Studies on certain medicinal plants for therapeutic management of cardiovascular disorders in type II diabetic rat

1. Studies on certain medicinal plants for therapeutic
management of cardiovascular disorders in type II
diabetic rat
Chandan Patil (V-1661/16)
MVSc Student
Department of Veterinary pharmacology & Toxicology
Synopsis seminar on
U.P. Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwa Vidhyalaya
Evam Go-Anusandhan Sansthan, (DUVASU) Mathura - 281 001 (U.P.)

2. Introduction
Diabetes mellitus is a metabolic disorder of diverse etiology
manifested as hyperglycaemia, polyuria, polydipsia and polyphagia
characterized by disturbed carbohydrate, fat & protein metabolism
due to defects in insulin secretion and/or insulin action.
(Mohan et al.,2013)
W.H.O and American Diabetes Association(ADA) classified diabetes
mellitus as-
 Type I Diabetes mellitus
 Type II Diabetes mellitus
 Gestational diabetes
 Other specific types.

3. WHO global report on diabetes 2016
• The number of people with diabetes has risen from 108 million in 1980 to 422
million in 2014 and is expected to increase to 438 million by 2030.
• The global prevalence of diabetes among adults over 18 years of age has risen
from 4.7% in 1980 to 8.5% in 2014.
• In 2012 alone diabetes caused 1.5 million deaths.
• Cardiovascular disease accounted for 44% of deaths in type 1 diabetes and 52% of
deaths in type 2 diabetes
• Diabetes prevalence has been rising more rapidly in middle- and low-income
countries.

4. Courtesy-:Understanding Diabetes-What is diabetes? By Dr.John Morris,2014
Diabetes zone

5. Courtesy-:Times Of India; March 4 2016

6. Normal Insulin Secretion
Source-:Wikipedia

7. Normal Insulin Signalling Pathway
Source-:Wikipedia

8. β-cell Dysfunction in Diabetes mellitus
Source-: https://doi.org/10.1371/journal.pone.0153017

9. Mechanism of insulin resistance
• Mutation of Insulin receptor substrate(IRS)
• Serine phosphorylation of IRS
Expression of intracellular serine kinases
1) Hyperinsulinemia
2) Hyperlipidemia
3) Mitochondrial dysfunction
4) Inflammation

10. Type II Diabetes and cardiovascular
Disorders

11. Type 2 diabetes and Cardiovascular complications
• Vascular complications
Macrovascular -Coronory artery disease(CAD)
-Peripheral arterial disease(PAD)
-Stroke
Microvascular -Nephropathy
-Neuropathy
-Retinopathy
• Cardiac complications - Cardiomyopathy
- Myocardial infarction

12. Pathogenesis of Diabetic Atherosclerosis

13. Dibetes and vascular dysfunction

14. Diabetic cardiomyopathy(DCM)
• Structural and functional abnormalities of myocardium in diabetic patients
without coronary artery disease/hypertension
• Factors influencing DCM –
> microangiopathy and related endothelial dysfunction
> autonomic neuropathy
> metabolic alterations
• Interstitial and perivascular
fibrosis is a histological hallmark of
diabetic cardiomyopathy
(Rubler et al.,1972;Factor et al.,1980;Hoeven
and Factorb 1990)

15. Pathogenesis of Diabetic Cardiomyopathy

16. Classical Biomarkers of type II Diabetes
Gymnema sylvestre
• Glucose
• HbA1c
• Lipid profile
• C-peptide
• miRNA

17. Role of microRNA in type II Diabetes
• Single stranded, non coding RNA molecules of 22-25 nucleotides
• Regulate gene expression by binding to untranslated region of their target
mRNAs, causing either translation inhibition and/or mRNA degradation
(Bartel et al.,2004)
• Can be appreciated in whole blood, urine, saliva, tears & breast milk
(weber et al.,2010)
• miRNAs regulating β-cell development and function
miR-375 in pancreatic islets of humans and mouse regulate insulin secretion
(Poy et al .,2004)
• miRNA and endothelial dysfunction in diabetes
miR-34a significantly increased in mouse microvascular endothelial cells in the
presence of hyperglycaemia, accompanied by a significant decrease in SIRT1 which
deacetylates and activates eNOS
(Arunachalam et al.,2016)

