Solvents and reagents used - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/12880/11/11_chapter...

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CHAPTER 5 EXPERIMENTAL

• Solvents and reagents used:

All the reagents used in the assay procedures were of analytical reagent grade

purchased from S. D. Fine Chemicals Ltd. Mumbai and analytical grade solvents

were purchased from Rankem, India.

Pre-coated HPTLC plates of silica gel GF254 were obtained from E-Merck,

Germany. Reference catechin was purchased from Sigma–Aldrich (Germany).

HPLC grade and analytical grade solvents were obtained from Merck (Mumbai,

India). Silica gel GF254 was obtained from (E-Merck, Germany). Reference

catechin was purchased from Sigma–Aldrich (Germany).

HPLC grade solvents were obtained from Merck (Mumbai, India).

Sodium nitroprusside, sulphanilic acid, α-naphthyl-ethylene diamine,

trichloroacetic acid (TCA), and DPPH (1,1-diphenyl-2-picrylhydrazyl), were used

from Sigma Aldrich (USA). Ferrous sulphate (FeSO4) and acetic acid were

obtained from S.D.Fine Chemicals, Mumbai. Folin–Ciocalteau reagent was

purchased from Fluka analyticals, Switzerland.

MCDB medium, Fetal Bovine serum, L-glutamine, and penicillin/streptomycin

mixture were obtained from GIBCO, Invitrogen (Grand Island, NY, USA). ROS

assay dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate,

acetyl ester (CM-H2DCFDA), trypsin (0.05%), tryphan blue stain, Endothelial

Cell Growth Medium (EGM-2) single Quots, and Endothelial Basal Medium-2

(EBM-2) were also purchased from Invitrogen. WST-1 dye (cell proliferation

reagent) was obtained from Roche Diagnostics (Indianapolis, USA).

Quant – iT protein quantification kit, EGM-2 single Quots, EBM-2 were also

purchased from Invitrogen. Page Ruler prestained protein was obtained from

Roche Diagnostics (Indianapolis, USA).

PCR primers for NF-E2-related factor-2 (Nrf2), NAD(P)H:quinone

oxidoreductase-1 (NQO1), heme oxygenase-1 (HMOX1), glutamate–cysteine

ligase catalytic subunit (GCLC) and glutamate–cysteine ligase regulatory subunit

(GCLM) were purchased from Invitrogen. Antibodies against Nrf2, were from

SantaCruz Biotechnology (Santa Cruz, CA, USA).

STZ was purchased from Sigma Aldrich, Germany, trisodium citrate and

nicotinamide, were purchased from Merck, India. The pellet diet was obtained

from Amrut animal feed suppliers, Mumbai, India.

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Avicel PH-102 (directly compressible micro crystalline cellulose- MCC) and

dibasic calcium phosphate (DCP) were purchased from FMC Biopolymers, India.

Talc was purchased from Norwegian GmbH, Germany and croscarmellose was

purchased from Maple Biotech Pvt. Ltd., India.

• Instruments and equipments used

UV- Visible spectrophotometer, Jasco V-530; Roche tablet friabilator, Dissolution

tester USP (XXIII) - Electrolab TDT-06T, Monsanto tablet Hardness tester -

Campbell electronics, Mumbai. Vernier Caliper-Mitutoyo, Japan were used in

analysis of tablet.

HPTLC studies were carried out using CAMAG LINOMAT 5 applicator and

CAMAG SCANNER III with WINCATS III software.

The HPLC analysis was done on a TOSOH-CCPM system equipped with UV-

Visible detector.

Chemwell Auto analyser supplied by Awareness Tech was used for analysis of

serum.

Densitometric analysis of western blotting was carried out with ChemiDoc XRS

(Bio-Rad). MyIQ PCR system (Bio-Rad, Hercules, CA) and MyiQ System

Software, Version 1.0.410 (Bio-Rad Laboratories Inc.) was used for PCR analysis.

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5.1 PROCUREMENT AND AUTHENTICATION OF PLANT MATERIAL

5.1.1 Collection of plant material

• Leaves:

The leaves of Anacardium occidentale L. (Cashew) were collected from

Tungareshwar forests of Vasai taluka, Dist. Thane; Maharashtra, India. The fresh

mature green leaves were collected in the month of January, 2008. The collection

site is geographically located at an altitude of 2177 ft. on the map of India.

• Testa:

The testa (Cashew nut skin) samples were obtained from a small scale cashew

manufacturing unit in Sawantwadi region of Sindhudurg, Maharashtra, India.

Sawantwadi is located at an altitude of 690m above sea level on the map of India.

The map indicating the geographical location of plant specimen collection is

depicted in Figure 5.1.

5.1.2 Authentication of Plant Material

� Preparation of Herbarium:

• In order to assist in accurate identification and to provide a species record a

herbarium specimen of cashew was prepared. The specimen material selected for

the purpose essentially consisted of fruit, seed, flowers, leaves and stem so that the

pattern of branching, leaf arrangement, and other features are readily discernible.

• The plant specimen was preserved by fixing the tissues in a preparation of

formalin-acetic acid-alcohol (Dikison, 1986 and Smith, 1971). The composition of

formalin-acetic acid-alcohol solution used for fixing is mentioned in Table 5.1.

Table 5.1: Composition of solution used for fixation of plant material

Solvent/reagent Volume in 100 ml of the mixture

Ethyl alcohol (70%) 90.0 ml

Formalin (commercial strength) 5.0 ml

Glacial Acetic acid 5.0 ml

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Figure 5.1: Geographical location of the region of plant collection

Source: Google - Map data ©2011 Basarsoft, Europa Technologies, Geocentre

Consulting, MapIT, SK M&C, Tele Atlas

• The plant specimen was dipped in the fixing solution for about 3 hours and then

dried and pressed between layers of newspapers.

• This fixed and dried specimen was then mounted on cardboard and packed with a

transparent gelatin sheet so as to expose certain characters advantageously (e.g.

upper and lower surfaces of the leaves being exposed, flowers to show as many

surfaces or views as possible thereby reducing the need for dissection of the

finished specimen). The prepared herbarium was then submitted for

authentication.

• The botanical identity of the plant specimen was confirmed by a taxonomist at

Department of Botany, Botanical Survey of India, Pune; (M.S).

• A voucher specimen number YOGA1/No.BSI/WC/Tech/2008/69, was

obtained. A copy of the authentication certificate is attached as Appendix - I.

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5.2 STANDARDIZATION OF PLANT MATERIAL

5.2.1 Introduction

The Ayurvedic system of medicine has been prevalent in India since a number of

decades, and still remains the mainstay of medical relief to over 60 per cent of the

population of the nation. In earlier times, the practitioners of Ayurveda collected

herbs and other ingredients for preparing medicines by themselves. For the

purpose of acquiring raw materials the Ayurvedic practitioners now depend on

commercial organizations trading in crude herbal drugs. Since past few decades a

number of Ayurvedic pharmaceutical units have come up for the manufacture of

Ayurvedic drugs and formulations on commercial scale.

Under the circumstances and responding to opinions of the scientific community,

the Govt. of India began a series of measures to introduce a quality control system

for western medicine. The Government of India introduced an amendment in 1964

to the Drug and Cosmetics Act 1940, to control to a limited measure the

Ayurvedic, Siddha and Unani drugs. Gradually, the development of standards for

the identity, purity and strength of single drugs and those of formulations at a later

stage, assumed importance for the effective enforcement of the provision of the

Act. If the raw materials to be used in a medicine and stage-by-stage processes of

manufacturers are standardized, the final product namely, the compound

formulation could be expected to conform to uniform standards. Arrangements to

evolve and lay down physical, chemical and biological standards, wherever even

necessary, to identify the drugs and ascertain their quality and to detect

adulterations are an urgent necessity of the health care related profession

(Harbone, 1998).

Ayurvedic, Unani and Homoeopathic Pharmacopoeias published by the Govt. of

India have prescribed various standards to be followed for herbal drugs. In 2002

Govt. of India published Good Laboratory Practices (GLP) guidelines to guide the

drug analysts in maintaining high scientific and professional standards for

ensuring that, only drugs of the highest quality are produced and marketed. In

2003, government issued notification of Good Manufacturing Practices (GMP) to

ensure authentic, contamination free quality raw material, manufacturing process

and product with desired quality standards.

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5.2.2 Background of the study

Standards for quality control of herbal products are based on pharmacognostic,

physicochemical, phytochemical and biological parameters. Although cashew has

been explored for varied pharmacological and phytochemical investigations, there

have been no reports based on the standardization of cashew (Konan, 2007; Abas,

2006; Kudi, 1999; Goncalves, 2005 and Kamtchouing, 2001).

5.2.3 Tests for phytochemical analysis

The process and parameters employed in standardization of cashew leaves and

testa are described below: (The Ayurvedic Pharmacopoeia of India, 2008;

Khandelwal, 1999; Trease, 1983 and Harbone, 1998)

A. Sampling of plant material:

• The leaves obtained after collection were washed, cleansed and made free of any

foreign material. Only mature green leaves were selected for further processing

and kept in shade until dried.

• The dried testa obtained was also cleansed manually and made free of any foreign

material.

• The plant materials were then crushed to coarse powder mechanically, sieved

through sieve no. 44 and stored in air tight containers and used for further

analysis.

