Phytochemical & Biological Evaluation of Cascabela Thevetia with Special

. Presented by: Biman Bhuyan Assistant Professor Department of Pharmaceutical Sciences Dibrugarh University Dibrugarh-786004, Assam, India Email ID:[email protected]

Presented at: XIXth International Congress "Phytopharm 2015“ New Phytotherapeutics – Developments, Requirements and Success for Patients with Rational Phytotherapy. (Institute of Pharmacy, University of Bonn; July 21-24, 2015 ) . Diabetes mellitus is chronic disorder of carbohydrate, protein and fat metabolism resulting from insulin deficiency and abnormality in the use of insulin.

. Due to several problems with available conventional therapies, alternative approaches become essential to treat diabetes.

. Plant based medicine has become one of the most promising alternative strategy for treatment of diabetes.

. In this study the bark of Cascabela thevetia L. was selected for evaluation of antidiabetic activity based on its traditional use in Assam.

. T. Miyagawa et al. (J. Nat. Prod., 2009) in his article “Cardenolide Glycosides of Thevetia peruviana and Triterpenoid Saponins of Sapindus emarginatus as TRAIL Resistance-Overcoming Compounds” reported that cardenolide glycosides isolated from Thevetia peruviana (Cascabela thevetia) have significant reversal effect on TRAIL resistance in human gastric adrenocarcinoma cells.

. M.H. Khan et al. (Indian J Tradit Know., 2010) in his article “Antidiabetic plants used in Thoubal district of Manipur, North-East India” reported that bark of the plant is used by Meitei community for treatment of diabetes.

. V. Bandara et al. (Toxicon, 2010) in his article “A review of the natural history, toxinology, diagnosis and clinical management of Nerium oleander (common oleander) and Thevetia peruviana (yellow oleander) poisoning” reported the cardiac glycosides related toxicity and their clinical management in emergency. . J. Buragohain (Recent Res. Sci. and Technol, 2011) in his article “Ethnomedicinal plants used by the ethnic communities of Tinsukia District of Assam, India” reported that latex of stem bark is applied on boils. Infusion of the bark is used in malaria. Seeds are used as antifertility agent.

. N. Srivastava et al. (Biotechnol Res Int., 2012) in her article “Isolation and Characterization of Some Phytochemicals from Indian Traditional Plants” reported the antioxidant activities of polyphenols present in aqueous extract of different parts like stem, flower, and leaf of Thevatia peruviana.

. S. Kohls et al. (Phytochemistry, 2012) in his article “Cardiac glycosides from Yellow Oleander (Thevetia peruviana) seeds” reported about isolation of six thevetia glycosides with two unknown structure from seeds of the plant.

. P. Mandol et al. (Int. J. Pharm. Biol. Sci., 2013) in his article “Herbal Medicines useful for the treatment of diabetes in North-East India” reported that powdered bark of the plant Cascabela thevetia L. can be used in the treatment of diabetes. . S. Naz et al. (IJPCS, 2013) in his article “An updated review on the Genus Cascabela Plant Family: Apocynaceae” reported the general morphology and other members of the genus Cascabela along with the folk fore use of the plant Cascabela thevetia L.

. K. M. Sowjanya et al. (IJRPBS, 2013) in his article “Phytochemical Extraction and Antimicrobial Efficiency of Crude Leaf Extract of Medicinal Plant Cascabela thevetia” reported the profile of the plant along with the phytochemical screening results of different solvent extracts of leaf part. They also reported that different solvent extracts of leaves of Cascabela thevetia L. show antimicrobial activity against different pathogens.

