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American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org
Original Article
Antidiabetic Potential and Identification of
Phytochemicals from Tinospora cordifolia
Varsha V Sonkamble and Laxmikant H Kamble*
School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded, 431606, India
ABSTRACT
Objective: Tinospora cordifolia an ayurvedic herb has different
classes of phytochemicals with medicinal significance in diabetes
management. The hypothesis and possible mode of action of these
phytochemicals used as antidiabetic drug has been already reported.
So, we focused on identification of the T. cordifolia phytochemicals
as well as the compounds responsible for antidiabetic activities in
context of α-amylase inhibition.
Methods: Total phenol estimation and Thin layer chromatography
(TLC) of T. cordifolia extracts and assay of α-amylase inhibition was
done. Positively responding extracts were analyzed by liquid
chromatography mass spectrometry (LCMS). Obtained LCMS data
was processed using online databases like MassBank, ChemSpider
and Phenol Explorer for characterization of compounds.
Results: Total Phenolic content of T. cordifolia extracts showed
significant variations in their concentrations with highest phenolic
content in ethanol extract, while highest α-amylase inhibition was
showed by ethyl acetate extract. Extracts with more than 40%
inhibitory activity were subjected to LCMS; analyzed by MassBank,
ChemSpider resource and phenol explorer database for compound
identification. Identified compounds were searched in the literature
for reported antidiabetic activity and we found seven; Cyanidin 3-O-
sambubiosyl 5-O-glucoside, Hesperetin 7-Rhamnoglucoside,
quercetin 3-O-β-xylopyranosyl-(1→2)-O-β-galactopyranoside,
Blumenol C malonylglycosyl galacturonide [M+H]+, Verbascoside,
Quercetin-3-glucuronide, and Catechin/Epicatechin-(epi)
gallocatechin dimer.
Conclusion: The phytochemical profiling of T. cordifolia presented
in this study revealed a diverse range of bioactive phenolics. Also it
can be predicted that the potent antidiabetic activity of T. cordifolia is
due to presence of compounds inhibiting α-amylase and α-
glucosidase enzymes.
Keywords: Tinospora cordifolia, Phytochemicals, Antidiabetic
activity, TLC, LCMS.
Address for
Correspondence
School of Life
Sciences, Swami
Ramanand Teerth
Marathwada
University, Nanded,
431606, India.
E-mail:
lhkamble@gmail.com
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
INTRODUCTION
Diabetes mellitus (DM) is
characterized by hyperglycemia which is
induced by decreased cellular glucose
uptake and metabolism. Patients with this
kind of issues trying to control in its early
treatment suffers a very critical conditions
1
.
One of the therapeutic approach is to
prevent absorption of carbohydrates after
food uptake which induces for reducing
postprandial hyperglycemia in patients with
DM. Only monosaccharides, such as glucose
and fructose, can be transported out of the
intestinal lumen into the bloodstream except
complex starches, oligosaccharides, and
disaccharides. Before absorption in the
duodenum and upper jejunum, they must be
broken down into individual
monosaccharides. This digestion is
facilitated by enteric enzymes, like
pancreatic α-amylase and α-glucosidases
which are attached to the brush border of the
intestinal cells
2
. Commonly practiced
treatments include oral hypoglycemic agents
or insulin injections which have certain
limitations. To manage diabetes without any
side effect is still a challenge to the workers
so, research is been switched towards the
natural resources like plants and herbs which
possess antidiabetic activities with low or no
side effects
3
.
In traditional practices medicinal
plants were used to control diabetes mellitus
in many countries. The ethnobotanical
information reports about 800 plants world-
wide which have been documented as
beneficial in the treatment of diabetes
4
. The
majority of traditional antidiabetic plants
await proper scientific and medical
evaluation for their ability to improve blood
glucose control. However, a few
comprehensive studies of traditional
antidiabetic plants have been carried out
1
.
This has caused an increase in the number of
experimental and clinical investigations
directed toward the validation of the anti-
diabetic properties, which are empirically
attributed to these remedies
5
. The
antidiabetic activity of several plants has
been confirmed along with their studies of
mechanisms of hypoglycemic activity.
