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

ISSN: 2349-865X


International Journal of Innovation in Pharma
Biosciences and Research Technology
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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.

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:
Assistant professor
Department of Chemistry,
Dhanalakshmi Srinivasan
College of Arts and science for
women. Perambalur – 621
212, Tamilnadu, India.

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
Keywords. Hypoglycemic, anti diabetic, medicinal plants, diabetes
mellitus, in silico docking

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.

Diabetes mellitus is a common and very prevalent disease affecting the citizens of both developed and developing countries.IJIPBART (2015) Volume2. The ethno botanical information reports about 800 plants that may possess anti diabetic potential (Alarcon-Aguilara. Many indigenous Indian medicinal plants have been found to be useful to successfully manage diabetes. 2000). The dried sample is finely powdered in a mixture (Table. Despite the introduction of hypoglycemic agents from natural and synthetic sources. diabetes and its secondary complications continue to be a major medical problem. 2004). SAMPLE COLLECTION The samples were collected in the local market of puducherry. 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. The collected samples is washed and cut it into fine pieces.1). Several herbs have shown ant diabetic activity when assessed using presently available experimental techniques (Jafri.1. 137 . even though they have been acclaimed for their therapeutic properties in the traditional systems of medicine (Wadkar. 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. then kept in a shadow place for 2 hours and dried it in a hot air oven at 40°C for two days. Issue (1). and non-insulin dependent diabetes mellitus is treated with synthetic oral hypoglycemic agents like sulphonyl urea’s and biguanides. 2008). pp:136-143 ISSN: 2349-865X Conventionally insulin dependent diabetes mellitus is treated with exogenous insulin. 2. Herbal drugs are considered free from side effects than synthetic one (Gupta. One of the great advantages of medicinal plants is that these are readily available and have very low side effects. 2008). Plants have always been an exemplary source of drugs and many of the currently available drug have been derived directly or indirectly from them. Different medicinal systems are using the active plant constituent which discovered as natural hypoglycemic medicine came from virtue of traditional knowledge. 1998). MATERIALS AND METHODS 2. Synthetic oral drugs produce adverse health effects. It is estimated that 25% of the world population is affected by this disease. 2008). The herbal drugs with ant diabetic activity are yet to be commercially formulated as modern medicines.

4261 2. After drying this mass transferred to sieve no. No. pp:136-143 ISSN: 2349-865X 2.6225g 1. No.5022 1. 36 form uniform granules and added preservative and lubricants.FORMULATION OF POLY HERBAL DRUG Preparation of granules dry granulation method: Calculated amount of crude extract.3.5915 1.0593g 10.3443g 0. Put into the soxhlet extraction fixed it into the round bottom flask and heats it at 60-70 °C. Micro crystalline cellulose.8262g 138 .5175 2. 1 2 3 4 5 6 7 INGREDIENTS Crude formulation Palm sugar Mccp Edta disodium Talc Starch soluble Orange Average weight Weight/unit 10.0374 1.0558g 30.7224g 55.5252 3.5.2004 3.7. 2.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. 2.0227 1.PREFORMULATION STUDIES OF POLY HERBAL DRUG Table-2: Formulation of poly herbal drug S.PHYTOCHEMICAL SCREENING Chemical tests were carried out using extract to identify various constitutes using standard methods of Trease and evans (1989).0271 0.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. Issue (1). SOURCES FOR FORMULATION OF POLY HERBAL DRUG Table-1: Sources for formulation of poly herbal drug S.2071g 2.3709 1. 2.2.IJIPBART (2015) Volume2. Talc was weighed and mixed properly and kept drying.0737 20.4.8741g 0.5338 2.0237 0.6. 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.SOXHLET EXTRACTION 20g of powder sample was taking in muslin cloth fold it and closely pour 170ml methanol was added.

Absorbance was measured at 540 nm 139 .15M NaCl).02Msodiumphosphate buffer (pH 6. These test tubes were then incubated in a boiling water bath for 5 minutes and cooled to room temperature.9g 0.IJIPBART (2015) Volume2. at 250C. Issue (1).22mM glucose solution and 1mL of distilled water. pp:136-143 ISSN: 2349-865X 2.22mM in 0.9with 0.6g 0.PREPARATION OF SYRUP Table-3: Formulation of poly herbal drug Syrup S.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. The beaker was then placed in an orbital shaker.8.9 with 0. 300. NaCl containing 0. The external solution was monitored every half an hour.3g 10g 0.006MNaCl) containing ߙ-amylase solution (0. Mumbai) along with a glucose solution (0. 200. 1mLof DNSA colour reagent was added to stop the reaction. The control contained 1mL of 0. 2. Then it was tied at both ends and immersed in a beaker containing 40mL of 0.15M Nacl and 10mL of distilled water. Four different concentrations (100. After preincubation.INHIBITION ASSAY FOR ߙ-AMYLASE ACTIVITY. A total of 500 ߤL of sample and 500ߤL of 0.9. Three replications of this test were done for 3hrs. and 400 ߤg/mL) of samples were prepared. 1mL of the sample was placed in a dialysis membrane (12000MW.GLUCOSE DIFFUSION INHIBITORY Test Sample Preparation.5mg/mL) were incubated for 10 minutes.1. % of inhibition by ߙ-amylase can be calculated by using the following formula. Finally this reaction mixture was again diluted by adding 10mL of distilled water. 500 ߤL of 1% starch solution in 0. 300.IN-VITRO STUDIES OF ANTI-DIABETIC ACTIVITY 2.02M sodium phosphate buffer (pH 6.006M NaCl) was added to each tube.16g 16. Four different concentrations (100. Himedia laboratories.6g 0.2.3). This reaction mixture was then incubated for 10 minutes at 250C. 1 2 3 4 5 6 7 Ingredients Crude formulation Palm sugar Mccp Edta disodium Talc Starch soluble Orange Average weight Weight/unit 3. 200. and 400 ߤg/mL) of samples and standard drug acarbose were prepared and made up to 1mL with DMSO.4g 1. No.

