CHINTAN.PPT

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DESIGN DEVELOPMENT AND EVALUATION OF NOVEL POLYHERBAL ANTIDIABETIC

TABLET FORMULATION

Pharmacy Department, Faculty of Technology & Engineering

The Maharaja Sayajirao University of Baroda

PRESENTED BY:

Chintan Chauhan

GUIDED BY:

Prof. S. H. Mishra

OVERVIEW

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1) Introduction2) Herb that used traditionally in diabetes3) Literature review4) Objective5) Experimental design6) Procurement of raw material7) Evaluation of raw material8) Phytochemical studies of raw material9) Thin layer chromatography(TLC) of Each extract10) Selection of Dose for formulation11) Anti hyperglycemic activity of each combination12) Future prospects

Herbs used traditionally in diabetes

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1. Allium cepa (Onion bulbs)2. Adiantum capillus-veneris

(Adiantum plant)3. Allium sativum (Garlic cloves)4. Anacardium occidentale (Cashew

leaves)5. Anacardium occidentale (Cashew

leaves)6. Andrographis paniculata (Kirata

leaf)7. Catharanthus roseus (Periwinkle

leaves)8. Cuminum cyminum (Cumin seed)9. Brassica oleracia (Cabbage)10. Cuminum cyminum (Cumin seed)11. Syzygium jambolanum (Jambul

seeds)12. Zingeber officinale (Ginger

rhizome)13. Pterocarpus marsupium (Indian

kino bark)14. Momordica charantia (Bitter

Melon fruit)

15. Hygrophila auriculata (Barleria plant)

16. Inula helenium (Elecampane root)17. Olea europaea (Olive leaves)18. Nymphaea lotus (Lotus roots)19. Oplopanax horridum (Devil’s Club

root bark)20. Ocimum sanctum (Sacred Basil

plant)21. Oenothera biennis (Evening

Primrose leaf)22. Panax ginseng (Chinese Ginseng

root)23. Urtica dioica (Stinging Nettle

plant)24. Trigonella foenum-graecum

(Fenugreek seeds)25. Tinospora cordifolia (Gulancha

plant)26. Galega officinalis (Goat’s Rue

seeds)27. Cucumis sativus (Cucumber fruit)28. Gymnema sylvestre (Gymnema

leaves)

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Plant Name Botanical name and family

Part use Chemical constituents

Biological use

Gudmar Gymnema sylvestre,Asclepediaceae

Leaves Gymnemosides,gymnemic acid(I-IV),resin stigmasterol etc

Anti-Diabetic agent

Bitter gourd(Karela)

Momordica charantia,Cucurbitaceae

fruit Momordicin,charantin,ascorbic acid

Anti-Diabetic agent

Indian Kino(Vijaysar/Bijasar)

Pterocarpus marsupium,Fabaceae

Bark, heartwood Pteroside ,epicatechin,sitosterol,lupeol

Anti-Diabetic Agent

Jamun Syzygium cumini(Eugenia jambolana),Myrtaceae

Seed,fruit,leaves,kernel

Anti-arthritic,Anti-Diabetes,Gastric Ulceration

Ginger Zingeber officnale,Zingeberaceae

Rhizome Gingerol,Shagoal,sesquiterpene

Anti-Arthritic,Antidaibetes,cardiovascular disease

Outline of plants selected for further studies

Literature review

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Gymnema sylvestre: Antidiaretic effect of a leaf extract from gymnema sylvestre in non-insulin-dependent diabetes mellitus

patients, K. BASKARAN, Journal of Ethno pharmacology, 30 (1990) 295 – 305

In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate –cells regeneration and anti-diabetic activity in Wistar rats, A. Bakrudeen Ali Ahmeda, journal of Phytomedicine,

Momordica charantia: A medicinal potency of momordica charantia, D. Sathish Kumar, International Journal of