Chemical studies directed to the isolation,
purification and identification of the
substances responsible for the hypoglycemic
activity are being conducted. One of the
examples is Tinospora cordifolia which is
widely used in veterinary folk
medicine/ayurvedic system of medicine for
its general tonic, anti-periodic, anti-
spasmodic, anti-inflammatory, anti-arthritic,
anti-allergic and anti-diabetic properties
6
.
The plant mainly contains alkaloids,
glycosides, steroids, sesquiterpenoid,
aliphatic compound, essential oils, mixture
of fatty acids and polysaccharides. The
alkaloids include berberine, bitter gilonin,
non-glycoside gilonin gilosterol
7
. Glycosyl
composition of a polysaccharide shown
terminal-glucose, 4-xylose, 4-glucose, 4, 6-
glucose and 2, 3, 4, 6-glucose
8
. These
chemical composition studies were done
earlier but their relation with antidiabetic
activity was not studied. Earlier literature
also states the aqueous, alcoholic, and
chloroform extracts of the leaves of T.
cordifolia in doses of 50, 100, 200 mg/kg
body weight to normal and alloxan-diabetic
induced rabbits exerted significant
hypoglycaemic effect
9
. Histological studies
of pancreas did not reveal any evidence of
regeneration of β-cells of islets of
Langerhance. The possible mode of action
of the drug has been discussed projecting a
hypothesis related to control of glucose
metabolism. So, our study is based on
identification of the chemical composition
of T. cordifolia as well as compounds
responsible for antidiabetic activities
resembling α-amylase and α-glucosidase
inhibition.
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
MATERIALS AND METHODS
Chemicals
Folin-Ciocalteu reagent, Sodium
Carbonate, Gallic acid, Starch, Potassium
Dihydrogen Phosphate, Dipotassium
Hydrogen Phosphate, α-amylase, and
Gram’s Iodine was purchased from Hi-
Media Laboratories, Mumbai, India.
Solvents like petroleum ether, ethyl acetate,
chloroform, acetone, ethanol, cyclohexane,
formic acid, acetic acid, toluene, butanol and
methanol were from Qualigens. TLC Silica
gel 60 F254 plates were purchased from
Merck.
Plant material
Dried powder of T. cordifolia plant
was purchased from the local market of
Nanded city, India which was used for the
further study.
Preparation of plant extracts
20 gm of sample was successively
extracted in an order of non-polar to polar
solvent based on increasing degree of
polarity. The different extracts obtained
sequentially were with petroleum ether,
ethyl acetate, chloroform, acetone, ethanol
and distilled water, respectively. Extractions
were performed using soxhlet apparatus.
The temperature maintained in each
extraction was 10- 20
0
C lower than the
melting point of the solvents used and the
time period of each extraction was fixed i.e.,
six hours. The extracts obtained were then
filtered and concentrated to a volume of 5ml
by boiling and were stored in a refrigerator
until further use.
Total phenolic content
The total amount of phenol in each
extracts was determined by Folin-Ciocalteu
reagent method with some modifications.
2.5ml of 10% Folin-Ciocalteu reagent and
2ml of 7.5% solution of sodium carbonate
was added to 10µl of plant extract. The
resulting mixture was incubated for 15
minutes at room temperature. The
absorbance of the sample was measured at
750nm. Gallic acid was used as standard
(1mg/ml). All the tests were performed in
triplicates. The results were determined from
the standard curve and were expressed as
gallic acid equivalent (GAE mg/g of
extracted compound).
TLC autography
Thin-layer chromatography was
performed on the TLC Silica gel 60 F254
plates (Merck KGaA, Germany). The
extracts were spotted on the plates using a
micropipette and allowed to dry. One
dimensional TLC analysis was performed
with different solvent systems depending on
respective solvent extracts (table 1). Spots
were observed under Ultra-Violet light (UV
light) at 254 nm and 366 nm. The TLC
plates were then incubated in the amylase
solution for 30 min for primary reaction
between the enzyme and inhibitor. After
incubation, the plates were taken out of the
amylase solution and incubated in 1 %
starch buffer of pH 6.9 for 10-20 min for
enzyme-substrate reaction. The plates were
then washed with Gram’s Iodine solution
and observed
10
.
