Upload
vwr-international
View
1.343
Download
4
Embed Size (px)
Citation preview
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!