Pharmaceutical Sciences Review and Research. Pharmacological actions and potential uses of Momordica charantia: a review, J.K. Grover, Journal

of Ethno pharmacology 93 (2004) 123–132

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Eugenia jambolana Anti-diabetic activity of Syzygium cumini and its isolated compound against streptozotocin-induced diabetic

rats, A. Kumar, Journal of Medicinal Plants Research Vol. 2(9), pp. 246-249 Hypoglycemic and hypolipidemic effects of flavonoid rich extract from Eugenia jambolana seeds on

streptozotocin induced diabetic rats, Bhavna Sharma, Food and Chemical Toxicology, 46 (2008) 2376–2383

Pterocarpus marsupium: Evaluation of anti-hyperglycemic and hypoglycemic effect of Trigonella foenum-graecum Linn,

Ocimum sanctum Linn and Pterocarpus marsupium Linn in normal and alloxanized diabetic rats, V. Vats, Journal of Ethnopharmacology 79 (2002) 95–100

Zingeber officinale: Effect of Ginger Extract Consumption on levels of blood Glucose, Lipid Profile and Kidney Functions in Alloxan Induced-Diabetic Rats., Abd-Elraheem A. Elshater, Egypt.

Acad. J. biolog. Sci., 153-162 (2009)

Literature Review

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Literature review

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Plant Phytochemical Reference Biological Reference

Gymnema sylvestre Amino acids V. A. Khramov et al Stimulate β-cellsregeneration

A. Bakrudeen Ali Ahmeda et al

dihydroxy gymnemic triacetate

Pitchai Daisya et alRegeneration of Islets of langerhans

E.R.B. shanmugasundaram et al

Triterpenoid saponins Niranjan p. Sahu et al

Momordica charantia Cucurbitane-type triterpenoids(charantin)

Sook Young Lee et al Pharmacological Review J.K. Grover et al

Medicinal potency of momordica charantia

D. Sathish Kumar et alMansoor ahmed et alTriterpenes, a sterol and a monocyclic alcohol

Pterocarpus marsupium pterostilbene Ajanta Chakraborty et al Hypoglycemic activity of Red kino tree

T.Radhika et al

aurone glycosides Achyut Narayan Kesariet al

Upregulation of Glut-4 and PPAR

R. Anandharajana et al

Pterocarposide S. S. Handa et al Efficacy of P.Marsupium in newly diagnosed patients

ICMR group

Eugenia jambolana mycaminose A. Kumar et al effects of flavonoid rich extract

Bhavna Sharma et al

Betulinic acid and 3,5,7,4 –tetrahydroxy flavanone

K.Karthic et alKasiappan Ravi et alantioxidant defense

system in STZ induced diabetes

Zingeber officinale Volatile oils, gingerols, shogaols

Natural Remedies Consumption on levels of blood Glucose, Lipid Profileand Kidney Functions in Alloxan Induced-Diabetic Rats

Abd-Elraheem et al

OBJECTIVE

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Evaluation of selected herbal extracts according to WHO guidelines and modern parameters

Optimization of different combinations of selected extracts by Oral Glucose Tolerance Test in Rats

Optimization of excipients in selected combination used in the formulation

Development and evaluation of herbal tablet Phytochemical analysis of developed herbal tablet by

HPTLC Anti-diabetic evaluation of optimized combination and

formulation of extracts by STZ-NAD induced experimental model in rats

EXPERIMENTAL DESIGN

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Procurement of Raw material.1) Evaluation of Raw material2) Phytochemical studies of each extract Optimization of Combination:1) Acute toxicity study as per OECD guidelines2) Oral Glucose Tolerance Test in normal Rats Development of dosage form(tablet)1) Method of preparation2) Selection of excipients3) Optimization of excipient using factorial design4) Evaluation of the dosage form5) Stability study Quality Assurance1) HPTLC of the final dosage form2) Total tannins evaluation3) Total alkaloids evaluation4) Total Flavanoids evaluation5) Proximate analysis Anti Diabetic Study1) STZ-NAD induced Anti-Diabetic Study

PROCUREMENT OF RAW MATERIAL

Herbal extracts were obtained from AMSAR INDUSTRIES LTD,INDORE. As a gift sample.