Inhibition assay for α-amylase activity
A total of 20 µl of plant extract and
500 µl of 0.02 M sodium phosphate buffer
(pH 6.9 with 0.006 M sodium chloride)
containing a-amylase solution (0.5 mg/ml)
were incubated at 25 °C for 10 min. After
pre-incubation, 500 µl of a 1% starch
solution in 0.02 M sodium phosphate buffer
(pH 6.9) was added to each tube at 5 s
intervals. The reaction mixtures were then
incubated at 25° C for 10 min. The reaction
was stopped with 1.0 ml of dinitrosalicylic
acid color reagent. The test tubes were then
incubated in a boiling water bath for 5 min
and cooled to room temperature. The
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
reaction mixture was then diluted after
adding 10 ml distilled water and absorbance
was measured at 540 nm
11
.
% Inhibition = [(A
540
Control - A
540
Extract)] x100/A
540
Control.
LC-MS analysis
The chemical constituents of extracts
showing positive results for α-amylase
inhibition assay were determined using
LCMS analysis. The MS instrument was
equipped with Turbo-Ionspray (electrospray
ionisation) interface. 1.0 ml of sample
extracts was diluted five times with
chloroform and filtered with 0.2 µM nylon
filter prior to analyses. Full scan spectra
from 100 to 1000 amu in the positive ion
mode were recorded. The resolved
compounds were then identified using
online software i.e., MassBank
12
which is a
public repository for sharing mass spectral
data. The identification was based on mass
and intensity obtained via records.
RESULTS AND DISCUSSION
The total Phenolic content of six
extracts from powdered sample of T.
cordifolia which was determined by Folin-
Ciocalteu method shows variations in their
concentrations as recorded in table 2 and
presented in fig. 1. Among the six extracts,
Ethanol extract had the highest
concentration of phenolic content GAE i.e.,
0.244 mg/ml followed by water extract
0.2167 mg/ml, ethyl acetate 0.176mg/ml,
petroleum ether 0.125mg/ml, acetone extract
0.087mg/ml and chloroform extract 0.042
mg/ml. For the screening of α-amylase
inhibitors, extracted samples were run on
thin layer chromatography and allowed for
treatment of autography. Each extract gave
various separated molecules with respective
solvent system and also showed starch-
iodine complex reaction indicating presence
of α-amylase inhibitors. Highest separated
molecules with blue color of starch-iodine
complex reaction were observed in
Petroleum ether and Ethyl acetate,
Chloroform and Acetone (fig. 2a & 2b).
Comparatively, ethanol and aqueous extracts
failed to show blue color bands.
When, inhibition assay for α-amylase
activity was done, ethyl acetate extract
showed highest percentage of α-amylase
inhibition followed by chloroform,
petroleum ether, acetone, ethanol and water
extract (table 3) which is represented by the
given graph (fig. 3). The extracts showing
more than 40% inhibition were further
considered for LCMS analysis.
Spectrum analysis was done of
samples containing petroleum ether, ethyl
acetate, chloroform and acetone extracts.
Full scan spectra from 100 to 1000 amu in
the positive ion mode recorded was obtained
as a raw data. The numbers of detected
peaks in petroleum ether extracts were three,
ethyl acetate extracts were nine, chloroform
extracts were seven and acetone extracts
were two. This raw data was then processed
via online software MassBank where
specific parameters were set for metabolite
prediction i.e., for peak-substructure
relationship, the raw data obtained from
LCMS analysis was copied in text format
and sent to the software as a query file, later
commanded to read the file. The spectrum
list appears on the page and then other
parameters were set like, cut off value was
50, tolerance was maintained at 0.005
whereas specificity was set up as 0.4 and the
ion mode was set as +ve. The values of mass
and intensity is provided to the software
which it reads and gives results in form of
number of matched formulae which includes
mass (m/z) values and its chemical
formulae. Thus, each peak had shown
different number of matched formulae
which are represented in table 4. Obtained
chemical formulae were then checked via
ChemSpider
13
an online chemical
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
information resource and phenol explorer
database
14
to identify the compound name.
