PRESENTEDBY

SUMAIYA NAHIDM.PHARM

USTC

Histopathological changes of alloxan induced diabetic rat after administration of methanolic extracts of Syzigium cumini (L) Skeels seeds and isolation & characterization of compounds

from ethyl acetate fraction.

Introduction

Historically, plants have provided a source of inspiration for novel drug compounds.

Plant derived medicines have made large contributions to human health and well being.

Cutler, Stephen J.; Cutler, Horace G. (2000) Used as anticancer agent

Plant ProfilePlant Profile:Scientific Classification

Kingdom: Plantae

Order: Myrtales

Family: Myrtaceae

Genus: Syzygium

Species: S cumini

Scientific Name:

Syzygium cumini (L.) Skeels. Tree

Leaves

FruitsFresh seeds Dry seeds Coarse powderHerbarium sheet

Fruits

Literature Review • Significant blood glucose lowering activity was observed in fasted, fed,

glucose loaded and diabetic rats at a single oral dose of 250mg/kg body weight. (Rahul Gupta and A. M. Saxena, 2011)

• Syzygium cumini seed kernel (aqueous suspension) was screened for its antidiabetic activity at the dose levels of 1g, 2g, 4g and 6 g/kg body weight.(R.Bhaskaran Nair and G.Santhakumari, 1986)

• The antibacterial and DPPH radical scavenging activities of the leaf extracts and leaf essential oil of Syzygium cumini Linn were investigated ( L.joji reddy, Beena jose, 1986)

• Isolation and identification of the putative antidiabetic compound, Mycaminose from the S. cumini seeds. ( A. Kumar, R. Ilavarasan, T. Jayachandran et al, 2008)

• 7-hydroxycalamenene , methyl-β-orsellinate , β-sitosterol and oleanolic acid were isolated from the pet-ether and carbon tetrachloride soluble fractions of seeds of Syzygium cumini. (Md. Al Amin Sikder, Mohammad A. Kaisar et al 2012)

To evaluate the effectiveness of Syzygium cumini” (L.) Skeels seeds in the management of Type 2 diabetes by following experiments-

1. Determination of phytochemical constituents of the crude extract

2.Evaluation of hypoglycemic activity

3. Estimation of lipid profile contents

4. Histopathological examination of Pancreas, Kidney, Liver, Heart, and Spleen.

5. Isolation and Characterization of compounds from Syzigium cumini (L.) Skeels seeds

Aim of the Research

Phytochemical Screening

Fehling Test

Flavonoid test

Shake Test Phenolic Test

Alkaloid Test

PolyPhenol test

Steroid Test

Glycoside Test

Tannin Test

Chemical constituent

Name of Test Positive Results

Alkaloids Wagner`s test +

Glycosides General test ++

PolyPhenol Ferric Chloride test +++

Steroids Salkowski`s test ++

Tannins Acetic anhydride Test

+

Flavonoids Conc. HCl &

alcoholic test

++

Saponins Shake test (aq.

Solution)

+

Reducing

sugar

Fehling`s test ++

Determination of Total Phenolic Content (Danijela Bursac, kivacavic et al.2009)) Materials & Method:

• Folin-Ciocalteu Reagent• Sodium carbonate• Methanol• Test tubes• Volumetric flask • pipette• Incubator• UV spectrophotometer• Standard- Gallic acid

0 50 100 150 200 2500

0.2

0.4

0.6

0.8

1

1.2

1.4

f(x) = 0.00552493150684931 x + 0.147356164383562R² = 0.950349385372383

Calibration curve of Gallic acid

Concentrationµg/ml

Abso

rban

ce a

t 750

nm

Addition of FCR Addition of Sodium carbonate

Absorbance measurement

Fig: Total Phenolic Content of different extracts Danijela Bursac kivacavic et al.2009

n-hex DCM EtoAc n-Bu Aqu0

20

40

60

80

100

120

140To

tal p

heno

lic c

onte

nt (µ

g/m

l)