B. Identification Tests

a) Organoleptic characterization:

• In order to determine the organoleptic characters of the drug, the colour, odour

and taste of the plant material were estimated by visual and sensory evaluation.

b) Macroscopic characteristics:

• To study the macroscopic characters of fresh leaves the following characteristics

were noted: size and shape, colour, surfaces, venation, presence or absence of

petiole, the apex, margin, base, lamina, texture, odour and taste.

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c) Microscopic analysis and powder characteristics:

• Microscopic analysis shows the unit structures in distinct manner and helps to

draw conclusions about the drug characteristics. The recognition of discrete and

disoriented tissue components helps to ascribe them to their correct source.

• In order to perform the microscopy studies of cashew leaves the green fresh

mature leaves were boiled in chloral hydrate solution. The sections of treated

leaves were stained with phloroglucinol and concentrated HCl and mounted with

glycerin and observed under a compound microscope with suitable magnification

(Tatke, 2009).

• Powder characteristics of the material were studied by making it free from any

cellular debris by treatment with suitable reagents. A part of the treated material

was then mounted upon slides and observed under the microscope.

• Fluorescence characters of powdered plant material with different chemical

reagents such as phluroglucinol were determined under ordinary and ultraviolet

light.

C. Physicochemical analysis

a) Determination of Moisture Content (Loss on Drying):

• The procedure mentioned below is used for substances appearing to contain water

as the only volatile constituent.

• About 10.0 g of drug (i.e. leaves and testa powder) (without preliminary drying)

were placed separately after accurately weighing in tared evaporating dishes.

• After placing the above said amount of the drugs in the tared evaporating dishes,

the drugs were dried at 1050 in a Hot Air Oven for 5 hours and weighed.

• The drying and weighing procedure was continued at one hour interval until

difference between two successive weighing corresponded to not more than 0.25

per cent w/w.

• When two consecutive weighings after drying and cooling for 30 mins intervals in

a desiccator, showed not more than 0.01 g difference in weight, then it was

considered that a constant weight.

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b) Determination of total ash:

• Ash is the inorganic residue remaining after the water and organic matter have

been removed by heating in the presence of oxidizing agents, which provides a

measure of the total amount of minerals within a sample. The most widely used

methods are based on the fact that minerals are not destroyed by heating, and that

they have a low volatility compared to other components.

• The total ash was determined by incinerating about 2.0 g accurately weighed

powdered drug in a silica dish. The heating was performed at a temperature not

exceeding 4500 in a muffle furnace until free from carbon.

• The contents were then cooled and the charred mass was exhausted with hot

water. The residue was collected on an ashless filter paper and the residue and

filter paper were incinerated.

• The filtrate was added to the incineration of filter paper and residue, evaporated to

dryness, and ignited at a temperature not exceeding 4500

C in a muffle furnace.

• The percentage of ash with reference to the air-dried drug was calculated.

c) Determination of acid-insoluble ash:

• To the crucible containing 1.0 g of total ash, 25.0 ml of dilute hydrochloric acid

was added. The insoluble matter was collected on an ashless filter paper

(Whatman 41) and washed with hot water until the filtrate was neutral.

• The filter paper containing the insoluble matter was transferred to the initial

crucible, dried on a hot-plate and ignited to constant weight. The residue was

allowed to cool in a desiccator for 30 minutes and weighed immediately. The

content of acid insoluble ash with reference to the air-dried drug was calculated.

d) Determination of water soluble ash:

• About 1.0 g of the ash obtained from total ash value determination was boiled for

5 minutes with 25 ml of water.

• The insoluble matter was collected on an ashless filter paper, washed with hot

water, and ignited for 15 minutes at a temperature not exceeding 4500 C in a

muffle furnace.

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• The weight of the insoluble matter from the weight of the ash was subtracted. The

difference in weight represents the water-soluble ash. The percentage of water-

soluble ash was calculated with reference to the air-dried drug.

e) Determination of sulphated ash:

• A silica crucible was heated to redness for 10 minutes, and allowed to cool in a

desiccator and weighed. About 2.0 g of the substance was accurately weighed,

placed into the crucible, and ignited until the substance was thoroughly charred.

• The crucible was then cooled, and the residue was moistened with 1.0 ml of

sulphuric acid.

• The crucible was then heated again until white fumes no longer evolved and the

residue was ignited at 8000

C ± 25

0 C until all black particles disappeared.

• The crucible was allowed to cool, and few drops of sulphuric acid were added to it

and heated. The ignition procedure was repeated as before, until two successive

weighing did not differ by more than 0.5 mg.

f) Determination of alcohol soluble extractive:

• About 5.0 g of the coarsely powdered air dried drug, was macerated with 100 ml

of alcohol in a closed flask for twenty-four hours, shaking frequently during six

hours and allowed to stand for eighteen hours.

• The contents were filtered, and from the total volume of solvent, 25.0 ml of the

filtrate was evaporated to dryness in a tared flat bottomed shallow dish, and dried

at 1050 C, to constant weight.

• The dish was then weighed and the percentage of alcohol soluble extractive with

reference to the air-dried drug was calculated.

g) Determination of water soluble extractive:

• The procedure performed for the determination of water soluble extractive was

same as that of alcohol-soluble extractive, except for the solvent used was

chloroform-water instead of ethanol.

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h) Determination of ether soluble extractive (fixed oil content):

• About 100.0 g of the air dried, coarsely powdered drug was transferred to an

extraction thimble and extracted with 500.0 ml of solvent ether in a continuous

extraction apparatus (Soxhlet extractor) for 6 hours.

• The extract was filtered and a 10.0 ml of the extract was transferred to a tared

evaporating dish. The solvent was evaporated off on a water bath and the residue

was dried at 1050 C to constant weight.

• The percentage of ether soluble extractive with reference to the air-dried drug was

calculated.

i) Determination of pH values:

• The pH value of a filtrate obtained from 1% w/v suspension of the drugs in water

was determined potentiometrically by means of a digital pH meter.

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5.3 EXTRACTION OF PLANT MATERIAL

5.3.1 Introduction

Extraction, as the term is used pharmaceutically, involves the separation of

medicinally active portions of plant or animal tissues from the inactive or inert

components by using selective solvents in standard extraction procedures. The

purposes of standardized extraction procedures for crude drugs are to attain the

therapeutically desired portion and to eliminate the inert material by treatment

with a selective solvent known as ‘menstruum’. The extract thus obtained can be

used as a medicinal agent in the form of tinctures and fluid extracts, it can be

further processed to be incorporated in any dosage form such as tablets or

capsules, or it can be fractionated to isolate individual chemical entities. Thus,

standardization of extraction procedures contributes significantly to the final

quality of the herbal drug (Handa, 2008 and Tandon, 2008).

5.3.2 Background

Based upon the literature survey for cashew and the pharmacological activity

envisaged in the project, the solvents were selected for each of the plant part, viz.

testa and leaves of cashew.

5.3.3 Methodology

Based upon the nature of the solvents conventional and Microwave assisted

extraction technique was also applied for extraction of leaves and testa with

various solvents. A comparison of the conventional extraction technique i.e.

Soxhlet extraction and Microwave Assisted Extraction Process (MAEP) was

carried out based upon the extractive yields of extracts. The further processing

(i.e. drying and concentration of the extract) was based upon the nature of the

solvent used. The drug : volume of solvent ratio was optimized so as to obtain

maximum extractive yield.

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A. Hot Continuous Extraction (Soxhlet Extraction)

a) Principle:

• In this method, the finely ground crude drug is placed in a porous bag or

“thimble” made of muslin cloth, and is placed in the Soxhlet apparatus. The

extracting solvent in flask is heated, and its vapors condense in condenser. The

condensed extractant drips into the thimble containing the crude drug, and extracts

it by contact. This process is continuous and is carried out until a drop of solvent

from the siphon tube does not leave residue when evaporated, indicating

completion of the extraction process.

• The advantage of this method is that large amounts of drug can be extracted with a

much smaller quantity of solvent. This affects economy in terms of time, energy

and consequently financial inputs (Tandon, 2008).

b) Procedure:

• The dried and coarsely powdered drug, was passed through sieve no. 44 was used

for extraction.

• The temperature range for extraction was 40-450C using a calibrated heating

mantle for heating.

• The drug was continuously extracted for a period of 18 hours and the resultant

solution was filtered. The marc was discarded and the filtrate was concentrated on

a rotary evaporator under vacuum.

• Several ratios of drug: solvent ratios were used to optimize the extraction

procedure. Drug: solvent ratios of 1:1, 1:3 and 1:5 were tried for leaves and testa

in order to obtain maximum extractive yield.

• The ratio of 1:5 and 1:3 (drug: solvent) was found to give maximum yield for

cashew leaves, and testa, respectively. These optimized proportions were used for

further extractions.

• Ethanol extract of leaves and ethanol and methanol extract of testa were prepared

by this process.

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B. Decoction

a) Principle:

In this process, the crude drug is boiled in a specified volume of water for a

defined time; it is then cooled and strained or filtered. This procedure is suitable

for extracting water-soluble, heat-stable constituents. The starting ratio of crude

drug to water is fixed. The filtrate obtained is then concentrated and used further.

b) Procedure:

• The dried and coarsely powdered drug, passed through sieve no. 44 was extracted

at 40-450C in a round bottom flask with distilled water as the solvent for

extraction.

• The drug was continuously extracted for a period of 3 hours and the resultant

solution was filtered through muslin cloth and then through filter paper to avoid

any suspended particles in the extract.