The compounds were single or attached with
the sub-groups. The compounds were then
checked for the existing literature showing
positive antidiabetic activity. And hence, our
finding reveals some compounds like
Cyanidin 3-O-sambubiosyl 5-O-glucoside,
Hesperetin 7- Rhamnoglucoside, quercetin
3-O-β-xylopyranosyl-(1→2)-O-β-galactopy-
ranoside, Blumenol C malonylglycosyl
galacturonide [M+H]+, Verbascoside,
Quercetin-3-glucuronide, and Catechin/
Epicatechin-(epi) gallocatechin dimer
showing antidiabetic activities.
CONCLUSION
The phytochemical profiling of T.
cordifolia presented in this study revealed a
diverse range of bioactive phenolics. Some
of the identified bioactive phenolics were
reported by several authors for antidiabetic
activities. So this was an attempt to identify
the potent antidiabetic compounds from T.
cordifolia. Thus from this study, it can also
be predicted that the potent antidiabetic
activity of T. cordifolia is due to presence of
compounds inhibiting α-amylase and α-
glucosidase enzymes.
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Table 1. Optimized TLC solvent systems used for separation of compounds
S. No. T. cordifolia extracts Solvent system used Ratio of solvent system
1
Petroleum ether
Cyclohexane: Ethyl acetate: Formic acid
6:3:1
2
Ethyl acetate
Cyclohexane: Ethyl acetate: Formic acid
6:3:1
3
Chlorofor
m
Toluene: Chloroform: Methanol
4.5:5:0.5
4
Acetone
Acetic acid: Acetone: Water
6:3.5:0.5
5
Ethanol
Toluene: Chloroform: Methanol
5:4:1
6
Distilled water
Butanol: Acetic acid: water
4:1:5
Table 2. Total phenol concentration in extracts of T. cordifolia
S. No. Sample extract Conc. (ppm) Conc. (mg/ml) GAE
1
Petroleum ether
125.15
0.125
2
Ethyl acetate
175.63
0.175
3
Chloroform
41.93
0.041
4
Acetone
87.5
0.087
5
Ethanol
243.67
0.243
6 Distilled water 216.77 0.216
Table 3. α-amylase inhibition by extracts of T. cordifolia
S. No. Sample Extract % of inhibition
1
Pet. Ether
42.29
2
Ethyl acetate
64.04
3 Chloroform 57.6
4
Acetone
46.28
5
Ethanol
13.29
6
Water
7.56
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
Table 4. LC-MS data representing tentatively identified phytochemicals in T. cordifolia extracts
with retention time, mass, molecular formulae and reported antidiabetic activity
Sample
extract Peak
Retent
ion
time
Accurate
mass
Proposed
molecular
formula
Phenolic compound
Anti
-
diabetic
Activity
Ref.