TYPE OF EXTRACT PART USED SOLVENT USED FOR EXTRACTION

GYMNEMA SYLVESTRE DRY EXTRACT

LEAVES HYDRO-ALCOHOLIC

MOMORDICA CHARANTIA DRY EXTRACT

FRUITS AQUEOUS

PTEROCARPUS MARSUPIUM DRY EXTRACT

BARKS ALCOHOLIC

EUGENIA JAMBOLANA DRY EXTRACT

SEEDS AQUEOUS

ZINGEBER OFFCINALE DRY EXTRACT

RHIZOME AQUEOUS

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Sr. No.

Evaluation Parameter

Gymnema sylvestre Extract

Momordica charantia Extract

Pterocarpus marsupium Extract

Syzygium cuminiExtract

Zingeber officnale Extract

1 Nature Hygroscopic Hygroscopic Free flowing Hygroscopic Free flowing

2 Color Dark Green Light Brown Brown Brown Pale yellow

3 Odour Characteristic Characteristic Characteristic

Characteristic Pungent

4 pH 6.04 5.10 5.10 3.68 4.7

5 Total Ash value(%)

4.28 % 1.56 % 3.17 % 6.89 % 5.84 %

6 Loss on drying(%)

3.08 % 1.87 % 1.28 % 4.77 % 3.8

EVALUATION OF HERBAL EXTRACTS

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Sr.No.

Type of Constituents

Gymnema sylvestre Extract




Zingeber officinale Extract

1 Carbohydrate - + - + +

2 Glycoside + + - + +

3 Fixed oil and Fats

- - - - -

4 Protein and Amino acid

+ + - - -

5 Saponin + - - - -

6 Phytosterol + - + + -

7 Alkaloid - + + - -

8 Flavanoid - + - + +

9 Resin - - - -

10 Tannins + - + + +

PHYTOCHEMICAL STUDIES

+=Present,-= Absent

TLC profile of each extract

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Phytoconstituent

Mobile Phase Detection Gymnema sylvestre Extract




Zingeber officnale Extract

Rf Values

Alkaloid Toluene:EA:Diethylamine(7:2:1)

Dragendorff 0.51,0.21,0.15

0.40

Steroid Toluene:EA(9.3:0.7),#(12:2:0.7)2

U.V 254 and *Anisaldehyde sulphuric agent

0.25(red color)**,0.125(blue color)**

0.48(violet color)

0.6,0.4,0.3,0.18

Flavanoid CHCl3 :MeOH(9:1),#EA:Acetic Acid:Formic Acid, H2O(10:1.1:1.1:2)

U.V 365 #0.73,#0.44 0.52,0.44,0.39,0.36,0.21

0.86,0.79

Tannins N-Butanol:Acetic acid :H2O(14:1:5)

Alcoholic FeCl3

0.84,0.53,0.28,0.15

0.88,0.67,0.58,0.5,0.26

0.75 0.32,0.47

TLC PROFILE OF EACH EXTRACT

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Plant Extract Reported Acute toxicity study (LD50)

Reference

Gymnema extract 3990 mg/kg in rats http://www.mdidea.com/products/new/new020.html

Momordica extract 1200 mg/kg in rats Abalaka, M. E et al.

Pterocarpus extract >2000 mg/kg in rats T.Radhika et al.

Eugenia extract >2000 mg/kg in rats A. Kumar et al.

Ginger extract >2500mg/kg in rats Natural remedies document

SELECTION OF DOSE FOR FORMULATION

Acute toxicity study of each plant had been reported and they are as under.

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Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method).Rats of either sex selected by random sampling technique were used for the study. The animals were kept fasting for overnight providing only water, after which the combination of extracts at a ratio of (1:1:1:1:1) was administered orally at the dose level of 100 mg/kg body weight by intragastric tube and observed for 14 days. If mortality was observed in 2 - 3 animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher dose 500, 1000 and 2000 mg/kg body weight.