• The marc was discarded and the filtrate was concentrated by lyophilisation.

• The drug: solvent ratio of 1:3, was found to be optimum for extraction of leaves

and testa by decoction to obtain the maximum extractive yield.

• Aqueous extract of leaves and aqueous extract of testa were prepared by this

process.

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C. Microwave-assisted Extraction

a) Principle:

• Microwave radiation interacts with dipoles of polar and polarizable materials.

Polar molecules try to orient in the changing field direction and hence get heated.

In non-polar solvents without polarizable groups, the heating is poor (dielectric

absorption only because of atomic and electronic polarizations).

• This thermal effect is practically instantaneous at the molecular level but limited

to a small area and depth near the surface of the material. The rest of the material

is heated by conduction.

• In microwave-assisted extraction (MAE) the extraction takes places by five basic

steps:

i) The heat of the microwave irradiation being directly transferred to the solid

without absorption by the microwave-transparent solvent;

ii) The intense heating in step 1 causing instantaneous heating of the residual

microwave - absorbing moisture in the solid;

iii) The heated moisture evaporates, creating a high vapor pressure;

iv) The vapor pressure generated by the moisture breaks the cell; and

v) Breakage of cell walls releases the trapped constituents within it.

• The major advantages of microwave heating are increased extraction / recovery,

reduced processing costs, significantly faster extraction, lesser energy usage, and

less solvent consumption.

b) Procedure:

The coarsely ground powders of leaves and testa were extracted with methanol

and water as extracting solvents in a microwave synthesizer at low (140 Watts)

and high power (700 Watts). The drug:solvent ratio used for extraction of testa

and leaves was 1:2. The mass thus obtained after extraction was filtered,

concentrated and dried.

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c) Optimization of Microwave-assisted extraction (MAE)

• Influential parameters of MAE namely, microwave power, irradiation time, and

amount of extracting solvent were studied for optimization of extraction protocol.

The experiments were carried out in triplicates and the results were represented as

Mean ± SEM.

• The experiments were carried out separately for leaves as well as testa powder.

However, the extractive yield of testa did not show any change in the extractive

yield as compared to the conventional processes. Whereas, the leaves of cashew

exhibited considerable change in the extractive yields and thus was used for

further experiments.

• Aqueous and methanol extracts of leaves of cashew were prepared by MAE.

• These extracts were further analysed for total phenolics and catechin content to

estimate the effect of microwave irradiation on the same.

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D. Preparation of Polyphenol fraction

Polyphenols have been reported to be potent anti-oxidants (Larrauri, 1997). The

testa and leaves of cashew contain considerable amount of polyphenols which also

exhibit strong antidiabetic properties (Sabu, 2002). Hence, an attempt was made to

separate the phenolic and tannin fraction from the whole extract.

a) Procedure:

• Ethanol extract of leaves and ethanol extract of testa of Cashew were used for

extraction of polyphenols and the following procedure was followed for the same

(Figure 5.2). The presence of polyphenols in the extracted Chloroform layer was

confirmed by blue and black colored spots after derivatising with FeCl3 reagent.

Extract + Methanol:Water (4:1)

Filter

Residue

Filtrate

Discard

Evaporate to 1/10 volume

Acidify with 2M sulphuric acid and extract with chloroform

Chloroform Layer Aqueous acid Layer

Wash {Chloroform:Methanol} (3:1)

Polar phenolics Treatment with NaOH

Chloroform layer Aqueous basic layer

Alkaloids N-Oxides/Quarternary

Alkaloids

Figure: 5.2 Schematic representation of extraction of polyphenol fraction from

extracts

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5.4 PRELIMINARY PHYTOCHEMICAL SCREENING OF EXTRACTS

5.4.1 Introduction

Phytochemicals are non-nutritive secondary plant metabolites that have protective

or disease preventive properties. There are many known phytochemicals quoted in

scientific publications. It is well-known that plants produce these chemicals to

protect themselves but recent research demonstrates that these phytochemicals can

protect not only plants but also humans against diseases. Some of the well-known

phytochemicals are lycopene in tomatoes, isoflavones in soy and flavonoids in

fruits. Phytochemicals have a number of effects in humans like

antioxidant activity, Hormonal action, Anti-bacterial effect etc.

Only a few years ago, the term "phytochemical" was barely known. But doctors,

nutritionists, and other health care practitioners have long advocated a low-fat diet

that includes a variety of fruits, vegetables, legumes, and whole grains.

Historically, cultures that consume such a diet have lower rates of certain cancers

and heart disease. Since the passage of the Dietary Supplement Health and

Education Act (DSHEA) in the United States in 1994, a large number of

phytochemicals are being sold as dietary supplements (Chang, 2000).

a) Significance of phytochemical screening approaches

• The goal in surveying plants for biologically active or medicinally useful

compounds should be to isolate one or more constituents responsible for a

particular activity. Hence phytochemical screening techniques can be a valuable

aid in selection of a specific plant for pharmacologic approaches (Tatke, 1999).

• Certain investigators feel that an initial selection of investigational plants should

be made not on evidence that extracts elicit a particular and interesting biological

activity, but rather on the basis that certain chemicals are present in the plant, and

compounds/constituents closely related to them can usually be associated with

biological activity.

• Tests for the presence of these compounds in plants are simple, can be conducted

rapidly, and are reasonably reliable, and they help in making extraction and

isolation procedures easier.

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• In addition, economics, as well as other factors associated with biological testing,

often force the investigation to pursue a phytochemical group that can be selected

for investigation.

5.4.2 Background

There are few reports stating the pharmacological effects of some extracts of

cashew leaves and testa (Kamath, 2007; Konan, 2007; Abas, 2006 and Gonçalves,

2005). However, there are no reports published for the phytochemical

investigations of the extracts selected in this study. Hence a detailed and

systematic phytochemical screening of the prepared extracts of leaves and testa of

cashew was carried out by qualitative chemical tests.

5.4.3 Procedure

One gram of each of the extracts of testa and leaves of cashew were dissolved in

100 ml of respective solvents used for extraction to obtain a stock of concentration

1% (v/v). The extracts thus obtained were subjected to preliminary phytochemical

screening following the methodology described below (Harborne, 1998 and

Khandelwal 1999).

A. Test for Carbohydrates

a) Molisch's test

The test solution is treated with few drops of alcoholic alpha-naphthol. About 0.2

ml of conc. Sulfuric acid was slowly added through the sides of the test tube.

Formation of violet ring indicates the presence of carbohydrates.

b) Benedict's test

The test solution is treated with few drops of Benedict's reagent (alkaline solution

containing cupric citrate complex) and boiled on water bath, to check the presence

of reducing sugars.

c) Fehling's test

Equal volume of Fehling's A (Copper sulfate in distilled water) and Fehling's B

(Potassium tartarate and Sodium hydroxide in distilled water) reagents are mixed

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and few drops of sample are added and boiled. A brick red precipitate of cuprous

oxide forms, if reducing sugars are present.

d) Barfoed’s test

Equal volumes of Barfoed’s reagent and test solution are mixed. The solution is

heated in a boiling water bath for 1-2 mins and cooled. Red precipitate indicates

the presence of monosaccharides.

e) Test for pentoses:

An equal amount of test solution is mixed with HCl and a crystal of

phloroglucinol was added to it. If red colour appears it indicates the presence of

pentoses.

f) Selwinoff’s test

About 1 ml of the test solution is added to 3ml of Selwinoff’s reagent and boiled

in a boiling water bath for 1-2 mins. Fructose gives red color within half minute.

The test is sensitive to 5.5mmol / liter if glucose is absent, but if glucose is present

it is less sensitive and in addition of large amount of glucose can give similar

color. Hydrochloric acid reacts with ketose sugar to form derivatives of

furfuraldehyde, which gives red color indicating the presence of ketoses.

g) Tests for non-reducing polysaccharides

About 3.0ml of the test solution is taken and few drops of dilute iodine solution

were added to it. A blue color disappears on boiling and develops on cooling

indicating the presence of starch.

h) Test for Gums

The test solution is hydrolysed using dilute HCl and Fehling’s test was performed.

A red color development indicates the presence of gums.

i) Test for mucilage

The powdered drug is treated with aqueous KOH. If the solution of powdered

drug swells, it indicates the presence of mucilage.

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B. Tests for Proteins

a) Millons test

Test solution is mixed with 2ml of Millons reagent (Mercuric nitrate in nitric acid

containing traces of nitrous acid). If a white precipitate appears, which turns red

upon gentle heating, it indicates the presence of proteins.

C. Tests for Amino Acids

a) Ninhydrin test

About 3.0 ml of test solution is boiled with few drops of with 5% solution of

Ninhydrin (Indane 1, 2, 3 trione hydrate), in a boiling water bath. The

development of violet color indicates the presence of proteins.

D. Test for Fats and Fixed Oils

a) Stain test

A small quantity of extract is pressed between two filter papers. If the filter paper

is stained then it indicates the presence of fixed oils.

b) Saponification test

Few drops of 0.5N of alcoholic potassium hydroxide is added to small quantities

of various extracts along with a drop of phenolphthalein separately. The mixture

is heated on a water bath for 1-2 hrs. The formation of soap or partial

neutralization of alkali indicates the presence of fixed oils and fats.

E. Test for Sterols and Triterpenoids

a) Libermann- Buchard test

The test sample is treated with few drops of acetic anhydride, boiled and cooled.