Petroleu
m ether
extract
1 7.51-
9.86
447.13
C
22
H
23
O
10
Petunidin 3
-
O
-
rhamnoside
-
-
559.60
-
-
-
-
2 17.58-
20.49
559.80
-
-
-
-
951.67 - - - -
3 27.21-
28.99
561.87
-
-
-
-
743.19 C
32
H
39
O
20
Cyanidin 3
-
O
-
sambubiosyl 5
-
O
-
glucoside Present
(14,
18,
26, 27)
Ethyl
acetate
extract
1 7.09-
10.27
373.133 C
20
H
21
O
7
4
-
(2,
4
-
dimethoxy
-
3,
6
-
dimethylbenzoyl) oxy-2-hydroxy-3, 6-
dimethylbenzoate
- -
447.133
C
22
H
23
O
10
Petunidin 3
-
O
-
rhamnoside
-
-
462.066
C
14
H
17
N
5
O
11
P Adenylosuccinate - -
462.266
C
26
H
40
NO
4
S - - -
479.332
C
24
H
50
NO
6
P lysoplasmenylcholine - -
2 10.48-
12.18
109.066
C
5
H
7
N
3
Imidazole
-
Pyrazole
-
-
109.066
C
7
H
9
O
1
-
cyclohexenylmethanone
-
-
112.933
HO
3
S
2
(Hydroxysulfonothioyl)oxidanyl
-
-
113.066
C
5
H
9
N
20
(2S)
-
2
-
Pyrrolidinecarboximida
te
-
-
295.133 C
14
H
19
N
2
O
5
(1E)
-
3
-
[2
-
(Dimethoxymethyl)
phenyl]
-
1-ethoxy-1, 3-dihydroxy-1-propene-2-
diazonium
- -
295.199
C
16
H
27
N
2
O
3
2
-
[(3
-
Amino
-
4
-
propoxybenzoyl)
oxy]
-
N, N-diethylethanaminium - -
313.066 C
17
H
13
O
6
(4
-
Cinnamoyl
-
3,
5
-
dihydroxyphen
oxy)
acetate - -
341.266
C
20
H
37
O
4
2
-
Hydroxy
-
3
-
oxoicosanoate
-
-
343.266
C
23
H
35
O
2
Cholic acid
-
-
343.66
H
341
-
-
-
349.066 C
20
H
13
O
6
2
-
Hydroxy
-
5,
10
-
dioxo
-
4
-
phenyl
-
3,
4,
5, 10-tetrahydro-2H-
benzo[g]chromene-2-carboxylate
- -
351.199 C
23
H
27
O
3
4
-
ethoxy
-
2
-
(4
-
isopentyloxyphenyl)
-
6
-
methyl-chromene - -
611.199
C
28
H
35
O
15
Hesperetin 7
-
rhamnoglucoside
P
resent
(15
-
17)
821.399
C
42
H
61
O
16
Monopotassium Glycyrrhizinate
-
-
933.266 C
43
H
49
O
23
Peonidin 3
-
caffeoyl
-
rutinoside 5
-
glucoside - -
3
15.41
-
611.199
C
28
H
35
O
15
Hesperetin 7
-
rhamnoglucoside
P
resent
(15
-
17)
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
16.25
810.133
C
23
H
39
N
7
O
17
P
3
S - - -
4 16.87-
17.48
595.133 C
26
H
27
O
16
quercetin 3
-
O
-
β
-
xylopyranosyl
-
(1
→2)
-
O-β-galactopyranoside Present (18-20)
595.199 C
28
H
35
O
14
Blumenol C malony
lglycosyl
galacturonide [M+H]+ Present
(21,
22)
810.133
C
23
H
39
N
7
O
17
P
3
S - - -
5
19.06
-
20.85 810.133
C
23
H
39
N
7
O
17
P
3
S - - -
6
21.87
-
22.77 810.133
C
23
H
39
N
7
O
17
P
3
S - - -
7
26.26
-
27.22 810.133
C
23
H
39
N
7
O
17
P
3
S - - -
8 27.53-
29.33
274.866
C
8
H
5
Br
2
O
-
-
-
604.066
C
16
H
24
N
5
O
16
P
2
GDP-L-galactose
623.199
C
29
H
35
O
15
Verbascoside
P
resent
(22
-
24)
810.133
C
23
H
39
N
7
O
17
P
3
S - - -
933.266
C
43
H
49
O
23
petanin
-
-
9
32.19
-
33.15 798.53 - - - -
Chlorofor
m extract
1
4.33
-
5.26 973.73 - - - -
2 8.26-
9.88
438.266
C
20
H
41
NO
7
P - - -
477.066
C
17
H
21
N
2
O
10
S
2
4-methyoxyglucobrassicin - -
477.066 C
21
H
17
O
13
Quercetin 3-glucuronide Present
(20,
25)
3 15.14-
16.38
291.133
C
10
H
19
N
4
O
6
N-(L-Arginino) succinate5’ - (12)
291.199
C
18
H
27
