IJIPBART (2015) Volume2, Issue (1), pp:136-143

ISSN: 2349-865X

OPEN ACCESS

International Journal of Innovation in Pharma

Biosciences and Research Technology
Journal home page :http://www.Refsynjournal.com

Original Research Article

Invitro and insilico investigation of antidiabetic activity

of poly herbal formulation
R. Sarala, G. Vanaja*
Department of Chemistry, Dhanalakshmi Srinivasan College of Arts and science for women.
Perambalur 621 212, Tamilnadu, India.

ABSTRACT
Article received
February 07, 2015
Article accepted
February 23, 2015
Article published
March 12, 2015

Diabetes Mellitus is a metabolic disorder characterized by high blood

sugar level caused due to deficiency of insulin secretion or insulin
action. The objective of this study was to develop combinational
herbal oral anti-diabetic suspension containing indigenous ethno
medicinal plants extract.

In present investigation poly herbal

formulation (PHF) composed of medicinal plants having anti-diabetic

property were selected and evaluated for in vitro alpha-amylase
inhibitory activity. Poly herbal formulation prepared by Soxhlet
method. Phytochemical constituents in the extract were analyzed
qualitatively as well as by GC-MS. The results revealed the presence
of Alkaloids and Saponins. In vitro study indicates that PHF1 showed
*Corresponding Author:
G.Vanaja
Assistant professor
Department of Chemistry,
Dhanalakshmi Srinivasan
College of Arts and science for
women. Perambalur 621
212, Tamilnadu, India.
vanaja.dhana@gmail.com

maximum percentage inhibition of alpha amylase activity and

Glucose Diffusion Inhibitory than control Metformin HCl. Moreover
the compound satisfies the Lipinski rule and hence it may be a
potential lead compound in the treatment of Type II Diabetes
Mellitus.
Keywords. Hypoglycemic, anti diabetic, medicinal plants, diabetes
mellitus, in silico docking

1. INTRODUCTION
Diabetes mellitus is a global metabolic epidemic affecting essential biochemical activities in
almost every age group. Diabetes mellitus is not a single disease but rather a group of metabolic
disorders. Hyperglycemia in diabetes results from defect in insulin secretion and or insulin action.
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Conventionally insulin dependent diabetes mellitus is treated with exogenous insulin, and non-insulin
dependent diabetes mellitus is treated with synthetic oral hypoglycemic agents like sulphonyl ureas
and biguanides. Synthetic oral drugs produce adverse health effects. Different medicinal systems are
using the active plant constituent which discovered as natural hypoglycemic medicine came from
virtue of traditional knowledge. Herbal drugs are considered free from side effects than synthetic one
(Gupta, 2008).

Diabetes mellitus is a common and very prevalent disease affecting the citizens of
both developed and developing countries. It is estimated that 25% of the world population is
affected by this disease. Diabetes mellitus is caused by the abnormality of carbohydrate
metabolism which is linked to low blood insulin level or insensitivity of target organs to
insulin (Maiti, 2004).
The herbal drugs with ant diabetic activity are yet to be commercially formulated as modern
medicines, even though they have been acclaimed for their therapeutic properties in the traditional
systems of medicine (Wadkar, 2008). Traditional medicine (herbal) is used for treatment of diabetes
in developing countries where the cost of conventional medicines is a burden to the population
(Saravanan, 2008). Despite the introduction of hypoglycemic agents from natural and synthetic
sources, diabetes and its secondary complications continue to be a major medical problem. Many
indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes. One
of the great advantages of medicinal plants is that these are readily available and have very low side
effects. Plants have always been an exemplary source of drugs and many of the currently available
drug have been derived directly or indirectly from them. The ethno botanical information reports
about 800 plants that may possess anti diabetic potential (Alarcon-Aguilara, 1998). Several herbs have
shown ant diabetic activity when assessed using presently available experimental techniques (Jafri,
2000).

2. MATERIALS AND METHODS

2.1. SAMPLE COLLECTION
The samples were collected in the local market of puducherry. The collected samples is
washed and cut it into fine pieces, then kept in a shadow place for 2 hours and dried it in a
hot air oven at 40C for two days. The dried sample is finely powdered in a mixture (Table.1).