. R. Bhatta et al. (J Anim Physiol Anim Nutr, 2013) in his article “Effects of medicinal and aromatic plants onn rumen fermentation, protozoa population and methanogenesis in vitro” reported the potential of tannins from 21 plants leaves including Cascabela thevetia L. as antimethanogenic additives in ruminant feed.  Botanical Name: Cascabela thevetia . KINGDOM: Plantae . SUB KINGDOM: Tracheobionta . DIVISION: Magnoliophyta . CLASS: Magnliosida . ORDER: Gentianales . FAMILY: Apocynaceae Fig: leaf and flower of Cascabela thevetia L. . TRIBE: Rauvofioideae . GENUS: Cascabela . SPECIES: Thevetia  Synonym: . Thevetia peruviana  Identification: Identification and authentication is done at BSI, Shillong via letter of ref. no. BSI/ERC/Tech//Identification/2014/361 on 26 August 2014 Fig: Dried barks  Macroscopic evaluation

Sl. No. Characteristics Stem bark

1. Colour of outer surface Greenish brown in fresh form and of bark dark brown in dried form

2. Colour of inner surface of Brownish white in fresh form and bark blackish brown in dried form.

3. Odour Characteristic and woody. 4. Taste Characteristic. 5. Shape Hard quills 6. Texture Rough on the outer side and smooth in texture in the inner side.  Microscopic evaluation (powder microscopy)

(a) Cr-Crystal (b) CC-Cork cell

(a) PhR-Phloem ray, (a) VB-Vascular bundle, Cork cell

(a) CCh-Collenchyma, (a) SG-Starch globule (b) Rossete calcium oxalate Fig: Transverse section of bark  Physicochemical evaluation

Sl. No. Parameters Resultant value (%)*

1. Ash Values Total ash 8.67±0.24 Acid insoluble ash 0.83±0.24

Water soluble ash 5.33±0.24 2. Extractive Values Alcohol soluble 4.53±0.38 extractive Water soluble extractive 12.27±0.38

3. Moisture content 7.83±0.24

*Average of three reading ± standard deviation  Physical characters of extracts

Sl. Extracts Yield Consistency Colour Odour No. (%w/w)

1 Petroleum ether 3.32 Sticky solid Greenish Characteristic extract yellow 2 Chloroform extract 1.86 Solid Dark brown Characteristic

3 Ethyl acetate extract2.38 Semi-solid Dark brown Characteristic

4 Methanol extract 13.40 Semi-solid Dark brown Characteristic

5 Hydro-alcoholic 2.52 Semi-solid Dark brown Characteristic extract  Preliminary phytochemical screening data Plant Petroleum Chloroform Ethyl Acetate Methanol Hydro-alcoholic constituents ether extract extract extract extract extract Alkaloid - - + + - Flavonoid - - - + + Triterpenoid - - + + + Phytosterol + + - - - Tannin - - - + + Saponin - - - + + Free - - + + + anthraquinone Coumarin - - - - - Carbohydrate - - + + + Protein - - - - - Amino acid - - - - - Fatty acid + + - - -  Fluorescence analysis

Sl. No. Particulars of the Under ordinary Under UV light treatment light (366nm) 1 Powder as such Saddle brown Sandy brown

2 Powder + 1N NaOH (Aq) Black Bluish black

3 Powder + 1N NaOH (Alc) Light brown Dark brown

4 Powder + 1N HCL Light brown Blackish brown

5 Powder + 50% H2SO4 Black Brackish brown

6 Powder + 50% HNO3 Reddish brown Black

7 Powder + Ammonia Greenish brown Dark brown

8 Powder + 5% Iodine Greenish brown Black

9 Powder + 5% FeCl3 Greenish brown Blackish brown

10 Powder + Acetic acid Reddish brown Dark brown  Thin layer chromatography

(a) Pet ether (b) Chloroform (c) Ethyl acetate (d) Methanol (d) Hydro Extract Extract Extract Extract alcoholic (4 spots) (4 spots) (4 spots) (4 spots) Extract (3 spots)

Fig: TLC plates showing the spots of compounds  Detection of Rf values

Extract Solvent system Number of Rf values Visualizing agent spots

Petroleum Tolune: Acetone = 7:3 4 0.28, 0.4, Anisaldehyde- ether 0.56, 0.94 Sulfuric acid chloroform Tolune: Acetone = 6.6: 4.4 4 0.20, 0.55, Anisaldehyde- 0.69, 0.89 Sulfuric acid