82 µg/ml

30 µg/ml

122 µg/ml

42 µg/ml

120 µg/ml

Total Flavonoid Content DeterminationMaterials :• Aluminum chloride• NaNO2

• NaOH• Vortex and Incubator• UV spectrophotometer• Standard- Quercetine

Aluminum chloride MethodExtracts solutions prepared for absorbance measurement after the addition of NaNO2, AlCl3 & NaOH respectively

0 2 4 6 8 10 120

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

f(x) = 0.0410636363636364 x + 0.024590909090909R² = 0.98372624611756

absorbance

concentration (µg/ml)

abso

rban

ce

Fig: Total flavonoid content of different extractsDanijela Bursac kivacavic et al.2009

Calibration curve for Quercetine

n_Hex DCM EtoAc n-But Aqu0

0.5

1

1.5

2

2.5

3

3.5

Tota

l flav

inoi

d co

nten

t (µg

/ml)

0.04 µg/ml

1.2 µg/ml

3 µg/ml

0.9 µg/ml 0.9 µg/ml

Modified Kupchan partitioning of Methanolic crude extracts

Ethyl Acetate Extract

Powdered Plant Material

N-Hexane Extract

Methanolic Extract (5 gm)

Aqueous Methanolic Solution

Marc (Reject)

DCM Extract

Aqueous Methanolic Solution + 12.5ml water

Aqueous Methanolic Solution + 16ml Water

Aqueous Methanolic Solution+ 16ml Water

90ml Methanol+ 10ml Water

Extraction with hexane(100×3ml)

Extraction with DCM(100×3ml)

Extraction with Butanol (100×3ml)

Butanol extract Aqueous extract

Extraction with EA (100×3ml)

N-Hexane fraction

DCM fraction

EA fraction Butanol fraction

Fractions for EvaporationFig: Modified Kupchan partitioning of Methanolic

crude extracts ( Hu, X. 1999).

Evaluation of Hypoglycemic Activity

a) Evaluation of In vitro hypoglycemic activity – - α-amylase inhibition activity 1. Iodine-starch method 2. DNSA method

b) Evaluation of In vivo hypoglycemic activity – 1. Alloxan induced method

Addition of PPA

Fig- comparative α-amylase inhibition activity study of Ethyl acetate extract with standard

Discussion: The ethyl acetate extract showed an increasing in vitro α-amylase inhibition activity with the concentration in comparison with the Acarbose as standard and is an indication of its hypoglycemic action.

* Xiao Z, Storms R, 2006

0.1 0.3 0.5 0.7 1.1 1.3 1.50

20

40

60

80

100

120

Acarbose (standard) Ethyl acetate extract

concentration (µg/ml)

% in

hib

itio

n

*

Mean ± SEM, n = 3

Iodine-Starch Method *

Addition of Iodine solution Blue-Violet complex Absorbance

at 620nm

Preparation of extracts solution

Addition of starch sol

* P < 0.07, ** p<0.005

**

Addition of starch h solution

samples with DNSA solution for absorbance at 540 nm

Fig- comparative α- amylase inhibition study of Ethyl acetate extract with standard

Discussion: The ethyl acetate extract of syzigium cumini seeds showed significant α-amylase inhibition activity as compared to Acarbose as standard which indicated the significant hypoglycemic activity of extract.

0.1 0.3 0.5 0.7 0.9 1.1 1.2 1.3 1.4 1.50

20

40

60

80

100

120Acarbose (standard) Ethyl acetate extract

concentration (µg/ml)

% in

hib

itio

n

* P < 0.08, ** p<0.003

*

Mean ± SEM, n = 3

DNSA Method

Materials

Addition of prepared enzyme solution

Preparation of DNSA solution

**

Alloxan Induced Diabetes Method *

IP Injection of Alloxan Glucose level measurement

Fig: Comparison hypoglycemic activity of methanolic extract with normal, Diabetic control and standard Glycazide

Discussion: This result showed a significant in vivo hypoglycemic activity of methanolic extract at 200mg /kg of Syzigium cumini seeds in comparison to Glycazide at 25mg/kg.

