Tan, Elaine D.Biochem 198 – Research Methods in Biochemistry

January 21, 2011Professor Luciana V. Ilao

Diabetes Mellitus and The Need for Alternative Therapies • Diabetes Mellitus Type 2:

– metabolic disorder in which the body has high blood sugar

– Insulin-resistant: insulin is needed to absorb glucose; thus blood glucose levels rise

• Has associated co-morbidities:– Cardiovascular diseases– Obesity

• Difficult to cure & requires expensive lifetime management

• Developing countries: need alternative therapies

Spices and Their Use In Developing Countries

• Spices: – collection of a wide variety of dried aromatic parts

of plants• Developing countries:

– spices are used to treat common ailments

However, these countries are not familiar with the biochemistry and pharmacology of these spices.

Flavonoids: Natural Substances in Herbs

• Belongs to a group of natural substances with variable phenolic structures

• 4000+ varieties identified, abundantly found in flowers, fruits and leaves

Flavonol

Review of Related Literature

In vitro, these Cameroonian spices show radical scavenging activity.

They also have a high polyphenolic content.

Flavonoids are direct radical scavengers and can therefore neutralize the harmful effects of free radicals in the body.

Review of Related Literature

In vitro, flavonoids were found to be competitive inhibitors of cytochrome P450.

In vitro, flavonoids were found to be potent topoisomerase II inhibitors.

Objectives:

To investigate and record the polyphenol content, anti-amylase, anti-lipase and antioxidant activities of nineteen different spices commonly used in cooking in different parts of Cameroon

Materials and Methods

Collection of plant materials

Preparation of aqueous extract

Determination of phenolic content (3x)

Determination of alpha-amylase I.A.

Determination of lipase I.A.

Antioxidant assay using ABTS (3x)

1.) Collection of Plant Materials

19 spices were collected

washed, sun-dried, ground into fine powder

seeds with husks:

1. with husk

2. without husk

kept and stored in dry, airtight container

The Plant Materials Used

2.) Preparation of Aqueous Extracts

2.5% (w/v) suspension was prepared

centrifuged

residue(discarded)

filtrate(was subject to succeeding tests)

3.) Determination of Phenolic Content

Folin – Ciocalteu Method(standard: catechin)

10 dilutions were made

1 mL Folin-Ciocalteu + 20 uL filtrate

30 minutes

Lambda Max = 750 nm

Concentration = CAE / gm

4.) Alpha-amylase Inhibitory Activity Starch-iodine Method

(Komaki, 2003)

20 uL alpha-amylase solution, 1.3 mL Tris-HCl buffer and 80 uL of the spice filtrate

Incubation time: 20 minutes, optimum temperature of 37 0C

100 uL of 0.1% starch, re-incubation (20 mins), 0.01% acidic iodine

Blank: Without filtrateControl: Without starch

Lambda Max: 450 nm

5.) Lipase Inhibitory Activity Assay

(Smeltzer, 1992)

Suspension:1% (v/v) of triolein and 1% (v/v) of Tween 40 in 0.1 M phosphate buffer with pH=8

emulsification

800 uL emulsion + 200 uL of porcine pancreatic lipase (0.033% (w/v) pancreatin in phosphate buffer at pH=8)

Blank: Without filtrateControl: Without suspension

Resulting mixture + 200 uL spice filtrate

Incubation Time: 30 minutes; Optimum Temperature: 37 0C

Lambda Max: 450 nm

1st reading: T0

2nd reading: T30

6.) Antioxidant AssayABTS assay or 2,2-azinobis (3-ethyl-

benzothiazoline-6-sulfonic acid)-(ABTS)(Re, 1999)