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2.2. SOURCES FOR FORMULATION OF POLY HERBAL DRUG

Table-1: Sources for formulation of poly herbal drug
S. No.
1
2
3
4
5
6
7
8
9
10
11
12
Average

Botanical name
Phyllantium embilca
Annona muricatta
Psidium guajava
Azadirachta indica
Enicostemma littroale
Murraya konigii
Allium sativum
Allium cepa
Phyllantus amarus
Punica granatum
T.foenum graecum
Boerhavia diffusa
weight

English name
Amla
Sugar apple
Guava
Neem leaf
Vallari
Curry leaf
Garlic
Onion
Keezhar neeli
Pomegranate peel
Fenugreek
Red spider ling

Weight/g
2.5252
3.0374
1.5175
2.0237
0.5022
1.0227
1.5338
2.0271
0.2004
3.3709
1.5915
1.0737
20.4261

2.3.SOXHLET EXTRACTION
20g of powder sample was taking in muslin cloth fold it and closely pour 170ml methanol
was added. Put into the soxhlet extraction fixed it into the round bottom flask and heats it at 60-70 C.
2.4.PHYTOCHEMICAL SCREENING
Chemical tests were carried out using extract to identify various constitutes using standard
methods of Trease and evans (1989).
2.5.GC-MS ANALYSIS
The crude sample is carried out GC-MS analysis for identification of phytoconstituents
present in the extract with the following conditions:
2.6.FORMULATION OF POLY HERBAL DRUG
Preparation of granules dry granulation method: Calculated amount of crude extract, Micro
crystalline cellulose, Talc was weighed and mixed properly and kept drying. After drying this mass
transferred to sieve no. 36 form uniform granules and added preservative and lubricants.
2.7.PREFORMULATION STUDIES OF POLY HERBAL DRUG
Table-2: Formulation of poly herbal drug
S. No.
1
2
3
4
5
6
7

INGREDIENTS
Crude formulation
Palm sugar
Mccp
Edta disodium
Talc
Starch soluble
Orange
Average weight

Weight/unit
10.0558g
30.2071g
2.8741g
0.6225g
1.3443g
0.0593g
10.7224g
55.8262g

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2.8.PREPARATION OF SYRUP
Table-3: Formulation of poly herbal drug Syrup
S. No.
1
2
3
4
5
6
7

Ingredients
Crude formulation
Palm sugar
Mccp
Edta disodium
Talc
Starch soluble
Orange
Average weight

Weight/unit
3.3g
10g
0.9g
0.6g
0.4g
1.6g
0.16g
16.96g

All the active ingredients sweeteners and preservatives table and allowed to dissolve in
10ml distilled water with vigorous shaking until the clear solution not prepared then,40ml of
buffer solution and 10ml of aqueous solution, was mixed properly the prepared solution was
used to formulate and get sterile in the autoclave(Table.3).
2.9.IN-VITRO STUDIES OF ANTI-DIABETIC ACTIVITY
2.9.1.GLUCOSE DIFFUSION INHIBITORY
Test Sample Preparation. Four different concentrations (100, 200, 300, and 400 g/mL) of
samples were prepared. 1mL of the sample was placed in a dialysis membrane (12000MW, Himedia
laboratories, Mumbai) along with a glucose solution (0.22mM in 0.15M NaCl). Then it was tied at
both ends and immersed in a beaker containing 40mL of 0.15M Nacl and 10mL of distilled water. The
control contained 1mL of 0.15M NaCl containing 0.22mM glucose solution and 1mL of distilled
water. The beaker was then placed in an orbital shaker. The external solution was monitored every
half an hour. Three replications of this test were done for 3hrs.
2.9.2.INHIBITION ASSAY FOR -AMYLASE ACTIVITY.
Four different concentrations (100, 200, 300, and 400 g/mL) of samples and standard drug
acarbose were prepared and made up to 1mL with DMSO. A total of 500 L of sample and 500L of
0.02Msodiumphosphate buffer (pH 6.9 with 0.006MNaCl) containing -amylase solution (0.5mg/mL)
were incubated for 10 minutes, at 250C. After preincubation, 500 L of 1% starch solution in 0.02M
sodium phosphate buffer (pH 6.9with 0.006M NaCl) was added to each tube. This reaction mixture
was then incubated for 10 minutes at 250C. 1mLof DNSA colour reagent was added to stop the
reaction.
These test tubes were then incubated in a boiling water bath for 5 minutes and cooled to room
temperature. Finally this reaction mixture was again diluted by adding 10mL of distilled water. % of
inhibition by -amylase can be calculated by using the following formula. Absorbance was measured
at 540 nm