Con. Sulfuric acid is added from the sides of the test tube. A brown ring at the

junction of two layers and the upper green colored layer shows the presence of

steroids and formation of deep red color indicates the presence of triterpenoids.

F. Test for Glycosides

The extract is tested for free sugars. The extract is hydrolyzed with dilute HCl and

then tested for the glycone and aglycone moieties.

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a) Legal’s test for Cardiac Glycosides

About 1.0 ml of the extract is treated with 1.0 ml pyridine and 1.0 ml alkaline

sodium nitroprusside solution. Pink to red color appears indicating presence of

cardiac glycosides.

b) Keller Killiani test [for deoxy sugars]

The test solution is treated with 0.4ml of glacial acetic acid containing a drop of

5% ferric chloride and 0.5ml of concentrated sulphuric acid is added by the sides

of the test tube. The appearance of blue color in the acetic acid layer indicates the

presence of deoxy sugars.

c) Froth Test for Saponin Glycosides

About 1ml of aqueous solution of extract in water is shaken well and noted for a

stable froth. A stable froth indicates the presence of saponins.

d) Hemolysis test for Saponin Glycosides

About 0.2ml solution of drug solution (prepared in 1% normal saline) is added to

0.2ml of v/v blood in normal saline on a glass slide. If hemolytic zone appears it

indicates the presence of saponins.

e) Sodium picrate test (grignard reaction) for Cyanogenetic Glycosides

About 200mg of drug is placed in a conical flask and moistened with few drops of

water. A piece of picric acid paper is moistened with sodium carbonate solution

(5% aqueous) and suspended by means of cork in the neck of the flask. The flask

is then warmed gently at about 37°C. The change in color is observed. Hydrogen

cyanide is liberated from cyanogenetic glycoside (if present) by the enzyme

activity and reacts with sodium picrate to form the reddish purple sodium

isopicrate.

f) Tests for Coumarin Glycosides

The test solution is made alkaline and the colour change is observed. The

development of blue or green color fluorescence indicates the presence of

Coumarin Glycosides.

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G. Test for Flavonoids

a) Shinoda test (Magnesium Hydrochloride reduction) To the test solution, few

fragments of Magnesium ribbon are added and concentrated hydrochloric acid is

added drop wise. A pink scarlet, crimson red or occasionally green to blue color

appears after few minutes if flavanoids are present.

b) Alkaline reagent test for Flavonoids

To the test solution few drops of sodium hydroxide solution are added. The

formation of an intense yellow color, which turns to colorless on addition of few

drops of dil. acid, indicates presence of Flavonoids.

H. Tests for Alkaloids

a) Dragendorff’s test

To 2-3 ml of test solution, few drops of Dragendorff’s reagent [Potassium bismuth

iodide solution] were added. Alkaloids give orange brown precipitate if present.

I. Test for Tannins and Phenolic Compounds

a) Ferric chloride test: To the test solution few drops of 5% FeCl3 solution are

added. The development of blue black color indicates the presence of tannins and

phenolics.

J. Tests for organic acids

a) Calcium chloride test

To 2.0 ml of test solution, few drops of 5% CaCl2 solution are added and color

changes are observed.

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5.5 ISOLATION OF CATECHIN

5.5.1 Introduction

Plants contain a large amount of structurally and functionally diverse components.

Medicinal plants serve as an important source to invent potential and safe drugs.

Numerous novel bioactive compounds have been isolated and identified from

plants. Considerable efforts have been directed towards the isolation and

identification of compounds from medicinal plants, which are most likely to be

responsible for the reported bioactivities. However, isolation and purification of

pure compounds from plants is usually difficult, tedious and expensive process.

Reports on the identification of novel compounds from plants are available in

significant numbers; however research publications on the quantitative analysis of

novel bioactive compounds are relatively few, due to the lack of standard

compounds. Thus, isolation of bioactive compounds is of great significance in the

field of phytochemistry.

The quality control of active constituents or marker compounds in the herbal

extract is of great importance in medicinal and dietary applications. The isolation

and identification of marker compounds in herbal medicines is a prerequisite in

quality control since most of these compounds are not commercially available.

Extraction and isolation methods including various chromatographic methods to

obtain marker compounds from herbal medicines have been extensively reported

(Hendriks, 2005).

5.5.2 Background

Catechin is a potent bioactive antioxidant compound present in a number of

plants. Testa of cashew is a rich source of polyphenols and tannins. Testa of

cashew is a byproduct of cashew manufacturing industry and hence can serve as

an economical and low cost source for isolation of catechin. Moreover, isolation

through Preparative Thin Layer Chromatography (P-TLC) method, used in this

research work can serve as an economic and alternative method to currently

available isolation techniques (Deore, 2010 and Mahajan, 2010).

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5.5.3 Procedure

A. Preparation of solutions of extracts and catechin standard

Solutions (50 mg/ml) of ethanol extract of testa were prepared in methanol and

used for isolation of catechin by thin layer chromatography. Working solution of

catechin (1mg/ml) was used for location of catechin spot from the extracts. All

solutions were prepared freshly prior to analysis.

B. Preparative Thin layer Chromatography technique

• A slurry of silica gel GF254 was prepared by addition of Silica gel GF254 in distilled

water and mixing it well to form a slurry with pourable consistency. The slurry

was poured on the glass plates while avoiding the entrapment of air bubbles and

spread to form a uniform layer of optimum thickness.

• Preparative TLC plates of optimum layer thickness were prepared. The plates

were air dried for 30 minutes and then dried in an oven at 1100 C for 30 minutes

before use.

• A concentrated band of ethanolic extract of testa (previously defatted with

hexane) was then applied at a distance of 1cm from bottom edge, by using glass

capillaries. A band of standard catechin was also applied on the same plate to

serve as a reference for detection and separation of catechin band.

• After drying of the applied bands, the plate was placed in developing chamber

pre-saturated with mobile phase for 15mins. The solvent system used for

chromatography was toluene: ethyl acetate: methanol: formic acid (6:6:1:0.1).

After the chromatographic run, the plate was air dried.

• The band which corresponded to marker catechin was scrapped out. Several plates

were prepared in similar manner and the band corresponding to catechin was

collected.

• The silica gel containing the component thus obtained was, sonicated with

methanol as solvent with minimum exposure to heat and light. The suspension

was then allowed to stand and the supernatant was collected. The supernatant was

then concentrated to 1/3rd

of its volume, filtered and evaporated to obtain crude

catechin. Crude catechin thus obtained was then recrystallised with hot water and

the yield was calculated.

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• The isolated catechin was confirmed for its identity by co-chromatography with

marker catechin on precoated TLC plates and HPLC analysis.

Ethanol extract (500 mg) + methanol (10 ml) + 50 ml of hexane

Add hexane (50 ml) and separate

Hexane layer Ethanolic layer

evaporate

Discard residue

Preparative TLC

Crude catechin

recrystallisation

Pure catechin

(%yield= 5.0%) , (Purity by HPLC was 99.65%.)

Figure 5.3: Scheme for isolation of catechin from ethanol extract of cashew testa

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5.6 CHROMATOGRAPHIC STUDIES

5.6.1 Introduction

With gaining popularity of herbal remedies worldwide, the need of assuring safety

and efficacy of these products increases as well. By nature they are complex

matrices, comprising a many compounds, which are prone to variation due to

environmental factors and manufacturing conditions. Furthermore, many

traditional preparations compose of multiple herbs, so that only highly selective,

sensitive and versatile analytical techniques will be suitable for quality control

purposes. Recently, chromatographic fingerprint technique has been accepted by

WHO as a strategy for the quality assessment of herbal medicines (WHO, 2005).

Chromatographic techniques such as HPLC and HPTLC have recently gained

increasing importance due to their emphases on the characterization of the

complete sample composition (Liang, 2004). The methods developed by use of

these techniques can also be applied to determination of standard compounds as

markers, bioactive components and enhancement of herbal medicinal product

quality (Mahady, 2001).

The analysis of constituents in plants is a challenging task because of

their chemical diversity, usually low abundance and variability even within the

same species. Considering the fact that many traditional herbal preparations

contain not one but several medicinal plants, only highly selective and sensitive

methods will be suitable for controlling their composition and quality. Sensitivity

is the major issue when using various analytical techniques for detection of

phytoconstituents. Thus, most commonly chromatographic techniques in

combination with different detectors are employed for this purpose. Due to

extremely small sample volumes and the attributes mentioned above, high

performance liquid chromatography (HPLC) and high performance thin layer

chromatography (HPTLC) are still the preferred separation techniques for the

analysis of natural products (Liang, 2004).

Chromatographic methods, especially HPTLC and HPLC help in the quality

control of botanicals. Identification of phytoconstituents can be carried out by

these techniques by comparison of a sample with a reference. It is an advantage of

HPTLC that not only the entire sample can be seen but also several samples can

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easily be compared at the same time. The prevailing value of HPTLC fingerprints

is the visual impression, which can be further expanded by multiple detection

(visualization of compounds prior to and after derivatization). A broad spectrum

of constituents can be detected at the same time in a single run in an experiment.

Therefore, the use of HPLC and HPTLC for the qualitative and quantitative

analysis of constituents in medicinal plants steadily has gained importance in the

last few decades (Liang, 2004).