For docking. Van der Waal’s and electrostatic. The active sites of the molecules were detected using the software applications Pocket-Finder and Q-Site Finder.3.AUTODOCK All selected phytochemicals were docked into the binding site of the protein using the open access software application tool. Each compound in the library was docked individually and the result page contained interactions such as energy. AutoDock 4. all the parameters were kept as default including population number.CHEMSKETCH 140 .10.iGEMDOCK iGEMDOCK tools provide post-analysis through k-means and hierarchical clustering methods based on the docked poses and compound properties (Su et al. pp:136-143 ISSN: 2349-865X 2. 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. 2. 2..2. Distribution.. freely available online tools for binding site prediction. 2009). 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. Metabolism and Excretion (ADME). Issue (1).10. The scores obtained were statistically analyzed following Dempster-Shafer Theory (DST) and identified best leads.10.1. such as proteins and nucleic acids.10.10.PDB The Protein Data Bank (PDB) was a repository for the 3-D structural data of large biological molecules. 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. 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 grid spacing of 0. 2011). Active residues of were available in Uniprot database and the same residues were used for docking. The docking was performed following AutoDock procedure (Morris et al. 2.MOLECULAR DOCKING 2.4. Patchdock and HEX Server.IJIPBART (2015) Volume2.375 A0 with 40×40×40 points for each enzyme was defined and centered on the active site.2.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 tool provides an interactive inter phase to prepare the protein binding site and ligand library.

Thus due to existence of Hexadecanoic acid. Phytol and other compounds PHF methanol extract showed maximum inhibitory activity on AlphaAmylase activity.3-dimethyl-4-(3-methylbut-2-enyl)-cyclohexene. The result indicates that PHF methanol extract showed maximum inhibitory effect on Alpha-Amylase with 53. Table. Then pdb file of this molecule was saved for further use in docking process.6. Phytochemical screening of the plant extract was carried out by using the standard methods. Saponins. sample was subjected to GC-MS. tannins etc.1.5).4.4. Issue (1).4). pp:136-143 ISSN: 2349-865X Alpha-Glucosidase (PDB ID2QMJ) was downloaded from the protein data bank already complexed with the inhibitors. These compounds are known to be biologically active. 870 long amino acid sequence protein. The results of phytochemical screening of Poly Herbal Formulation (PHF) showed the presence of various phytochemical (Table.3-trimethyl-2hydroxymethyl-3. 2. This is due the presence of phytochemical that acts as potential alpha-amylase inhibitors such as Alkaloids. Then this PDB file of Alpha-Glucosidase is opened in chimera 1.IJIPBART (2015) Volume2. Alkaloids and phenols have been reported to possess anti-diabetic activity.4-trimethyl-3-hydroxymethyl-5a-(3-methyl-but-2-enyl)- cyclohexene. It was noted that in PHF methanol extract.8 % which is less than Metformin HCL as control. 3. To analyze which phytochemical compound in PHF methanol extract was responsible to inhibit the enzyme alpha-amylase. Phytochemical screening of extracts PHYTOCONSTITUENTS Alkaloid Tannin& phenol Flavanoids Triterpenoids (or) Terpenoids Steroid Saponins Volatile oil Glycosides NAME OF THE TEST Wagner’s test Gelatin test Zinc-HCl test Salkowski test Sulphuric acid test Froth test NaOH Test Liebermann’s test OBSERVATION + _ _ _ + _ _ The phytochemical analysis of Poly Herbal Formulation reveals the presence of various important compounds like alkaloids. Phenolics. In this study PHF was evaluated for their respective alpha-amylase inhibitory activity.1 where the inhibitor molecule that is complexed with Alpha Glucosidase was removed and we get pure form of alpha glycosidase. It is well known that certain alkaloids and phytol exhibit hypoglycemic activity and 141 . Alpha-Glucosidase is human intestinal maltase-glucoamylase made up of single chain A. 11 major compounds were detected which is shown in (Table.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. suggesting that PHF extract could be a promising lead extract.3. Saponins.

