Tan, Elaine D.

Biochem 198 – Research Methods in Biochemistry

January 21, 2011
Professor 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 Determination of alpha-
materials amylase I.A.

Preparation of Determination of lipase

aqueous extract I.A.

Determination of Antioxidant assay using

phenolic content (3x) 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 filtrate
(discarded) (was subject to
succeeding tests)
3.) Determination of Phenolic
Content Lambda Max = 750 nm
Folin – Ciocalteu Method
(standard: catechin)

10 dilutions were made

1 mL Folin-Ciocalteu + 20 uL filtrate

30 minutes

Concentration = CAE / gm
 4.) Alpha-amylase Inhibitory Activity
Blank: Without filtrate
Starch-iodine Method Control: Without starch
Lambda Max: 450 nm
(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

 5.) Lipase Inhibitory Activity
Blank: Without filtrate
Assay Control: Without suspension
Lambda Max: 450 nm
(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)

Resulting mixture + 200 uL spice filtrate

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

1st reading: T0
2nd reading: T30
 6.) Antioxidant Assay
Control: Ethanol
ABTS assay or 2,2-azinobis (3-ethyl-
benzothiazoline-6-sulfonic acid)-(ABTS)
(Re, 1999) Lambda Max: 734 nm

7 mM ABTS + 140mM potassium persulfate

1 mL activated ABTS + 50 uL spice filtrate

Incubation time: 30 minutes, room temperature

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

No Potency
26.09% 17.39%
Low Potency

Moderate
T
26.09% Potency
30.43% High Potency
 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%
Figure 2. Spices and Potency of Lipase Inhibitory Activity

No Potency
4.35% each
Low Potency

17.23%% Moderate
Potency
69.57%
T
High Potency

Not known
 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 Ser
Acts 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!