5.6.2 Background

There have been no reports published for the HPTLC fingerprinting and HPLC

profiling of the extracts of cashew testa and leaves. Moreover performing HPTLC

and HPLC analysis helps in identifying various phytoconstituents present in the

extracts. Thus, HPTLC and HPLC methods were developed and optimised for

various extracts of leaves and testa of cashew.

5.6.3 Procedure

A. Preparation of solutions of extracts and catechin

Stock solutions (1mg/ml) of reference catechin were prepared in methanol.

Working solutions of catechin were prepared by appropriate dilutions of the stock

solution with methanol. All solutions were prepared freshly prior to analysis.

Working solutions of extracts (5mg/ml) of cashew leaves and testa were prepared

with methanol. All solutions were prepared freshly prior to analysis.

B. Development and optimization of TLC parameters

a) Preparation of TLC plates

Preparative TLC plates were prepared by pouring the silica gel GF254 slurry on the

glass plates of 10x 20 cm dimension. Prepared TLC plates were then made free of

the moisture associated with thin layers by drying the thin layer plates, for 30

minutes in air and then in an oven at 1100C for another 30 minutes. The extracts

of testa and leaves dissolved in methanol were applied in a row along one side of

chromo plate, about 1 cm from the edge, by using sealed glass capillaries.

b) Selection of mobile phase

To make a choice of suitable solvent system, initially the elutropic series of

different solvents was tried by running on the TLC plates. Neat solvents of

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varying polarity and solvents in different combination ratios were used to

optimize elution of various components and a combination of solvents that gave

better resolution of maximum number of components in extracts was selected.

Formic acid was used as a modifier to affect better resolution of bands.

c) Optimization of saturation time

Various time periods from 10-25 minutes (10, 15, 20, 25 mins) were attempted to

select the optimum saturation time, suitable for maximum resolution and faster

development of the TLC plate.

d) Development of plates

The samples were applied at 1cm distance from the bottom on the TLC plates and

the solvent front was marked at 8 cm distance from the application position. The

plates were allowed to dry and then placed in chambers saturated with the solvent

system (mobile phase) for a period of 20 min prior to placement of plates. The

qualitative evaluation of the plate was done by determining the migrating behavior

of the separated substances by calculating Rf value.

e) Derivatisation/visualization of plates

Derivatising agent was selected based upon the class of phytoconstituents found in

the preliminary phytochemical screening tests. The derivatising reagent helps in

visualization as well as confirmation of the identity of the phytoconstituents.

C. High performance thin layer chromatography (HPTLC) analysis

a) Pre-conditioning of plates

Precoated HPTLC plates used for analysis were preconditioned by overnight

washing with methanol in a twin trough chamber. Preconditioning in methanol has

been shown to be effective for layer cleaning. The prewashed plates were then

heated at 1050C for 5 minutes before use.

b) Optimized chromatographic parameters

The optimized TLC conditions to be used in HPTLC analysis were as follows:

Stationary Phase: Precoated, aluminum backed HPTLC plates (20cm×20

cm, 0.2mm thickness, 5–6µm particle size.

Mobile phase: toluene:ethyl acetate:MeOH:formic acid (6:6:1:0.1v/v/v/v)

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Saturation time: 15 mins.

Development distance: 80 mm

Derivatising agent: 5% alcoholic FeCl3 solution

c) HPTLC analysis

The analysis was performed in air-conditioned room maintained at 220C and 55%

humidity. TLC was performed on precoated silica gel GF254 aluminum backed

HPTLC plates (20cm×20 cm, 0.2mm thickness, 5–6µm particle size, E-Merck,

Germany). Five microlitres of the sample solutions were spotted as bands of 6mm

width by using a 100µl Hamilton syringe. The plates were developed using

toluene: ethyl acetate: methanol: formic acid (6:6:1:0.1v/v/v/v) as the solvent

system with saturation time of 15 minutes in a CAMAG twin-trough plate

development chamber. The developed plates were air dried and scanned. A

spectro-densitometer (Scanner 3, CAMAG) equipped with ‘win CATS’ planar

chromatography manager (version 1.3.0) software was used for the densitometry

measurements, spectra recording and data processing. Absorption/remission were

done in the measurement mode at a scan speed of 20mm/s. Densitograms were

recorded at the wavelength of 254 nm for catechin and various components of

extracts.

D. Development and optimization of HPLC parameters

a) Preparation of sample

The extract samples (0.1 g) were dissolved in 10mL methanol by sonication. The

resultant solutions were filtered through a 0.45 µm PVDF filter into an amber

sample vial for HPLC fingerprinting analysis. These stock solutions were further

diluted with appropriate dilutions for analysis.

b) Selection of stationary phase

The aqueous, and ethanolic extracts of cashew leaves and aqueous, ethanolic and

methanolic extracts of testa contained majority of tannins and polyphenols as their

constituents, which are polar in nature. Hence a nonpolar stationary phase would

to be required to elute the constituents. Thus, a reverse phase C18 (octadecylsilane)

column was selected for analysis.

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c) Selection of mobile phase

The HPLC separation conditions, such as choice of mobile phase and

isocratic / gradient program, were further optimized. To make a choice of suitable

solvent system, initially neat solvents with relatively polar nature were tried. Then

mobile phases of solvents in different combination ratios were used to optimize

retention of various components. A number of mobile phases with different ratios

were screened in order to obtain a reliable chromatogram with most peaks at

acceptable resolution and balance for the HPLC fingerprinting and to obtain

baseline separation of catechin in a relatively short analytical time for the HPLC

quantitation.

d) Selection of detection wavelength

The choice of detection wavelength is crucial for developing a reliable fingerprint

and for accurate quantitative analysis of marker compounds in the herb.

Chromatographic detection was carried out at 254 nm, 273 mn (λ max of catechin)

and at 280nm (~λ max of catechin). Optimal signal-to-noise ratios for UV

detection was obtained at 254 nm with a good resolution for maximum number of

peaks. Hence, the optimal detection wavelength in the HPLC analysis was

determined to be 254 nm. At this wavelength, more characteristic peaks in the

chromatogram were observed, with a good resolution for catechin that was used as

a marker (Fracassetti, 2011).

E. HPLC analysis

The HPLC profiling was carried out on a C18 column (Phenomenex C18,

4.6mm×250 mm, 5µm) equipped with an extended guard column at ambient

temperature with a sample injection volume of 10µL. An isocratic elution was

carried out with methanol:water (90:10 v/v). Flow rate was 1ml/min flow rate.

The fingerprint chromatograms were recorded at a wavelength of 254 nm.

The optimized parameters for HPLC analysis were:

Solvent system: methanol:water (90:10 v/v)

Flow rate: 1ml/min

Column: C18 column (Phenomenex)

Detection wavelength: 254 nm

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F. Calibration curve of catechin

Standard stock solutions were prepared by dissolving the reference standard in

methanol to obtain a concentration of 1mg/mL for catechin. The concentrations of

catechin reference standard used for calibration were in the range 0.3 – 1.4µg/µL

in methanol, respectively. The peak in HPLC chromatograms were identified by

comparing the retention times in the chromatograms of extracts with those of

reference standard catechin peak.

G. Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ were determined as signal to noise ratio using the equations

LOD= 3.3 σ/S and LOQ=10 σ/S where, σ is standard deviation of response and S

is the slope of calibration curve. The LOD and LOQ were 0.1 and 0.3 µg

respectively for catechin.

H. Quantitation of catechin in various extracts by HPTLC analysis

The extracts were dissolved in methanol and the solution of concentration 5µg/µL

was filtered through 0.45 µm PVDF filter and HPTLC was performed under the

conditions optimized for the reference compound. The plates were scanned at 254

nm and the UV–vis spectra of the bands corresponding to catechin were recorded.

The amount of catechin in the extracts was quantified by comparison with

catechin bands from solutions of known concentration. After scanning at 254 nm,

the plates were dipped in 5% alcoholic FeCl3 solution for 5 s and then kept at

1000C for 5 min for visualization of tannins and polyphenols. Bluish black colored

bands indicate the presence of tannins and polyphenols.

I. Quantitation of catechin in various extracts by HPLC

Standard stock solutions were prepared by dissolving the reference standard in

methanol to obtain a concentration of 1µg/µL for catechin. The concentrations of

catechin reference standards used for calibration were 0.6, 0.7, 0.8, 0.9, 1.0 µg/µL,

respectively. The peaks in HPLC chromatograms were identified by comparing

the retention times in the chromatograms of extracts with those of reference

standard catechin peak.

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5.7 EFFECT OF VARIOUS DRYING METHODS ON THE POLYPHENOL

CONTENT AND ANTIOXIDANT ACTIVITY OF CASHEW LEAVES

5.7.1 Introduction

The leaves of plants are often dried before extraction to reduce moisture content.

Dehydration of herbs can be performed using different methods. The most popular

method is convective drying. Extraction yields of phenolics from plant tissues

depend on the various extraction conditions like pH and temperature. The method

of drying usually has a significant effect on the quality and quantity of the

phytoconstituents from such plants (Yag, 1999).

5.7.2 Background

In recent years, the drying behaviors of different plants and culinary herbs have

been studied by many investigators. However, studies on the drying characteristics

of cashew leaves are not found in the literature, particularly the traditional sun

drying properties as well as microwave drying properties of plants are not

adequately investigated. The aim of the work was to determine the sun, oven and

microwave drying characteristics of cashew leaves and to compare traditional sun

drying and conventional oven drying methods to the microwave drying method,

which reduces drying time considerably, and to determine the effects of these

different drying techniques on total phenolic content.