* Fazil M.A, 2010

Wister Albino rat Extract feeding

day 0 day 1 day 3 day 5 day 7 day 9 day 11 day 140

5

10

15

20

25

30

35

40 diabetic control normal standard extract 200mg/kg

No of Days

Blo

od

Glu

cose

Lev

el (

mm

ol/

L)

Mean ± SEM, n = 3 *P <0.05, **P< 0.001

***

**

Results:

*

Estimation Of Lipid Profile *

Serum Collection Centrifuge machine Blood collection

Method:

Discussion: The low plasma total cholesterol and the significant increase in HDL-cholesterol level in the treated animals compared with the untreated diabetic control rats clearly demonstrated the presence of Antihyperlipidemic agents in the methanolic extract of Syzigium cumini (L.) Skeels.

* Eckel RH, 2002

Diabetic control normal control standard extract0

50

100

150

200

250

300

TC

HDL

LDL

TG

*

Lev

el o

f T

C,

HD

L,

LD

L

and

TG

(m

g/d

l)

Photometer

*P <0.01, **P< 0.007

**

Histopathological Examination of Pancreas, Liver, Heart, Kidney and Spleen of Rats

Materials and Method *

Tissue Collection Preservation Grossing

Section Cutting Prepared Block Blocking

Tissue Processing

Parafin Impregnation

Examination in Olympus microscope

Prepared slides

Section Collection in Slide Slide stainer machine

*Vessal M, Hemmati M, Vasei M 2003

Histopathological examination of rat pancreas

pancreatic tissue of the normal control pancreatic tissue of the diabetic control

pancreatic tissue of the Glycazide treated grouppancreatic tissue of plant extract treated group

Microscopic findings: The histopathological observations showed that the pancreatic islets from normal control group revealed normal histological features. In the diabetic group, marked morphological changes were observed. The pancreas of SCE 200mg/kg treated group showed that SCE administration improved the previous degenerative changes in most of the rats

Histopathological examination of rat Heart

Cardiac tissue of the normal.

Cardiac tissue of the Glycazide treated groupCardiac tissue of the plant extract treated group

Cardiac tissue of the Diabetic

Microscopic findings : The histopathological observations showed that the Cardiac tissue from normal control group displayed normal histological features. In the diabetic group, marked morphological were observed. The cardiac tissue of SCE 200mg/kg treated group showed that SCE administration improved the previous morphological changes in compare to the Diabetic control and Glycazide 25mg/kg.

Histopathological examination of rat Liver

Microscopic findings : The histopathological observations showed that, the diabetic control group revealed marked morphological changes like Necrosis in the hepatic muscle fibre and infiltration with few mixed inflammatory cells. Section of SCE 200mg/kg treated group revealed normal hepatic tissue compare to the diabetic control and Glycazide 25mg/kg group.

Hepatic tissue of the S. cumini treated group Hepatic tissue of the Glycazide treated group

Hepatic tissue of the diabetic control Hepatic tissue of the normal control

Histopathological examination of rat Kidney

kidney tissue of the Normal kidney tissue of the Diabetic control

kidney tissue of the group S. cumini treated group

kidney tissue of the Glycazide treated group

Microscopic findings: The histopathological observations showed that the kidney tissue from normal control group revealed normal histological features. In the diabetic group, marked morphological changes were observed.The treated group ,i.e Syzigium cumini 200mg/kg group and Glycazide 25mg/kg group did not show any positive effect in the diabetic kidney.

Histopathological examination of rat Spleen

Spleen tissue of the group S. cumini treated group Spleen tissue of the Glycazide treated group

Spleen tissue of normal control group Spleen tissue of Diabetic control group

Microscopic findings: The histopathological observations showed that the Spleenic tissues from normal control, Diabetic control, Syzigium cumini 200mg/kg and Glycazide 25mg/kg groups revealed normal histological features.