O
3
(15Z)
-
12
-
Oxophyto
-
10,
15
-
d
ienoate
-
-
335.066
C
11
H
16
N
2
O
8
P
3
-
Carbamoyl
-
1
-
(5
-
O
-
phospho
no
-
β
-
D
-
ribofuranosyl) pyridinium - -
593.133 C
30
H
25
O
13
Catechin/Epicatechin
-
(epi)
gallocatechin dimer Present
(20, 21,
26)
595.133 C
26
H
27
O
16
quercetin 3
-
O
-
β
-
xylopyranosyl
-
(1
→2)
-
O-β-galactopyranoside Present
(17
–
19)
595.199 C
28
H
35
O
14
Blumenol C
malonylglycosyl
galacturonide [M+H]+ Present
(21,
22)
651.799
C
15
H
13
I
3
N
O
4
Triiodothyronine - -
933.266 C
43
H
49
O
23
Petunidin
-
3
-
O
-
coumaroylrutinoside
-
5
-
O-glucoside - -
4
17.57
-
663.87
-
-
-
-
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
20.44
5
25.17
-
27.17 623.199 C
29
H
35
O
15
Verbascoside Present (22-24)
6 27.54-
28.98
274.866
C
8
H
5
Br
2
O
-
-
-
274.99
C
10
H
9
Cl
2
N
2
O
3
3,
5
-
dichloro
-
4
-
morpholin
-
4
-
ylpyridine-2-carboxylate
275.066
C
18
H
11
O
3
3
-
(1
-
Naphthoyl)
benzoate
-
-
275.199
C
18
H
27
O
2
6,
9,
12,
15
-
Octadecatetraenoatato
623.199
C
29
H
35
O
15
Verbascoside
P
resent
(22
-
24)
7 29.98-
31.48
274.866
C
8
H
5
Br
2
O
-
-
-
274.99
C
10
H
9
Cl
2
N
2
O
3
3,
5
-
dichloro
-
4
-
morpholin
-
4
-
ylpyridine-2-carboxylate
275.066
C
18
H
11
O
3
3
-
(1
-
Naphthoyl)
benzoate
-
-
275.199
C
18
H
27
O
2
6,
9,
12,
15
-
Octadecatetraenoatato
-
-
345.133 C
19
H
21
O
6
3,
3
-
Bis
(3,
4
-
dimethoxyphenyl)
propanoate - -
477.066
C
17
H
21
N
2
O
10
S
2
4-methoxyglucobrassicin - -
477.066 C
21
H
17
O
13
Quercetin 3-glucuronide Present
(20,
25)
623.199
C
29
H
35
O
15
Verbascoside
P
resent
(22
-
24)
Acetone
extract
1 16.11-
20.97
529
-
-
-
-
663.87
-
-
-
-
2
33.97
-
39.53 557.13 - - - -
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
1= petroleum ether extract, 2= ethyl acetate extract, 3=chloroform extract, 4= acetone extract, 5=
ethanol extract and 6= water extract.
Figure
1
.
Total phenol concentration in extracts of
T. cordifolia
Figure 2. TLC plates A. Separated bands present in the respective solvent extracts under
ultra-violet (254 nm) light source, B. Separated bands indicating presence of α-amylase
inhibitors (blue stains) in normal light source
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
Figure 3. α-amylase inhibition by extracts of T. cordifolia
F
igure
4
.
LCMS spectra of Petroleum ether extract
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
A. Spectra checked at retention time 10.48-12.18, B. Spectra checked at retention time 16.87-
17.48 (Detailed analysis is described in table 4).
Figure 5. LCMS spectra of Ethyl acetate extract
Kamble et al________________________________________________ ISSN 2321 – 2748
AJPCT[3][01][2015] 097-110
A. Spectra checked at retention time 15.14-16.38, B. Spectra checked at retention time 29.98-
31.48 (Detailed analysis is described in table 4).
Figure 6. LCMS spectra of chloroform extract