5.7.3 Procedure

• Fresh leaves of cashew were exposed to various drying conditions viz. sun drying,

shade drying, oven drying, and their aqueous extracts were prepared.

• The various conditions of drying are detailed as below:

A. Oven drying: The fresh green mature leaves of cashew were collected, cleaned,

washed and dried. The drying of the leaves was carried out in a Hot air oven at

800 C for about 30 minutes until dry.

B. Sun drying: The naturally dried, shredded mature leaves of cashew were

collected, cleansed and used for extraction.

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C. Shade drying: The fresh mature green leaves of cashew were collected and

cleansed. The leaves were dried in shade for 8-10 days until dry and were then

powdered and used for extraction.

D. Fresh leaves: The mature green leaves of cashew were collected, cleansed and

used for extraction without drying.

• The leaves obtained from various drying conditions were then powdered to coarse

size and extracted with water as solvent at 400 C for 3 hours. The mass was then

filtered and the filtrate was concentrated and extractive yield was calculated.

• The total polyphenol content in the extracts were determined by Folin - Ciocalteu

method and the antioxidant activity was determined by DPPH. radical scavenging

assay and Greiss assay. The procedure for the determination of total phenolic

content and antioxidant assays is detailed in subsequent chapters (Giustarini,

2008; Etsuo, 2010 and Vernon, 1999)

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5.8 EVALUATION OF ANTIOXIDANT ACTIVITY

5.8.1 Introduction

Aerobic organisms produce a number of reactive free radicals (molecules or atoms

having unpaired electrons) continuously in cells during respiration, metabolism

and phagocytosis. It has been found that fruits and vegetables, rich in

antioxidants, decrease the risk of oxidative stress. A number of herbal

formulations used in traditional Indian medicines are also some of the potent

antioxidants which need to be explored. The approach to the development of

antioxidants has in general been based on macroscopic biochemical changes by

both in vitro and in vivo studies and from such studies several phytochemicals

have been reported as potent antioxidants (Bisby, 1993; Tatke, 2011).

Figure 5.4: Role of reactive oxygen species in cell injury

Source: General Pathology, Chapter 1, pp-17, Second Edition, 2005, Elsevier.

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The effects of these reactive species are wide-ranging, but following three

reactions are particularly relevant to cell injury:

• Lipid peroxidation of membranes: Free radicals in the presence of oxygen may

cause peroxidation of lipids within plasma and organellar membranes. Oxidative

damage is initiated when the double bonds in unsaturated fatty acids of membrane

lipids are attacked by oxygen-derived free radicals, particularly by OH. The lipid–

free radical interactions yield peroxides, which are themselves unstable and

reactive, and an autocatalytic chain reaction continues (called propagation), which

can result in extensive membrane, organellar, and cellular damage.

• Oxidative modification of proteins: Free radicals promote oxidation of amino

acid residue side chains, formation of protein-protein cross-linkages (e.g.,

disulfide bonds), and oxidation of the protein backbone, resulting in protein

fragmentation.

• Lesions in DNA: Reactions with thymine in nuclear and mitochondrial DNA

produce single-stranded breaks in DNA. This DNA damage has been implicated

in cell aging and in malignant transformation of cells.

Cells have developed multiple mechanisms to remove free radicals and thereby

minimize injury: A series of enzymes acts as free radical–scavenging systems and

break down hydrogen peroxide and superoxide anion. These enzymes are located

near the sites of generation of these oxidants and include the following:

• Catalase, present in peroxisomes, which decomposes as given below:

H2O2 (2 H2O2 O2 + 2 H2O )

• Superoxide dismutases are found in many cell types and convert superoxide to

H2O2 (2 O2- + 2 H H2O2 + O2). This group includes both manganese

superoxide dismutase, which is localized in mitochondria, and copper-zinc–

superoxide dismutase, which is found in the cytosol.

• Glutathione peroxidase also protects against injury by catalyzing free radical

breakdown (H2O2 + 2 GSH GSSG [glutathione homodimer] + 2 H2O, or

2 OH + 2 GSH GSSG + 2 H2O).

The intracellular ratio of oxidized glutathione (GSSG) to reduced glutathione

(GSH) is a reflection of the oxidative state of the cell and is an important aspect of

the cell’s ability to detoxify reactive oxygen species.

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5.8.2 Assessment of Free Radical Scavenging Capacity in vitro

The free radical scavenging capacity of antioxidant in vitro has been evaluated by

several different methods under different conditions. The “antioxidant capacity”

often means different things at different occasions and to different people.

Recently, the capacity of antioxidants for scavenging free radicals has been

assessed more often and widely by either the reaction with stable reference radical

or by competition methods using conventional UV-Visible absorption

spectrophotometer. It is difficult to prove but the mechanisms and dynamics of

antioxidant action found in vitro may be applied to biological systems if the

factors which affect them are properly considered (Etsuo, 2010).

A. DPPH (1, 1, diphenyl 2-picryl hydrazyl) Assay

a) Principle:

The capacity of antioxidant compounds for scavenging free radicals should be

assessed by two factors, that is, rate of scavenging radicals and number of radicals

each antioxidant molecule can scavenge, which are determined inherently by the

chemical structure of the antioxidant compound and also the free radicals. These

two parameters can be measured by following the reaction with stable reference

free radical such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) ((Brand, 1995).

Many antioxidants react with DPPH by hydrogen atom transfer (Reaction 1) or

electron transfer, followed by proton transfer (Reaction 2), depending on the

antioxidant, radical, and also reaction environment. The reaction of antioxidants

with DPPH is followed from a decrease in their absorption at 520 nm. The relative

reactivity and stoichiometric number can be assessed easily from a rate of

decrease in absorption induced by the antioxidants or mixtures.

X· + IH XH + I· (Reaction - 1)

X· + IH X- + IH

.+ XH + I· (Reaction - 2)

Where, Free radical scavenging antioxidants - (IH)

Active free radicals - (X·)

Stable compound - (XH)

Antioxidant-derived radical (I·)

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This is probably the simplest way to assess the relative capacity for scavenging

radicals. However, this assay may not be used when the test compounds have

absorption overlapping that of DPPH. The hydrogen atoms or electron-donating

ability of the extracts was determined from the bleaching of purple-colored

methanol solution of DPPH. This activity is given as percent DPPH radical

scavenging, which is calculated with the equation:

% DPPH radical scavenging = (Control Absorbance - Sample Absorbance) × 100

----------------------------------------------------

Control Absorbance

b) Procedure

• Radical scavenging activity of the testa and leaf extracts was evaluated using

DPPH.(1,1-diphenyl-2-picrylhydrazyl) method.

• Varying volumes of 0.2 mg/mL the extracts of testa and leaves of cashew were

added to 200 µL of (0.36 mg/mL concentration) DPPH. solution in methanol. A

series of concentrations ranging from 2 to 16 µg of dried extracts were tested.

• The mixtures were shaken vigorously and incubated in the dark for 30 min after

which the reduction of DPPH. absorption was measured at 517 nm.

• Percent inhibition by sample treatment was determined by comparison with the

methanol-treated control group. The IC50 values denote the concentration of each

sample required to give 50% of the optical density shown by the control, using a

non-linear regression analysis. All test analyses were run in triplicate and average

values were reported. Ascorbic acid was used as positive control.

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B. Nitric Oxide Scavenging Activity

a) Principle

NO is very unstable in biological systems and has a physiological half life of only

1–40 s. A colorimetric simple and accurate method for measuring total NO

species is known as the Griess assay. This assay is based on reducing NO species

to nitrite and then detecting the nitrite. The Griess assay can be used for most

biological systems, including organs, tissues, cells, and subcellular compartments.

The Griess assay detects the red–pink color produced by the reaction of Griess

reagent with nitrites. Therefore, all nitrates in the sample should be reduced to

nitrite to be detected in this assay. After reduction to nitrite, samples are reacted

with the Griess reagent consisting of equal volumes of sulfanilamide solution and

N-(1- napthyl)ethylenediamine (NED) solution (Giustarini, 2008).

Figure 5.5: Griess reaction: formation of chromophoric diazo compound by the

Griess reaction

Source: Methods in Enzymology, Volume 440, 2008.

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b) Procedure

• Nitric oxide scavenging activity of testa and leaf extracts of cashew was evaluated

through Griess Assay method.

• Griess reagent was prepared by mixing equal volumes of reagents (a) and (b).

Reagent (a) is 1% sulfanilamide in 5% phosphoric acid , prepared by adding 3.5

mL of 85% H3PO4 to 100 mL with distilled water and then dissolving 1.0 g of

sulfanilamide.

Reagent (b) is 0.1% N-1-naphthylethylenediamine dihydrochloride, prepared by

dissolving 100 mg of NEDD in 100 mL of distilled water.

• Accurately 2.0 mL of 10 mM sodium nitroprusside and 5.0 mL of phosphate

buffer (pH 6.5) were mixed with 0.5 mL of different concentrations of the plant

extracts and incubated at 250C for 150 min.

• The samples were run as above but the blank was replaced with the same amount

of water. After the incubation period, 2mL of the above incubated solution was

added to 2 mL of Griess reagent and incubated at room temperature for 30 min.

• The absorbance of the chromophore formed was read at 540 nm. Ascorbic acid

was used as positive control and results were expressed as percentage inhibition of

nitric oxide. All determinations were performed in triplicates.