18. Role of microRNA in type II Diabetes cont…..
• Some of the miRNAs associated with diabetic
cardiovascular complications
• miR-16
• miR-133
• miR-492
• miR-373
• miR-223
• miR-320
• miR-503
• miR-1
• miR-504

19. Drugs used in type II Diabetes
Class Examples Mechanism of action
Sulfonylureas Glibenclamide,
Tolazamide,
glimepride
Stimulating insulin release from pancreatic
beta cells by inhibiting the KATP channel
Biguanides Metformin Acts on the liver to reduce gluconeogenesis
and causes a decrease in insulin resistance
via increasing AMPK signalling.
Alpha-
glucosidase
inhibitor
acarbose, miglitol,
voglibos
Inhibit Alpha-glucosidase enzyme needed
for carbohydrate digestion
Thiazolidinedion
es
Pioglitaxone,
rosiglitazone
Reduce insulin resistance by activating PPAR-
γ in fat and muscle
Dipeptidyl
Peptidase 4
inhibitors
Sitagliptin,
saxagliptin
Increase the activity of Incretins by inhibiting
DPP 4 enzyme

20. Drawbacks of currently used anti diabetic drugs
• Hypoglycaemia
• lactic acidosis
• Gastro intestinal problems
• Increased risk of cancer (Tokajuk et al.,2015)
• Expensive

21. Most commonly used indegenous plants in diabetes
• Allium cepa
• Allium sativum
• Aloe vera
• Ficus bengalensis
• Gymnema sylvestre
• Ocimum sanctum
• Tinospora cardifolia
• Coccinia indica
• Momordica charantia

22. Gymnema sylvestre
• Family-Asclepidaceae
• Location-South western India,
Australia & Tropical Africa
• Synonyms-Madhunashini, Gurmar,
Meshashrunga
• Part used-Leaf
• Extraction used-Ethanol extract
• Active ingredients –Gymnemic acid, Gurmarin

23. • The gymnemic acid components exhibit Inhibitory activity on sodium-dependent
glucose transporter 1 found in high levels in brush-border membranes of
intestinal epithelial cells.
(Wang et al.,2014)
• Immuno histochemistry of Pancreas showed few newly formed β-cells with no
surrounding α-cells in Gymnema treated rats.
(Hafizur et al.,2015)
• Ethanol extract offers cardiac protection by decreasing cardiac caspase-3 levels,
Na+/K+ ATPase activity, DNA laddering, oxidative stress, and maintaining normal
architecture of myocardium
(kumar et al.,2014)

24. Tribulus terrestris
• Family-Zygophyllaceae
• Location-Europe, southern Asia, Africa, and
Australia
• Synonyms-goat's-head, bindii,, burra gokharu,
bhakhdi,caltrop, puncturevine & tackweed.
• Parts used-seeds and fruits
• Active constituents-Protodioscin, terrestrosins
A-E, desgalactotigonin, F- gitonin, gitonin,
tigogenin, furostanol glycosides, β-Sitosterol,
stigmasterol, diosgenin, hecogenin, ruscogenin,
Kaempferol, quercetin

25. • Noted for diuretic, aphrodisiac, antiurolithic, immunomodulatory,
antihypertensive, antihyperlipidemic, antidiabetic, hepatoprotective,
anticancer, anthelmintic, antibacterial, analgesic, and anti-inflammatory
propertises
• TT produced dilation of coronary artery and improved the coronary circulation.
It is therefore recommended in Ayurveda for the treatment of angina pectoris
and other cardiac complications of diabetes.
• TT ethanolic extract was found to decrease cholesterol-induced
hyperlipidemia, with a decrease in cholesterol, triglycerides, low density
lipoprotein (LDL), very low density lipoprotein (VLDL), and atherogenic index
(AI), and an increase in high density lipoprotein (HDL) levels in the blood.