Ethyl Chloroform: Ethyl 4 0.10, 0.29, 10% H2SO4 acetate acetate: Methanol = 4:3:3 0.58, 0.85

Methanol Chloroform: Ethyal 4 0.12, 0.29, 10% H2SO4 acetate: Methanol = 0.47, 0.83 9:4:0.5

Hydro Ethyl acetate: Methanol: 3 0.36, 0.72, 10% H2SO4 alcoholic Glacial acetic acid = 4:8:3 0.85 The in vivo study was approved by Institutional Animal Ethical Committee, Dibrugarh University, vide registration number- 1576/GO/a/11/CPCSEA Dated: 17/2/2012 and Approval number- 1AEC/DU/87 dtd. 27/3/2015 Acute toxicity study

 For acute oral toxicity OECD guideline 425 was followed using female Wister albino rat.

 As per calculations from acute oral toxicity (guideline 425) statistical

program (version 1.0), the LD50 value of Cascabela thevetia L. bark extract was found to be more than 2000 mg/ kg body weight. Hence one tenth of this those was selected for calculations of the doses using in antidiabetic study.

 After 15 days of treatment, the liver and kidney of the test extract treated animals were collected and observed for toxic effects. (a) Group 4 (100 mg/kg b.w.) (b) Group 5 (200 mg/kg b.w.)

Fig: Histoarchitecture of liver tissue (a) Group 4 (100 mg/kg b.w.) (b) Group 5(200 mg/kg b.w.)

Fig: Histoarchitecture of kidney tissue . Experimental design (6 groups of albino Wister male rat consisting 6 animals in each group)

GROUP-I Administered with normal saline (Normal control)

GROUP-II Treated with metformin hydrochloride at 10 mg/kg (Standard drug treated) body weight per orally for 15 days

GROUP-III No treatment received (Negative control)

GROUP-IV Treated with test extract at 100 mg/kg body weight per (Low dose treated) orally for 15 days

GROUP-V Treated with test extract at 200 mg/kg body weight per (Medium dose treated) orally for 15 days

GROUP-VI Treated with test extract at 400 mg/kg body weight per (High dose treated) orally for 15 days . In vivo study protocol for antidiabetic activity  Body weight (g) variations Treatment day 0th day 5th day 10th day 15th day

Group 4 82.67 ± 2.73** 91.33 ± 2.06** 94.00 ± 1.78** 100.67 ± 2.73** (100 mg/kg)

Group 5 76.67 ± 1.36** 81.67 ± 2.58**## 94.67 ± 2.73** 105.00 ± 2.68 (200 mg/kg)

Group 6 76.67 ± 4.03** 90.67 ± 1.03** 94.67 ± 1.36** 103.67 ± 4.59 (400 mg/kg)

Normal control 100.67 ± 4.03 100.33 ± 4.22 104.00 ± 3.57## 106.67 ± 3.72

Negative control 79.00 ± 3.36** 81.00 ± 1.78**## 82.00 ± 0.89**## 79.00 ± 0.89**##

Standard drug 83.67 ± 2.73** 91.67 ± 4.03** 96.00 ± 2.36** 104.00 ± 2.68

100

60 Body (g) Body Weight 40

0 0th day 5th day 10th day 15th day Number of days

G-4 (100 mg/kg) G-5 (200 mg/kg) G-6 (400 mg/kg) Normal control Standard Drug Negative control  Body temperature (oF) variations

Treatment day 0th day 5th day 10th day 15th day

Group 4 97.00 ± 1.33# 97.9 ± 1.26 97.67 ± 1.64 97.17 ± 1.30 (100 mg/kg)

Group 5 97.33 ± 0.65 97.7 ± 0.47 98.26 ± 0.54 98.53 ± 0.44 (200 mg/kg)

Group 6 98.1 ± 1.03 97.96 ± 0.90 98.67 ± 0.99* 98. 57 ± 0.78 (400 mg/kg)