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2.10.MOLECULAR DOCKING
2.10.1.AUTODOCK
All selected phytochemicals were docked into the binding site of the protein using the open
access software application tool, AutoDock 4.2. Active residues of were available in Uniprot database
and the same residues were used for docking. The active sites of the molecules were detected using
the software applications Pocket-Finder and Q-Site Finder, freely available online tools for binding
site prediction. The docking was performed following AutoDock procedure (Morris et al., 2009).This
tool uses Monte Carlo Simulated Annealing and Lamarckian genetic algorithm for the generation of
possible orientations of ligand at the binding site of each venom protein. The grid spacing of 0.375 A0
with 404040 points for each enzyme was defined and centered on the active site. For docking, all
the parameters were kept as default including population number. The ligand bound complexes were
analyzed for its binding affinity and possible orientations were ranked according to their lowest
binding energy through cluster analysis. The top ranked molecules with free energy of binding 5
kcal/mol were considered as hit molecules and these molecules were further analysed by Lipinski's
rule of Five and Rule of Three for drug likeness characters such as Absorption, Distribution,
Metabolism and Excretion (ADME). To reduce the error in selection of leads the molecules showed
best hits in Autodock were subjected to further docking with other docking tools such as
iGEMDOCK, Patchdock and HEX Server. The scores obtained were statistically analyzed following
Dempster-Shafer Theory (DST) and identified best leads.

2.10.2.iGEMDOCK
iGEMDOCK tools provide post-analysis through k-means and hierarchical clustering
methods based on the docked poses and compound properties (Su et al., 2011). The tool provides an
interactive inter phase to prepare the protein binding site and ligand library. Each protein was
uploaded and binding site was defined using the option prepare binding site and population number
and generations were set as 100 and 50 respectively. Each compound in the library was docked
individually and the result page contained interactions such as energy, Van der Waals and
electrostatic.
2.10.3.PDB
The Protein Data Bank (PDB) was a repository for the 3-D structural data of large biological
molecules, such as proteins and nucleic acids.
2.10.4.CHEMSKETCH

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Alpha-Glucosidase (PDB ID2QMJ) was downloaded from the protein data bank

already

complexed with the inhibitors. Alpha-Glucosidase is human intestinal maltase-glucoamylase made up

of single chain A, 870 long amino acid sequence protein. Then this PDB file of Alpha-Glucosidase is
opened in chimera 1.6.1 where the inhibitor molecule that is complexed with Alpha Glucosidase was
removed and we get pure form of alpha glycosidase. Then pdb file of this molecule was saved for
further use in docking process.
3.RESULTS AND DISCUSSION
The chemical nature of the constituents of the crude extract and the one that predominates
over the others can be revealed by phytochemical screening. Phytochemical screening of the plant
extract was carried out by using the standard methods. The results of phytochemical screening of Poly
Herbal Formulation (PHF) showed the presence of various phytochemical (Table.4).
Table.4. Phytochemical screening of extracts
PHYTOCONSTITUENTS
Alkaloid
Tannin& phenol
Flavanoids
Triterpenoids
(or) Terpenoids
Steroid
Saponins
Volatile oil
Glycosides

NAME OF THE TEST

Wagners test
Gelatin test
Zinc-HCl test
Salkowski test
Sulphuric acid test
Froth test
NaOH Test
Liebermanns test

OBSERVATION
+
_
_
_
+
_
_

The phytochemical analysis of Poly Herbal Formulation reveals the presence of various
important compounds like alkaloids, Saponins, Phenolics, tannins etc. These compounds are known to
be biologically active. Alkaloids and phenols have been reported to possess anti-diabetic activity. In
this study PHF was evaluated for their respective alpha-amylase inhibitory activity. The result
indicates that PHF methanol extract showed maximum inhibitory effect on Alpha-Amylase with 53.8
% which is less than Metformin HCL as control, suggesting that PHF extract could be a promising
lead extract. This is due the presence of phytochemical that acts as potential alpha-amylase inhibitors
such as Alkaloids, Saponins.

To analyze which phytochemical compound in PHF methanol extract was responsible to

inhibit the enzyme alpha-amylase, sample was subjected to GC-MS. It was noted that in PHF
methanol extract, 11 major compounds were detected which is shown in (Table.5).Thus due to
existence

of

Hexadecanoic

acid,

2,4,4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)-

cyclohexene,1,3,3-trimethyl-2hydroxymethyl-3,3-dimethyl-4-(3-methylbut-2-enyl)-cyclohexene,
Phytol and other compounds PHF methanol extract showed maximum inhibitory activity on AlphaAmylase activity. It is well known that certain alkaloids and phytol exhibit hypoglycemic activity and

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is also known for their ability of beta-cell regeneration of pancreas. Thus the significant anti-diabetic
effect of the extract may be due to the presence of Alkaloids, Saponins.