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5.8.3 Determination of Antioxidant Capacity against Lipid Peroxidation

A. Thiobarbituric acid Reacting substances (TBARS) test

a) Introduction

The capacity of antioxidant for inhibition of lipid peroxidation can be assessed by

measuring the extent of suppression of lipid peroxidation by the test antioxidant.

Many oxidizable substrates have been used in thiobarbituric acid-reactive

substances (TBARS) determination, including free fatty acids, LDL and fluids

(urine, serum) from cells or tissues.

b) Principle

Malondialdehyde formed from the breakdown of polyunsaturated fatty acid reacts

with thiobarbituric acid to form thiobarbituric acid reacting substance (TBARS),

the end product of lipid peroxidation, a pink to red colour trymethionine complex

exhibiting an absorption maximum at 530-535 nm species (Laguerre, 2007).

Figure 5.6: Formation of TBA Chromophore

Source: Progress in Lipid Research, 46, 2007.

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c) Procedure

• Preparation of tissue homogenate: Mice (previously fasted overnight) were fixed

on the operation table with ventral side up and then dissected. Liver was perfused

with normal saline through hepatic portal vein. Liver was harvested and its lobes

were briefly dried between filter papers (to remove excess of blood) and were cut

thin with a heavy-duty blade. These small pieces were then transferred to the glass

Teflon homogenizing tube and the homogenate (1 g, w/v) was prepared in

phosphate buffer saline (pH 7.4) in cold condition. It was centrifuged at 2000g, for

10 min. Supernatant was collected and finally suspended in PBS to contain

approximately 0.8-1.5 mg protein in 0.1 ml of suspension to perform the in vitro

experiment.

• An incubation mixture containing 1 ml potassium chloride (150mM), 0.3 ml of

10% liver homogenate as lipid source and various concentration of test compound

(extracts of testa and leaves of cashew) in a volume of 0.5 ml.

• Peroxidation was initiated by adding 0.1 ml FeSO4. After incubating for 20

minutes of 37°C, reaction was stopped by adding 1 ml Trichloroacetic acid(TCA)

in 50 % acetic acid , followed by heating at for 30 mins in a boiling water bath,

cooled, centrifuged at 1000 rpm for 10 minutes and absorbance of the supernatant

liquid was recorded at 535 nm.

• The percentage of anti-lipid peroxidation effect (% ALP) or % inhibition was

calculated.

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5.8.4 Determination of Total Phenolic Content by Folin - Ciocalteu reagent

method

a) Introduction

Phenols occurring in nature and the environment are of interest from many

viewpoints (antioxidants, astringency, bitterness, browning reactions, color,

oxidation substrates, protein constituents, etc.). Phenols are responsible for the

majority of the oxygen capacity in most plant-derived products, such as wine. An

antioxidant effect can be from competitive consumption of the oxidant, thus

sparing the target molecules being protected, and from quenching the chain

reaction propagating free radical oxidation. Antioxidants become oxidized as they

interfere with the oxidation of lipids and other species. Free radicals are very

reactive molecules with an unpaired electron. Encountering another free radical

from any source the two combine to form a new covalent bond, terminating any

chain reaction caused by extraction by the free radical of an electron from an

intact molecule to generate another free radical. The unpaired electron in a

semiquinone can resonate among the former hydroxyl and the positions ortho and

para to it (two, four, or six of the ring). A mixture of dimerized products results as

the new bonds form. If the new bond is to one of the ring carbons, the phenolate is

regenerated. Oxidation may then not only be repeated, but the regenerated phenol

is often oxidized more easily than the original one. If the important property of

oxidizability is to be the basis for the quantitation of phenols, the reaction must be

brought quickly to a conclusion to minimize such regenerative polymerization.

That the phenolate ion is important is shown by the fact that the uptake of oxygen

by phenols can be rapidly complete near or above the pK of the phenol (usually

about pH 10). Reaction at alkaline pH is indicated for assay purposes (Vernon,

1999).

b) Principle

The Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin-Denis

reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture

of phosphomolybdate and phosphotungstate used for the colorimetric assay of

phenolic and polyphenolic antioxidants. It works by measuring the amount of the

substance being tested needed to inhibit the oxidation of the reagent.

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However, this reagent does not only measure total phenols and will react with any

reducing substance. The reagent therefore measures the total reducing capacity of

a sample, not just the level of phenolic compounds. Copper complexation

increases the reactivity of phenols towards this reagent.

Folin-Ciocalteu’s phenol reagent reacts with phenols and nonphenolic reducing

substances to form chromogens that can be detected spectrophotometrically. The

color development is due to the transfer of electrons at basic pH to reduce the

phosphomolybdic/phosphotungstic acid complexes to form chromogens in which

the metals have lower valence (Vernon, 1999).

b) Procedure

• Total phenolic content of extracts of leaves and testa was determined by Folin-

Ciocalteu reagent test.

• The reaction mixture was composed of 0.1 mL of extract, 7.9 mL of distilled

water, 0.2 mL of the Folin-Ciocalteu’s reagent, and 1.5 mL of 20% sodium

carbonate.

• The resultant solution was mixed and allowed to stand for 2 hours, the absorbance

was measured at 765 nm in a Shimadzu UV- Spectrophotometer.

• The total phenolic content was determined as gallic acid equivalents (GAE)/mg of

extract.

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5.8.5 Cellular defense mechanisms against oxidative stress

a) Introduction

Cultured cells have often been used as a substrate to elucidate the underlying

mechanisms of oxidative stress and also to evaluate the protective effects of

antioxidants against various oxidative stressors. The advantage of using cultured

cells is that various different stressors and cell types including model systems for

some specific disease can be used for evaluation of the antioxidant effects. The

effects of antioxidants have been measured against oxidative stress in cultured

cells for the suppression of ROS formation, oxidation of lipids, proteins and

DNA, and cell death.

b) The Nrf2-antioxidant response element signaling pathway and its activation

by oxidative stress

A major mechanism in the cellular defense against oxidative or electrophilic stress

is activation of the NF-E2-related transcription factor 2 (Nrf2) - antioxidant

response element signaling pathway, which controls the expression of genes

whose protein products are involved in the detoxication and elimination of

reactive oxidants and electrophilic agents through conjugative reactions and by

enhancing cellular antioxidant capacity. It is well established that Nrf2 activity is

controlled, in part, by the cytosolic protein Keap1, but the nature of this pathway

and the mechanisms by which Keap1 acts to repress Nrf2 activity remain to be

fully characterized (Kaspar, 2009). Nrf2 is a nuclear transcription factor that

controls the expression and coordinated induction of a battery of defensive genes

encoding detoxifying enzymes and antioxidant proteins.

c) Antioxidant-response element and Nrf2

Promoter analysis identified a cis-acting enhancer sequence designated as the

antioxidant-response element (ARE), which controls the basal and inducible

expression of antioxidant genes in response to xenobiotics, antioxidants, and UV

light. Nrf2 binds to the ARE and regulates ARE mediated antioxidant enzyme

gene expression and induction in response to a variety of stimuli. The importance

of this transcription factor in upregulating ARE-mediated gene expression has

been demonstrated by several in vivo and in vitro studies and results indicate that

Nrf2 is an important activator of phase II antioxidant genes (Kaspar, 2009).

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Figure 5.7: Nrf2 signaling in ARE-mediated coordinated activation of

defensive genes

Source: Free Radical Biology & Medicine, 47, 2009.

d) The vascular endothelium and its function

The endothelium, the largest organ in the body, is a single layer of cells that line

the luminal surface of blood vessels. Since the seminal discovery of endothelial

derived relaxing factor (EDRF) (Furchgott, 1980) it has become increasingly

apparent that the endothelium is far more than just a structural lining. It acts as a

direct interface between the components of circulating blood and regulates

numerous local blood vessel functions such as vascular tone, coagulation

and inflammation (Cooke, 2000) through the release of several mediators and/or

activation of transcription factors. These include endothelium derived relaxing

factors such as nitric oxide, endothelium derived hyperpolarizing factor (EDHF)

and prostacyclin and contracting factors (such as endothelin-1, thromboxane A2

and reactive oxygen species) as well as inflammatory modulators/mediators

(Rubanyi, 1986). Endothelial dysfunction, initially identified as impaired

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vasodilation to specific stimuli, is often associated with other abnormalities of

endothelial function (Deanfield, 2007). Inflammatory cytokines,

lipopolysaccharide, ischemia-reperfusion, physical trauma and diabetes are able to

induce endothelial dysfunction. Endothelial dysfunction is implicated in the

pathophysiology of several cardiovascular diseases such as hypertension

(Endemann, 2004), atherosclerosis (Ross, 1993) and type 2 diabetes mellitus

(Browne, 2003). Indeed, disturbances in the vascular endothelium are a

fundamental component in the development of diabetic microvascular and

macrovascular complications, although other cell types (mesangial cells,

podocytes) are also involved.

e) Sources of reactive oxygen species in endothelial cells

The important sources of generation of reactive oxygen species in endothelial

cells include the mitochondrial enzyme complexes and the electron transport

chain, NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide

synthase and cytochrome P450. The role of hyperglycaemia in generating reactive

oxygen species is supported by in vivo and in vitro studies (Sartoretto, 2007;

Gryglewski, 1986; Dixon, 2005; Ceriello, 2001; and Ding, 2007). Thus increased

superoxide production was demonstrated in endothelial cells grown under

hyperglycemic conditions (Hattori, 1991). Rat and human mesangial cells cultured

in the presence of high glucose concentrations showed increased lipid

peroxidation and also upregulation of a number of thiol antioxidant genes

(Morrison, 2004).

f) Effects of reactive oxygen species on endothelial function in diabetes

Endothelial dysfunction is clearly evident in both clinical (Morcos, 2001; and

Parthiban, 1995) and experimental diabetes. Its manifestations include impaired

endothelium-dependent vasodilatation (Mayhan, 1989) increased expression of

adhesion molecules (ICAM-1 and VCAM-1), adhesion of monocytes (Yorek,

2002), increased platelet adhesiveness and atherosclerosis. Reactive oxygen

species may contribute to this dysfunction in a number of ways but inactivation of

endothelial nitric oxide or inhibition of nitric oxide formation are important

mechanisms. Reactive oxygen species were also shown to inhibit nitric oxide

species as well as prostacyclin synthetase (Du, 2006), thus offering additional

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mechanisms for disruption of endothelium-dependent vasodilatation. (Hirsch,

2005; and El-Osta, 2008).