Normal control 97.06 ± 0.22 97.36 ± 0.84 97.17 ± 0.31 97.87 ± 0.36

Negative control 97.2 ± 0.26 96.93 ± 0.34# 97.00 ± 0.24 96.97 ± 0.28

Standard drug 98.2 ± 0.38 98.26 ± 0.40 98.00 ± 0.24 97.97 ± 0.27

98,5 F) o 98

97,5

97 Body Temperature ( Body Temperature

96,5

96 0th day 5th day 10th day 15th day Number of days

G-4 (100 mg/kg) G-5 (200 mg/kg) G-6 (400 mg/kg) Normal control Standard Drug Negative control  Blood glucose level (mg/dl) variations

Treatment day 1st day 5th day 10th day 15th day

Group 4 314.33 ± 9.85 257.33 ± 13.45 195.33 ± 9.13**## 171.00 ± 3.57**## (100mg/kg) Group 5 290 .00 ± 24.19 248.33 ± 11.67 186.67 ± 7.71**## 155.67 ± 11.63**## (200mg/kg) Group 6 297.00 ± 38.02 260.00 ± 12.29 194.00 ± 13.97**## 149.33 ± 6.71**## (400mg/kg) Normal 103.67 ± 6.59 103.00 ± 4.77 102.00 ± 0.89## 100.67 ± 1.36#

Standard 278.33 ± 24.35 225. 67 ± 25.95 165.00 ± 8.80** 115.00 ± 10.31* (Metformin hydrochloride 10 mg/kg) Negative control 304.00 ± 43.52 315.00 ± 25.98 313.67 ± 16.67**## 317.00 ± 8.62**

300

250

200

150

100 Blood Glucose Level Level (mg/dl) GlucoseBlood

0 0th day 5th day 10th day 15th day Number of days

G-4 (100 mg/kg) G-5 (200 mg/kg) G-6 (400 mg/kg) Normal control Standard Drug Negative control  Plasma lipid profiles

Groups Triglyceride (mg/dL) Cholesterol (mg/dL) HDL (mg/dL) LDL (mg/dL)

Group 4 124.68 ± 4.14**## 157.27 ± 6.93## 38.61 ± 4.41**## 93.98 ± (100 mg/kg) 11.78**##

Group 5 103.50 ± 7.31**## 88.73 ± 8.26 56.25 ± 5.04# 11.77 ± 4.58 (200 mg/kg)

Group 6 112.29 ± 14.35**## 131.45 ± 8.98**## 49.54 ± 1.13 59.44 ± 8.81**## (400 mg/kg)

Normal 78.37±3.30 92.47±3.92## 54.23±2.72 22.55 ± 7.08

Standard (Metformin 78.53 ± 3.83 79.72 ± 2.19** 51.53 ± 1.04 12.48 ± 1.43 Hydrochloride 10mg/kg) Negative control 139.82 ± 3.65**## 161.32 ± 3.13** 35.82 ± 1.16**## 97.53 ± 4.58**##

 Values are expressed as mean ± SD (number of animals, n=6) significantly different at **p<0.01, using Dunnett Compare all vs normal control and at #p<0.05, ##p<0.01, using Dunnett Comapare all vs standard treated. Triglyceride Choesterol 160 200 140 120 150 100 80 100 60 40 50 20 0 0 G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative Triglyceride level (mg/dl) level Triglyceride mg/kg) mg/kg) mg/kg) control Drug control Cholesterol (mg/dl)Level mg/kg) mg/kg) mg/kg) control Drug control Groups Groups

LDL HDL 120 60 100 50 80 40 60 30 40 20

LDL Level (mg/dl) Level LDL 20

HDL Level Level (mg/dl) HDL 10 0 0 G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative mg/kg) mg/kg) mg/kg) control Drug control mg/kg) mg/kg) mg/kg) control Drug control Groups Groups  Serum biomarker levels

Groups ALP (U/L) SGOT (U/L) SGPT (U/L) Total Protein (g/dL)