12TRIMETHYLTRIDECYL)-.4. Issue (1).CIS. Ion channel modular.876 16.7. 4.3. 3-trimethyl-2-hydroxymethyl-3.3-trimethyl-2142 . pp:136-143 ISSN: 2349-865X is also known for their ability of beta-cell regeneration of pancreas.691 8.549 13. 1-CHLORO ANDROSTAN-17-ONE.858 6 22.10.432 3.5.2. (5. thus lowering PPHG (Post prandial hyperglycemia) level in type II Diabetic patients.668 7. The binding affinity energy of the compound 1. 3-trimethyl-2-hydroxymethyl-3.8. The enzymes alpha-Glucosidase is an effectors target molecule because by blocking it we can get good inhibitors.131 4.H]CYCLOTETRADECENE.TRANS.8 (kcal/mol) is higher than control Metformin HCL.040 20. 3-dimethyl-4-(3-methylbut-2-enyl)-cyclohexene is 6.18-O 2. Saponins..3.ALPHA. 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.e.243 10 11 28. [2R] GAMMA.084 2.469 8 24.15.501 3.389 29.11.-OCTADECADIENOATE DIBENZO[A.8.3. 3. 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.6.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 1.286 2.3DIMETHYL-4-(3-METHYLBUT-2-ENYL)CYCLOHEXENE 2H-1-BENZOPYRAN-6-OL.13.4-DIHYDRO-2.175 19.9. 3.761 5 21. Nuclear receptor ligand and kinase inhibition we compare compound for the anti-diabetic activity. 3-ETHYL-3-HYDROXY-. ACETATE.) 1.11.195 16.IJIPBART (2015) Volume2.12TETRAETHENYL1.065 20. 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. 3.3-TRIMETHYL-2-HYDROXYMETHYL-3.410 N-HEXADECANOIC ACID PHYTOL ETHYL 9.4.-SITOSTEROL DIALLYLPHENYLVINYLSILANE On the basis of mechanism of action of 1. 3-dimethyl-4(3-methylbut-2-enyl)-cyclohexene i. 2.12.871 7 22.16.591 9 OCTADECANE.3. Thus the significant anti-diabetic effect of the extract may be due to the presence of Alkaloids.5.

Docking analysis has concluded involvement of hydrogen and hydrophobic interactions between the target enzyme and potential inhibitor molecule. Roman-Ramos R. 11th edn. Issue (1). AutoDock 4 and AutoDock Tools 4: Automated docking with selective receptor flexibility. pp:136-143 ISSN: 2349-865X hydroxymethyl-3. Magdum CS. provided the original work is properly cited. Aslam M. Antidiabetic potential and Indian medicinal plants. 6. 50: 6809-6836. Saravanan G. Study of the anti-hyperglycemic effect of plants used as antidiabetics. Maiti R. 70: 309314. Ghosh. Diaz-Tovar CA.. Huey R. Gani R. Naikwade NS. Patil SS. 5(1):1-17. 30: 2785-2791. Saxena AM. 8. Chem. Jafri MA. Hypoglycaemic and antihyperglycaemic effect of Syzygium cumini bark in streptozotocin-induced diabetic rats. Su. Javed K. J. Liu AL. Gupta R. which permits unrestricted use. D Jana. Journal of Computational Chemistry 2009. Copyright © 2015 by authors. Trease G. Alarcon-Aguilara FJ. J Ethnopharmacol 2000. The African Journal of Traditional 2008. Pharmacognosy. Bailliere Tindall. 2. Wadkar KA. 4.. Singh S. London. 5. Res 2011.E.C. Johri S.3:1-10. Selection of prediction methods for thermophysical properties for process modeling and product design of biodiesel manufacturing.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. Journal of Herbal Medicine and Toxicology 2008. 3. J Pharmacol Toxicol 2008. Ethnopharmacol 1998. 92: 85-91. Aguilar-Contreras A.. 7. et al. ContrerasWeber CC and Flores-Saenz JL. D. YC. Eng. UK Das. 5. 61: 101-110. An overview of Indian novel traditional medicinal plants with antidiabetic potentials. distribution. Perez-Gutierrez S. 45-50 (1989) CONFLICT OF INTERESTS The authors declare that they have no conflict of interests regarding the publication of this paper. 9. Ind. Antidiabetic effect of aqueous extract of seed of Tamarindus indica in streptozotocin-induced diabetic rats. Morris GM. 143 . and reproduction in any medium. 2(1): 45-50. Ethnopharmacol 2004.REFERENCES 1. This is an open access article distributed under the Creative Commons Attribution License. Bajpai KG. and Evans W. J. Pari L.IJIPBART (2015) Volume2. Lindstrom W. Effect of Punica granatum Linn (flowers) on blood glucose level in normal and alloxan-induced diabetic rats.