The present study indicates that methanol extracts of Polyherbal formulation(PHF) composed
of twelve medicinal plants can be useful in management of postprandial hyperglycemia because the
methanol extract possess important bioactive compounds which acts as alpha-amylase inhibitors, thus
lowering PPHG (Post prandial hyperglycemia) level in type II Diabetic patients.
Table-5: The compounds present in the methanol extract of Poly Herbal
Peak
No

Area %

Chemical Name

1
2
3
4

Retention
Time
(min)
18.175
19.040
20.065
20.691

8.761
3.195
16.084
2.963

21.501

3.858

22.131

4.871

22.286

2.469

24.432

3.591

26.668

7.243

10
11

28.389
29.549

13.876
16.410

N-HEXADECANOIC ACID
PHYTOL
ETHYL 9.CIS.,11.TRANS.-OCTADECADIENOATE
DIBENZO[A,H]CYCLOTETRADECENE, 2,3,11,12TETRAETHENYL1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18-O
2,4,4-TRIMETHYL-3-HYDROXYMETHYL-5A-(3METHYL-BUT-2-ENYL)-CYCLOHEXENE
OCTADECANE,
1-CHLORO
ANDROSTAN-17-ONE, 3-ETHYL-3-HYDROXY-,
(5.ALPHA.)
1,3,3-TRIMETHYL-2-HYDROXYMETHYL-3,3DIMETHYL-4-(3-METHYLBUT-2-ENYL)CYCLOHEXENE
2H-1-BENZOPYRAN-6-OL, 3,4-DIHYDRO-2,5,7,8TETRAMETHYL-2-(4,8,12TRIMETHYLTRIDECYL)-, ACETATE, [2R]
GAMMA.-SITOSTEROL
DIALLYLPHENYLVINYLSILANE

On the basis of mechanism of action of 1, 3, 3-trimethyl-2-hydroxymethyl-3, 3-dimethyl-4(3-methylbut-2-enyl)-cyclohexene i.e. Ion channel modular, Nuclear receptor ligand and kinase
inhibition we compare compound for the anti-diabetic activity. The binding affinity energy of the
compound 1, 3, 3-trimethyl-2-hydroxymethyl-3, 3-dimethyl-4-(3-methylbut-2-enyl)-cyclohexene is 6.8 (kcal/mol) is higher than control Metformin HCL.
The drugs those are accessible for the treatments Diabetes Mellitus have limitations like they
have low usefulness and can cause diverse effects to eyes and heart. The enzymes alpha-Glucosidase
is an effectors target molecule because by blocking it we can get good inhibitors.
4.CONCLUSION
In this study virtual screening of compound obtained from Poly Herbal Formulation is done
by using docking study with Alpha Glucosidase concluded that the compound 1,3,3-trimethyl-2142

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hydroxymethyl-3,3-dimethyl-4-(3-methylbut-2-enyl)-cyclohexene having highest binding energy and

bind to or near the active site can block the enzyme to bind with the substrate and can work as a
potential inhibitors of alpha Glucosidase for the treatment of Diabetes. Docking analysis has
concluded involvement of hydrogen and hydrophobic interactions between the target enzyme and
potential inhibitor molecule.
5.REFERENCES
1. Gupta R, Bajpai KG, Johri S, Saxena AM, An overview of Indian novel traditional medicinal
plants with antidiabetic potentials, The African Journal of Traditional 2008; 5(1):1-17.
2. Maiti R, D Jana, UK Das, D. Ghosh, Antidiabetic effect of aqueous extract of seed
of Tamarindus indica in streptozotocin-induced diabetic rats. J. Ethnopharmacol 2004; 92:
85-91.
3. Wadkar KA, Magdum CS, Patil SS,

Naikwade NS. Antidiabetic potential and Indian

medicinal plants. Journal of Herbal Medicine and Toxicology 2008; 2(1): 45-50.
4. Saravanan G, Pari L. Hypoglycaemic and antihyperglycaemic effect of Syzygium cumini bark
in streptozotocin-induced diabetic rats. J Pharmacol Toxicol 2008;3:1-10.
5. Alarcon-Aguilara FJ, Roman-Ramos R, Perez-Gutierrez S, Aguilar-Contreras A, ContrerasWeber CC and Flores-Saenz JL. Study of the anti-hyperglycemic effect of plants used as
antidiabetics. J. Ethnopharmacol 1998; 61: 101-110.
6. Jafri MA, Aslam M, Javed K, Singh S. Effect of Punica granatum Linn (flowers) on blood
glucose level in normal and alloxan-induced diabetic rats. J Ethnopharmacol 2000; 70: 309314.
7. Morris GM, Huey R, Lindstrom W, et al. AutoDock 4 and AutoDock Tools 4: Automated
docking with selective receptor flexibility. Journal of Computational Chemistry 2009; 30:
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8. Su, YC, Liu AL, Diaz-Tovar CA., Gani R. Selection of prediction methods for
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CONFLICT OF INTERESTS
The authors declare that they have no conflict of interests regarding the publication of this paper.
Copyright 2015 by authors. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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