Group 4 (100 126.80 ± 4.29**## 52.90 ± 4.16# 56.31 ± 5.27**## 5.17 ± 0.22**## mg/kg) Group 5 (200 80.94 ± 7.19**## 49.03 ± 5.76 48.36 ± 3.25# 4.15 ± 0.11 mg/kg) Group 6 (400 99.96 ± 7.85**## 58.06 ± 5.76 48.02 ± 4.66# 5.79 ± 0.18**## mg/kg) Normal 44.83±5.78## 54.19±4.16 45.26±2.82 4.38±0.45 Standard 61.72 ± 4.58** 47.74 ± 6.10 42.52 ± 1.67 4.08 ± 0.72 (Metformin Hydrochloride 10mg/kg) Negative control 142.56 ± 4.48**## 112.14 ± 2.37**## 91.99 ± 1.34**## 5.96 ± 0.43**##

Values are expressed as mean ± SD (number of animals, n=6) significantly different at **p<0.01, using Dunnett Compare all vs normal control and at #p<0.05, ##p<0.01, using Dunnett Comapare all vs standard treated. ALP SGOT 160 120 140 100 120 80 100 80 60

60 40 40 ALP Level (U/L) ALP SGOT Level Level SGOT(U/L) 20 20 0 0 G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative mg/kg) mg/kg) mg/kg) control Drug control mg/kg) mg/kg) mg/kg) control Drug control Groups Groups

SGPT Total Protein 100 7 90 80 6 70 5 60 4 50 40 3 30 2 SGPT Level Level (U/L) SGPT 20 10 1 Total Protein Level (g/dl) Protein Level Total 0 0 G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative G-4 (100 G-5 (200 G-6 (400 Normal Standard Negative mg/kg) mg/kg) mg/kg) control Drug control mg/kg) mg/kg) mg/kg) control Drug control Groups Groups (a) Group 4 (b) Group 5 (c) Group 6 (100 mg/kg b.w.) (200 mg/kg b.w.) (400 mg/kg b.w.)

(d) Normal group (e) Standard treated (f) Negative control

Fig: Histoarchitecture of pancreas tissue . From the above study different pharmacognostic parameters of the bark of Cascabela thevetia L. were determined which will be helpful in standardizing the plant.

. The phytochemical screening of the different extracts of bark revealed the compounds present and it will be helpful in isolation of the compounds.

. The in vivo antidiabetic study of bark shows that it is helpful in controlling blood sugar level as mentioned in the documented report of local use. But more depth study like molecular level, clinical level studies will be helpful for further exploration. . Kokate, C.K. Practical Pharmacognosy, 4th ed., Vallabh Prakashan, Delhi, 2011. . Khandelwal, K.R. Practical Pharmacognosy: Techniques and Experiments, 19th ed., Nirali Prakashan, Pune, 2008. . Quality control method for herbal materials, World Health Organization, Geneva, 2011. . The Ayurvedic Pharmacopoeia of India, Part-I, Appendix Volume(s)-II,III,IV, Govt. of India, Ministry of Health an Family Welfare, New Delhi, 1999. . Harborne, J.B. Phytochemical Methods- A guide to modern techniques of plant analysis, 3rd ed., Chapman & Hall, London, 1998. . Wagner, H.; Bladt, S. Plant Drug Analysis- A Thin Layer Chromatography Atlas, 2nd ed., Springer, Verlag, Berlin, Heidelbberg, New York, 2001. . Masiello, P.; Broca, C.; Gross, R.; Roye, M.; Manteghetti, H. D.; Novelli, R. G. Expermental NIDDM, Development of a New Model in Adult Rats Administered Streptozotocin and Nicotinamide, Diabetes, 1998, 47, 224- 229. . Rees, D.A.; Alcolado, J.C. Animal models for diabetes mellitus. Diabetic Med, 2005, 22, 359-370. . Gazanfar, K.; Ganai, B.A.; Akbar, S.; Khan, M.; Dar, S.A.; Dar, M.Y.; Tantry, M.A. Antidiabetic Activity of Artemisia amygdalina Decne in Streptozotocin Induced Diabetic Rats. BioMed Res Int, 2014, 1-10. . Bansal, M.P. Molecular Biology and Biotechnology: basic experimental protocol, The Energy and Resource Institute, New Delhi, 2013.