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