Column Chromatography of Ethyl acetate extract

Column Packing with silica gel

Materials and Method:• Beakers• Glass rod • Funnel suitable for wet

solids• Solvent (Chloroform)• Silica gel (60-120 mesh),

Column grade • Pasteur pipette Sampling

3 2

1

4

Sub-fractions SCE F 1- SCE F 129

Fig: 1-4 Sequence of band pattern observed during the column run.Amrita University, 2015

Observation of sufficient amount of Crystals from Sub-fractions

The following sub-fractions of ethyl acetate extract of Syzigium cumini (SCE F) samples formed crystal after the evaporation of mobile phase (eluent):

• SCE F-49• SCE F-50• SCE F-51• SCE F-52• SCE F-53• SCE F-55• SCE F-56

SCE F-42 – SCE F- 48 and SCE F-62- SCE F-80 are semisolid in nature.

Crystal Sub Fraction ( SCE F49- SCE F 56)

Semisolid like Sub Fraction ( SCE F62- SCE F 80)

Crystal Fraction (SCE F 55)

Thin layer chromatography (TLC) for characterization of sub-fractions

Materials and Method:

• TLC Plate• Beaker (Development

Chamber)• UV Lamp• Hot Plate• Chloroform ,Methanol• Para –Anisaldehyde• Ethanol, Acetic Acid• Sulfuric Acid• Pasteur pipette Sampling

Development of chromatogram Uv detection at 254nm

UV detection after staining with P- Anisaldehyde at 365nm

UV detection at 365 nmUV detection after staining with P-

Anisaldehyde at 254nm

Solvent system ratioCHCl3: Me-OH(ascending polarity)(1 drop of AA)

No. of

specimen

Solvent system

ratio

[CHCl3:Me-OH]

(1 drop of AA)

 

Fraction

No

Rf

Value

Colour changes observed

254 nm 365 nm

6 5:1 41- 43 0.475 Blackish yellow

colour

Yellow colour

7 5:1 44-46 0.48 Blackish yellow

colour

Yellow colour

8 5:1 47-48 0.4 Blackish yellow

colour

Yellow colour

9 1:1 49-53 0.77 Straw colour

intensifying

Straw colour

intensifying

11 1:1.5 54-56 0.846 Yellow colour Yellow colour

12 1:2 58 0.94 Straw colour

intensifying

Straw colour

intensifying

13 1:2 62-67 0.875 Yellow colour Yellow colour

14 1:3 68-72 0.85 Yellow colour Yellow colour

Result of TLC Analysis Of important Fractions

RESULT OF CHARACTERISATION AND IDENTIFICATION OF SUB-FRACTIONS OBTAINED FROM ETHYL ACETATE EXTRACT OF Syzigium cumini seeds.

Proton (1H) NMR Spectroscopy

1H proton NMR spectrum of SCE F-49 Sub fraction

1H proton NMR spectrum of SCE F-52 Sub fraction

1H proton NMR spectrum of SCE F-55 Sub fraction

1H proton NMR spectrum of SCE F-66 Sub fraction

Structure elucidation of Compound

The given proton NMR revealed that the compounds might be Polyphenols as all the compounds showed significant peaks between 7-8 ppm which indicated the presence of benzene ring and 3-4 ppm revealed the presence of hydroxyl group . However, further purification and other spectroscopic analysis is needed for structure elucidation.

Conclusion

The Ethyl acetate extract of syzigium cumini seeds showed-

1. Significant in vitro α-amylase inhibitory activity. 2. Significant in vivo hypoglycemic activity.

Estimation of lipid profile and histopathological examination of rat organs used in the in vivo study, were done to observe the recovery effect from the diabetic complications of syzigium cumini seeds .

Surprisingly , the result revealed the low LDL, TG and TC and high HDL for the treated group and recovery effect on diabetic heart and Liver in histopathological examination.

The probable polyphenol structures obtained from the 1H NMR spectra will be correlated with the 13 C and other spectroscopic methods for the confirmation of the structures.

Exploration of the other Antihyperlipidemic and other relevant compounds will be highly encouraged by observing the Microscopic findings in histopathological examination.

Future Plan

Thank You