Anti-diabetic Assays

Presented By:

- Ritesh Bhagea

- Rouksaar Buctowar

- Huda Nazeer

- Christabelle Cécile

- Keshavi Ghoorbin

Plant Biotechnology

BSc (Hons) Biotechnology

University of Mauritius

25.09.14

Contents

❏ Background

❏ Introduction

❏ Literature review

❏ Methodology

❏ Advantages

❏ Disadvantages

❏ Prospects

❏ Conclusion

Background

❏ Diabetes mellitus (DM) - rapidly emergingas global health problem

❏ Could reach pandemic levels by 2030

❏ One of the oldest common metabolic disorders affecting the whole body system

❏ Continues to be a leading cause of morbidity and mortality for the foreseeable future

❏ Essential to look for effective anti-diabetic agents to treat DM

❏ Currently available anti-diabetic agents: sulfonylureas, biguanides, thiazolidinediones and α-amylase and α-glucosidase inhibitors as well as medicinal plants

Introduction❏ DM is a chronic metabolic disorder of carbohydrate, fat and

protein metabolism, resulting from insulin deficiency

❏ Characterized by abnormal increase in the blood sugar level❏ Pathogenic processes in development of diabetes:

❏ autoimmune destruction of β-cells of pancreas❏ abnormalities in metabolisms

❏ There are 2 types of DM:❏ Type 1 - juvenile-onset or insulin-dependent diabetes❏ Type 2 - adult-onset or non–insulin-dependent diabetes

Introduction❏ Signs and symptoms:

❏ Weight loss❏ Blurred vision❏ Hypotension❏ Polyuria - production of

abnormally large volumes of dilute urine

❏ Polydipsia - abnormally great thirst

❏ Polyphagia - excessive eating or appetite

❏ Tachycardia - abnormally rapid heart rate

Literature Review❏ According to the International Diabetes Federation: [Surya et

al., 2014]

❏ There are currently 371 million people living with diabetes

❏ 28 million people are at high risks

❏ As of 2010, around 280 million people had diabetes globally

(Type 2)

❏ Diabetes is more common in developed countries, where

there is an increase in longevity and obesity

❏ Environmental and genetic factors play an important role in

the development of diabetes

❏ Bergenia crassifolia

Literature Review

❏ Syzygium cumini

As aforementioned, some medicinal plants and their phytochemicals have anti-diabetic properties and these include:

Literature ReviewBergenia crassifolia (L) Fritsch:

❏ From Saxifragaceae family

❏ Used as medicinal and ornamental plant

❏ Contains arbutin, tannins, bergenin as main

phytochemicals compounds,

❏ Ethnopharmacology → Rhizomes infused

❏ Cold,

❏ Gastritis,

❏ Bleeding gums

❏ Used as a beverage in Russia

Literature Review

Bergenia crassifolia (L) Fritsch (cntd)

❏ Has different effective compounds [Shikov et al.,2014]:

❏ Anti-cancer

❏ Anti-hypertensive

❏ Anti-microbial

❏ Anti-diabetic

Bergenin found to reduce BGL in normal rats which

were subjected to oral glucose tolerance test.

Literature Review

Syzygium cumini:

❏ From Myrtaceae family

❏ Widely used in traditional and folk medicine

❏ Leaves, fruits and seeds used to treat diabetes,

pharyngitis and ringworm infections

❏ Bark and seed extracts have shown to have

inflammatory, anti-bacterial and anti-diarrheal effects

❏ Contains vitamin C, gallic acid, tannins, malvidin-

glucoside and others.

Literature ReviewSyzygium cumini (L.):❏ 4 compounds with great anti-diabetic properties were

found (Alam et al., 2012):

❏ Stigmasterol: has significant effect on lowering serum

glucose concentration with increase in insulin level

❏ Lupeol: suppress the progression of diabetes. Serum

insulin level is elevated with lupeol treatment.

❏ 12-oleanen-3-ol-3ß-acetate

❏ ß-sitosterol

Literature Review

Ulva lactuca Sargassum polycystum

Gracilaria edulis Gracilaria corticata

❏ Seaweeds: Ulva lactuca, Sargassum polycystum, Gracilaria edulis and Gracilaria corticata

Literature ReviewSeaweeds: Ulva lactuca, Sargassum polycystum, Gracilaria edulis,and Gracilaria corticata

According to Kumar and Sudha (2012):❏ Contain inhibitory substances❏ Affect enzymes α-amylase and α-glucosidase❏ Inhibitors prevent liberation of glucose from dietary complex

carbohydrates❏ Glucose absorption is delayed and postprandial hyperglycemia

is suppressed❏ Glucose levels do not shoot up to extreme peaks in diabetic

people❏ Therefore is a good therapeutic pathway to treat diabetes

(decrease postprandial hyperglycemia)

Two assays used to measure the biological activity of a sample:

1) Alpha-amylase inhibition assay

2) Alpha-glucosidase inhibition assay

Aqueous extracts of four seaweeds collected from Gulf of Mannar coastal

waters were tested using those 2 assays (SenthilKumar and Sudha,

2012):

➔ Extraction of seaweeds were done with water using the Soxhlet

apparatus.

➔ Crude extracts were concentrated under reduced pressure to get the

corresponding residues.