This study showed no mortality up to the dose of 2,000 mg/kg body weight. So, the combination of extracts is safe for long term administration.

Sr. No. Dose of Combination

Mortality of Rats

1 100 mg/kg 0

2 500 mg/kg 0

3 1000 mg/kg 0

4 2000 mg/kg 0

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Combination 1 Combination 2 Combination 3 Combination 4

Gymnema sylvestre 2Momordica charantia 1Pterocarpus marsupium 1Eugenia jambolana 1 Zingeber officinale 1




Combination 5 Combination 6 Combination 7 Combination 8





Part

The total dose of the formulation was selected as 400 mg/kg in human and Dose for each extract in combination was selected on random trial bases that were divided into parts.

Preliminary Screening of combination Through Oral Glucose Tolerance Test.

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Above selected combination were screened for oral glucose tolerance test in normal rat for its anti-hyperglycemic activity.

Animals

Healthy female Sprague dawley rats weighing 250-350gm, procured from Zydus Cadilla Research Laboratory, Ahmedabad maintained under standard husbandry conditions (Temperature 23 ± 2 °C, relative humidity 55 ± 10% and 12 hrs light dark cycle).

The animals were fed with standard rat pellet diet and had free access to water. The experimental protocols were approved by the Institutional Animal Ethics Committee, The M.S.University of Baroda, Vadodara, Gujarat.

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Preparation of solutions for Administration: I Vehicle: 0.5 % CMC suspension was used as a vehicle. II Metformin suspension: Standard metformin was suspended vehicle and

administered at the dose of 150 mg/kg of body weight. III Suspension of test substances: Selected formulation were suspended in vehicle

and administered at their decided dose levels.

In vivo screening of different combinations for oral glucose tolerance test in normal rats.

The oral glucose tolerance test for different selected combinations were performed in overnight (18 hr) fasted normal rats.

Glucose (3 g/kg) was fed orally to each group 30 min after the administration of combinations and Metformin. Then each group was subjected to blood collection for 60,90,120 min.

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Collection of blood and estimation of serum glucose: Blood was withdrawn using haematocrit capillary from the retro orbital plexus

under ether inhalation anesthesia at -30, 0, 30, 60, and 120 min of glucose administration and glucose levels were estimated using glucose oxidase-peroxidase standard kit from Span Diagnostics Ltd. India.

Estimation of glucose

Calculation

Plasma Glucose mg/dl =Absorbance of Test

Absorbance of Standard

* 100

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Time Control standard Combination 1

Combination 2

Combination 3

Combination 4

Combination 5

Combination 6

Combination 7

Combination 8

-30 61.25 46.06 50.49 67.01 68.44 53.33 32.54 46.21 43.1 39.03

0 63.82 26.54 49.94 69.53 74.52 62.74 60.23 57.97 50.12 38.21

30 112.42 50.24 99.81 101.37 116.54 91.02 66.42 94.53 80.09 51.25

60 148.24 66.61 104.19 92.86 129.53 89.44 76.01 100.17 84.21 74.83

90 162.6 62.38 103.7 112.02 130.71 93.88 83.24 106.34 94.37 68.13

120 129.27 47.53 123.87 113.24 143.84 105.22 95.89 123.87 97.28 71.9

Glucose estimated(mg/dl)

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From the above study Combination 2 was showing good blood glucose lowering activity( Anti-Hyperglygemic) compared to other combinations during the OGTT study.

Combination 2 was showing decrease in glucose level at the time interval of 60 min .

Hence for further development in formulation and streptozotocin induce Anti-Diabetic study this combination along with optimized formulation were selected.

Future studies

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Optimization of excipients used in the formulation.Method of preparation of herbal tabletEvaluation of herbal tablet as per WHO guideline.Phytochemical analysis of the herbal tablet by

HPTLC.STZ-NAD induced Anti-Diabetic evaluation of the

selected combination and optimized formulation.

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