26. Type II Diabetes animal models
• spontaneous/genetically derieved (eg. Zucker fatty rat,Newzealand obese mouse)
• Diet or nutrition induced(eg. sand rat, Tuco tuco,C57BL/6J mouse)
• Chemically induced(chemicals used - streptozotocin, Alloxan )
• Surgical diabetic animals(Pancreatectomy)
• Transgenic/ knock-out
To mimic the human type 2 diabetes mellitus at low cost and with efficiency.
Type 2 diabetes model in rats by a combination of high-fat diet and
streptozotocin treatment
(Srinivasan et al., 2005; Reed et al., 2000)

27. Objectives
• To develop consistent type 2 diabetic Wistar rat model
• To evaluate antidiabetic activity of Gymnema sylvestre,
Tribulus terrestris and ITK formulation in type 2 diabetic
rats.
• To asses therapeutic activity of Gymnema sylvestre,
Tribulus terrestris and ITK formulation in type 2 diabetes
induced cardiovascular disease

28. Technical Programme

29. Phase I
• Identification, collection & Extract preparation
• Phytochemistry
• Invitro antioxidant activity of Gymnema sylvestre, Tribulus terrestris, ITK
formulation
# 2,2-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging activity
# 2,2’-azino-bis-3 ethylbenzothiozoline-6-sulphonic acid(ABTS) method
(Bhardwaj et al.,2015)

30. • Invitro antidiabetic activity of Gymnema sylvestre, Tribulus terrestris, ITK
formulation
i) α glucosidase inhibition method (Srianta et al.,2013)
ii) α amylase inhibition assay (Arunachalam et al.,2013)
iii) In-vitro glucose uptake assay
Based on above observations an appropriate dose of plant extract will be
decided

31. Phase II
• Procurement of experimental animals from licenced breeder.
• Wistar rat
• Number of animals-70
• Body weight 180-200gms
• Sex-Male
• Maintained in 12hr light-dark cycle
*Temperature-22± 2°c
*Feed & water-adlib
Experiment will begin after approval from IAEC

32. Group
No.
Number of
animals Type of treatment Duration
Group 1 10 Negative control 60 days
Group 2 10 Positive control 60 days
Group 3 10 Diabetic rats + Gymnema sylvestre 60 days
Group 4 10 Diabetic rats + Tribulus terrestris 60 days
Group 5 10 Diabetic rats+ Gymnema sylvestre +
Tribulus terrestris
60 days
Group 6 10 Diabetic rats + ITK formulation 60 days
Group 7 10 Diabetic rats + Standard antidiabetic drug 60 days
After an acclimatization period of 7 days, rats will be grouped into following
groups

33. Development of Type II diabetic model
• Streptozotocin + High fat diet will be used .
• High fat diet can be either prepared in our lab by using normal pellet diet, raw
cholesterol and mixture of vanaspati ghee and coconut oil(2:1) or it may be
commercially procured.
(Suman et al.,2013)
• Streptozotocin dose will be decided based on pilot study or from available
literature and single standard dose of streptozotocin dissolved in 0.1M citrate
buffer administered by intra peritonial(I/P) route.
• Rats showing Plasma glucose concentration of >200mg/dl after 72 hours of STZ
injection will be considered as diabetic.
• Plasma glucose concentration will be measured at 0,3,15,30,45,60 days.

34. Parameters
• Daily feed and water intake
• Weekly body weight
• Thoracic and abdominal circumference at 15 days intervals.
• Hematobiochemical
i) Glucose
ii) Insulin
iii) Glycated hemoglobin(HbA1C)
iv) Gamma-glutamyltransferase (GGT)
V) Alanine aminotransferase (ALT)
• Lipid profile(LDL,HDL,TG,Cholesterol)
• Electro Cardiogram(ECG) will be done ,if possible

35. • Hemodynamic parameters - Systolic, diastolic, mean arterial blood pressure and
heart rate will be measured, if possible
• Oxidative stress in heart
i) Lipid peroxidatio(LPO)
ii) Glutathione peroxidase(GPx)
iii) Reduced glutathione(GSH)
iv) Catalase(CAT)
v) Superoxide Dismutase(SOD)
vi) Glutathione-S-Transferase(GST)
• Biomarkers of Cardiac injury
i) Creatinine Kinase-MB(CKMB)
ii) Cardiac troponin I
iii) Lactate dehydrogenase(LDH) using commercially available kits.
• Gene expression study
i) GLUT 4
ii) Endothelin-1
iii) Insulin receptor substrate(IRS)
iv) Angiotensin converting enzyme-2(ACE 2) using RT-PCR

36. • Western Blot
i) AKT
ii) PKC delta
• Histopathological examination
i) Aorta
ii) Heart
• Statistical analysis
The data obtained will be analyzed statistically for level of significance
and differences in the values .