Methodology

Methodology

Alpha-amylase enzyme

1. Alpha-amylase inhibition assay1. Starch azure (2 mg) was suspended into tubes containing 0.2 ml of 0.5M

Tris-HCl buffer (pH 6.9) and 0.01 M CaCl2

2. Tubes boiled for 5 min

3. Incubate at 37oC for 5 min

4. Seaweed extract (0.2 ml) in each tube at different concentrations (10,

20, 40, 60, 80, 100 microgram/ml) of dimethyl sulfoxide (DMSO)

5. PPA dissolved in Tris-HCl buffer forming a conc. of 2 units/ml in another

tube

6. 0.1ml of PPA solution was added to the 6 different conc. of dimethyl

sulfoxide tubes

7. Reaction carried out at 37oC for 10 min

Methodology

8. Reaction stopped by addition of 0.5ml acetic acid in each tube

9. Reaction mixture centrifuged at 3000 rpm for 5 minutes at 4oC

10. Absorbance of supernatant measured at 595 nm

Calculation of alpha-amylase inhibitory activity:

= [(Ac+) - (Ac-)] – [(As- Ab)] / [(Ac+) - (Ac-)] x 100

Where;

Ac+ = the absorbance of 100% enzyme activity (only solvent with enzyme)

Ac- = 0% enzyme activity (only solvent without enzyme)

As = Test sample (with enzyme)

Ab = A blank (a test sample without enzyme).

Methodology

Methodology

Alpha- glucosidase

2. Alpha-glucosidase inhibition assay

1. Alpha-glucosidase reaction mixture contained:

- 2.9 mM pNPG

- 0.25 ml of seaweed extract (varying conc.) in DMSO and baker’s yeast

alpha-glucosidase in sodium phosphate buffer (pH 6.9)

2. Reaction mixtures incubated at 25oC for 5 min

3. Reaction stopped by boiling for 2 min

4. Absorbance of the resulting p-nitrophenol (pNP) was determined at 405

nm and was considered directly proportional to the activity of the enzyme

Control: DMSO, enzyme and substrate

Positive control: Seaweed extract is replaced by acarbose

Blanks: DMSO

Methodology

Calculation of glucosidase inhibition activity as percentage of

control:

= 100% - % activity of test as percentage control

Where;

% Activity of test = (corrected A405 of test x100%) / A405 of controls

Where;

corrected A405 test samples = A405 extract and substrate mixture - A405 extract

alone

Methodology

Advantages

Alpha glucosidase inhibition:

❏ Alpha glucosidase inhibitors lower the activity of alpha

glucosidase and thus decreasing blood sugar level

Alpha amylase inhibition:

❏ inhibitors also lower the breakdown of long chains of

carbohydrates

Disadvantages

Alpha-glucosidase inhibition:

❏ inhibitors like acarbose and voglibose cause severe gastrointestinal side effects as they prevent digestion of carbohydrates which are further broken down by bacteria

❏ may result in hypoglycemia (diminished level of blood glucose)

Prospects

1. Aqueous extract of Terminalia paniculata bark:❏ Has the potential to reverse

insulin resistance in type-2 diabetes (Subramaniam et al., 2013).

2. Aqueous extract from Actinidia kolomikta root:❏ Ability to decrease the

postprandial hyperglycemia (Xuansheng et al., 2013).

Prospects

3. Ghrelin O-acyltransferase (GOAT):

❏ Effective therapy for type-2 diabetes.

4. Extract of Hintonia latiflora (Vierling et al.,

2014):

❏ Has the ability to reduce blood

glucose levels

❏ Can also cause vasodilation which

promotes decrease in BGLs

Conclusion❏ Diabetes mellitus is one of the major health and development

challenges of the 21st century

❏ Many medicinal plants have anti-diabetic properties to treat

DM

❏ Two assays that can be used to measure the biological

activity are the α-amylase and α-glucosidase inhibition

assays

References❏ Alam. (2012). Evaluation of antidiabetic phytochemicals in Syzygium

cumini (L.)Skeels (Family: Myrtaceae). Journal of Applied Pharmaceutical Science.

❏ Senthilkumar, P. and Sudha, S. (2012). Evaluation of alpha-amylase and alpha- glucosidase inhibitory properties of selected seaweeds from Gulf of Mannar. International Research Journal of Pharmacy, 3 (8), pp: 128-130.

❏ Shikov, A. N., Pozharitskaya, O. N., Makarova, M. N., Makarov, V. G., and Wagner, H. (2014). Bergenia crassifolia (L.) Fritsch – Pharmacology and phytochemistry. Phytomedicine, 21(12), 1534–1542.

❏ Surya, S., Salam, A. D., Tomy, D. V., Carla, B., Kumar, R. A., and Sunil, C. (2014). Diabetes mellitus and medicinal plants-a review. Asian Pacific Journal of Tropical Disease, 4(5), 337–347.

❏ Vierling, C., Baumgartner, C. M., Bollerhey, M., Erhardt, W. D., Stampfl, A., and Vierling, W. (2014). The vasodilating effect of a Hintonia latiflora extract with antidiabetic action. Phytomedicine, 21(12), 1582–1586.

Thank you for your attention!

An apple a day keeps the doctor away!