7 mM ABTS + 140mM potassium persulfate

Incubation time: 30 minutes, room temperature

1 mL activated ABTS + 50 uL spice filtrate

Control: Ethanol

Lambda Max: 734 nm

Results

• Phenolic Content

• Alpha-amylase Inhibitory Activity

• Lipase Inhibitory Activity

• ABTS Scavenging Activity

1.) Phenolic Content

Range: 2.06 – 60.75 mg CAE / g extract

Table 1. Spice Filtrate and Its Corresponding Phenolic Content

Note:

High Phenolic Content

Low Phenolic Content

Phenolic Content and The Folin-Ciocalteu Method

• Folin-Ciocalteu Reagent– composed of phosphomolybdate and

phosphotungstate

• Reacts with reducing substances– Reducing substances: include phenols and non-

phenols

• Chromogens are formed – color dev’t due to transfer of electrons at basic pH to

reduce phosphomolybdic/phosphotungstic acid complexes

• Spectrometrically detected

The Limitations of The Folin-Ciocalteu Method

• Detects all reducing substances, not just phenols

• Phenolic content– Neither equal nor equivalent to flavonolic

content

phenol Flavonol

2.) Alpha-amylase Inhibitory Activity

Classification % Inhibition

High Potency 75-100%

Moderate Potency 50-75%

Low Potency 25-50%

No Potency 0-25%

T

Figure 1. Spices and Potency of Alpha-amylase Inhibitory Activity

No Potency

Low Potency

ModeratePotency

High Potency

17.39%

26.09%30.43%

26.09%

What is alpha-amylase?

• E.C. 3.2.1.1

• Cleaves internal alpha-1,4-linkages in starch to release glucose

Why inhibit alpha-amylase?

• Inhibiting alpha-amylase decreased release of glucose from starch

• As the release of glucose is decreased, glucose levels in the blood decrease

How is alpha-amylase inhibited by the spices?

• Starch blockers: substances that prevent dietary starch from being absorbed by the body

• Allosteric Inhibition– Regulation of an enzyme by binding an

effector molecule at a proteins’s allosteric site

– When inhibitor binds, conformational change occurs, active site is altered, catalysis does not occur

3.) Lipase Inhibitory Activity

Classification % Inhibition

High Potency 75-100%

Moderate Potency 50-75%

Low Potency 25-50%

No Potency 0-25%

T

Figure 2. Spices and Potency of Lipase Inhibitory Activity

No Potency

Low Potency

ModeratePotency

High Potency

Not known

69.57%

17.23%%

4.35% each

What is lipase?

• Catalyzes the hydrolysis of TAGs at C1 and C3 to yield 2-monoacylglycerols and free FA

Why inhibit lipase?

• Lipase inhibition– Prevents the hydrolysis of dietary fat in the form of

triglycerides into absorbable free fatty acids and monoacylglycerols

• Decrease of systemic absorption of dietary fat– Caloric deficit

• Caloric deficit– Lower risk of CV diseases, lower risk of obesity

How is lipase inhibited by the spices?

Lipase “Catalytic Triad”Active Site: Asp, His and SerActs cooperatively to provide the necessary acid and base catalysis

Table 2. Spice Filtrate and Corresponding Alpha-amylase and Lipase Inhibition

4.) Antioxidant Activity

Range: 8.09 – 90.55% Inhibition

Table 3. Spice Filtrate and Its Corresponding % ABTS Inhibition

ABTS.+

• ABTS.+:– Radical cation

• Antioxidant activity– Inhibition of ABTS.+

How do flavonols act as antioxidants?

Conclusions

• Folin-Ciocalteu Method: – a crude method of determining the

polyphenolic content

• Alpha-amylase inhibition: – Satisfactory: 26.09% are highly potent

• Lipase inhibition: – Poor: 69.57% are not potent

• Antioxidant Activity:– Only 4 of 23 filtrates contributed significantly

Recommendations

• Instead of using Folin-Ciocalteu, use mass spectrometry instead.

• Deduce the active components of the spices.

• Make further studies on the use of flavonoids – bioavailability, safety and toxicity.

Thank you!