Assay
The MTT assay is used to assess the viability and the proliferation of cells [46]. It can also be
used to determine cytotoxicity of potential medicinal agents and toxic materials, since those
agents would stimulate or inhibit cell viability and growth. The yellow colour MTT is
reduced to purple formazan by the enzyme reductase which present in the living cells. The
absorbance of the EA-Stem extract of TT treated with the L6 Muscle cell line at the different
concentrations (50-500 g/ml) compared with control (untreated L6 cells) shown in Table 2.
The results confirm that there was nil or low toxicity effect of EA-Stem extract at higher
concentrations.

30.

Table 2 Absorbance of EA-Stem treated L6 cell line in MTT Assay


Concentration(g/ml)

Percentage of viability (%)

Control
50
100
250
500

100
97.232.7
93.242.99
77.971.64
73.534.83

31.
32.
33.
34.
35.
36.
37.
38.

Results are expressed as Mean S.E; n=3

CONCLUSION
Our present investigation reveals the antioxidant and antidiabetic potentiality in the partial
fractions of stem extracts (EA) using in vitro model. Further investigations are in progress to
isolate the active principle and study the complete mechanism in both in vitro and in vivo
model.
ACKNOWLEDGEMENT
The corresponding author likes to thank the DST SERC, New Delhi, India for providing
financial assistants.

39.
40.
41.
42.
43.
44.
45.

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Source of support: DST SERC, New Delhi, India, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 3. March 2011

891-893