Figure 5.8: Role of hyperglycemia in endothelial dysfunction

Source: European Journal of Pharmacology 636, (2010).

g) Novel approaches to antioxidant therapy

The use of new antioxidant agents that penetrate specific cellular compartments

may provide a new approach to dealing with oxidative stress in diabetes

(Hausse, 2002). Xue et al. (2008) showed that activation of Nrf2 using

sulphoraphane, which increased ARE-linked gene expression, prevented

hyperglycaemia-induced reactive oxygen species formation.

These new strategies may suggest the potential for better treatment approaches to

reduce the burden of oxidative stress and to improve endothelial function in

diabetes (Fatehi, 2010).

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h) Hydrogen peroxide in regulating cellular signals

Most biological sources of hydrogen peroxide involve the spontaneous or catalytic

breakdown of superoxide anions (O2-), produced by the partial reduction of

oxygen during aerobic respiration and following the exposure of cells to a variety

of physical, chemical, and biological agents. Increased levels of hydrogen

peroxide in cells can result in oxidative stress and cause cellular damage. Indeed,

such damage is associated with the initiation and progression of many diseases,

including neurodegenerative disorders, diabetes, atherosclerosis, and cancer.

However, studies in higher eukaryotes have revealed that hydrogen peroxide is

also used as a signaling molecule to regulate many different cellular processes.

The association of oxidative stress with disease and the aging process has led to

great interest in utilizing antioxidants to protect against oxidative stress induced

damage. This should be taken into consideration in the design of future strategies

to treat and prevent oxidative stress-associated cell damage and disease.

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5.8.6 Effect of Cashew Extracts and Fractions on The Antioxidant Defense of

Cultured Endothelial Cells.

A. Background of the studies on HMEC Cells

Much evidence in literature suggests that oxidative stress represents a major

pathogenic mechanism underlying the development and the progression of

cardiovascular diseases. Hypertension, diabetes, hypercholesterolemia and

smoking are all independent risk factors for cardiovascular disease. In both

physiologic and pathophysiologic conditions, endothelial cells, vascular smooth

muscle cells, and monocytes/macrophages are subjected to a close interplay of

oxidant and antioxidant influences (Gomez, 2001). Keap1 has been identified as a

cytosolic binding protein for Nrf2 (Nguyen, 2004). Different countries have

distinct herbal traditions, each with their indigenous plants and unique practices.

Herbs contain compounds with remarkable properties that make them potentially

powerful medicines (Balogun, 2003; Lee, 2003; Na, 2008; and Kensler, 2000).

B. Aims and objectives

To study the effect of bioactive plant extracts on the antioxidant defence of

cultured endothelial cells, specifically, the intracellular mechanism that is

governed by Nrf2. Several compounds, including known Nrf2 activators,

bioactive extracts, of leaves and testa of cashew, phenolic fractions and catechin

were tested for their potential to reduce hydrogen peroxide-induced oxidative

stress and its detrimental effects.

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C. ROS Assay:

a) Principle

The cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), also

known as dichlorofluorescin diacetate, is commonly used to detect the generation

of reactive oxygen intermediates in neutrophils and macrophages. Upon cleavage

of the acetate groups by intracellular esterases and oxidation, the nonfluorescent

H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF).

Oxidation of H2DCFDA is reportedly not sensitive to singlet oxygen directly, but

singlet oxygen can indirectly contribute to the formation of DCF through its

reaction with cellular substrates that yield peroxy products and peroxyl radicals. In

a cell-free system, H2DCF has been shown to be oxidized to DCF by peroxynitrite

anion (ONOO–), by horseradish peroxidase (in the absence of H2O2) and by

Fe2+

(in the absence of H2O2). Furthermore, the oxidation of H2DCF by Fe2+

in the

presence of H2O2 was reduced by the HO• radical scavenger formate and the iron

chelator deferoxamine. In addition, DCF itself can act as a photosensitizer for

H2DCFDA oxidation, both priming and accelerating the formation of DCF.

Although other more specialized ROS probes have been and continue to be

developed, H2DCFDA and its chloromethyl derivative CM-H2DCFDA remain the

most versatile indicators of cellular oxidative stress.

The cell-permeable dye 2',7'-dichlorofluoresceindiacetate (H2DCFDA) is oxidized

by hydrogen peroxide, peroxinitrite (ONOO-), and

hydroxyl radicals (OH

•) to

yield the fluorescent molecule 2'7'-dichlorofluorescein. Thus, dye oxidation is an

indirect measure of the presence of these reactive oxygen intermediates, calculated

by difference in the mean fluorescence of a treated sample to that of the untreated

one.

b) Cell culture

HMEC cells were cultured in Molecular, Cellular, and Developmental Biology

(MCDB) medium supplemented with 5% 200mM L-glutamine, 10,000 Units/ml

penicillin/ 10,000 µg/ml streptomycin and Fetal Bovine Serum mixture at 37ºC in

a 5% CO2 atmosphere.

c) Procedure

Cultured HMEC cells were washed with PBS and incubated at 37ºC for 30

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minutes with ROS (CM-H2DCFDA) dye previously dissolved in DMSO and

diluted in PBS to a final concentration of 10 µM.

• After incubation, the dye mixture was replaced with 100 µl/well EGM-2 and the

cells were again incubated at 37ºC for 30 minutes. Background fluorescence was

measured in a Fluroskan Ascent fluorometer at 510nm.

• HMEC’s were treated with plant extracts, incubated for 1 hour at 37ºC and then

the plate was measured to obtain the baseline fluorescence measurement.

• Next, the cells were subjected to tert-butyl hydrogen peroxide (tbH2O2) or vehicle

and the plate was measured after incubation times of 3 hours.

• The final normalized values were obtained by subtracting the baseline

fluorescence values from the fluorescence values obtained after tert-butyl

hydrogen peroxide (tbH2O2) stimulation.

• The background noise subtraction was carried out by subtracting the fluorescence

values obtained after stimulation of cells with plant extracts from the initial

fluorescence values obtained by stimulation of cells with dye.

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D. Viability Assay:

a) Principle

The assay is based on the reduction of Water soluble tetrazolium dye -1 (WST-1)

by viable cells. The reaction produces a soluble formazan salt. Recently various

kinds of tetrazolium salts (e.g. MTT2,3,4, XTT5,6,7, MTS8) are available to

measure cell proliferation or cell viability. These tetrazolium salts are cleaved to

formazan dye by the succinate-tetrazolium reductase, which exists in

mitochondrial respiratory chain and is active only in viable cells. Total activity of

this mitochondrial dehydrogenase in a sample rises with the increase of viable

cells. As the increase of enzymes' activity leads an increase of the production of

formazan dye, the quantity of formazan dye is related directly with the number of

metabolically active cells in the medium. The formazan dye formed by

metabolically active cell can be quantitated by measuring its absorbance by

ELISA reader, which enables to measure cell proliferation activity and viability.

The absorbance of formazan dye solution is in direct proportion to the number of

viable cells.

Figure 5.9: Formation of formazan in cells via the mitochondrial

dehydrogenase system

Source: http://www.roche-applied-science.com

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Figure 5.10: Cleavage of the tetrazolium salt (WST-1) to formazan

(EC - electron coupling reagent, RS - mitochondrial succinate-tetrazolium-

reductase system )

Source: http://www.roche-applied-science.com

b) Cell culture

HMEC cells were cultured in MCDB medium supplemented with 5% 200mM L-

glutamine, 10,000 Units/ml penicillin/ 10,000 µg/ml streptomycin and Fetal

Bovine Serum mixture at 37ºC in a 5% CO2 atmosphere.

c) Procedure

• Cultured HMEC (Human Microvascular endothelial cells) cells were washed with

physiological buffer saline (PBS) and incubated at 37ºC for 1 hour with plant

extracts. After incubation the plate was measured in a Multiskan FC Photometer

to obtain a baseline absorption measurement at 450 nm.

• Next, cells were simultaneously incubated with tert-butyl hydrogen peroxide and

WST-1 dye treatment and incubated at 37ºC for 3 hours. Absorption at 450 nm

was measured after incubation time periods of 3 hours.

• The background subtraction was done by subtracting the absorbance values

obtained after stimulation of cells with plant extracts from the absorbance values

obtained by stimulation of cells with tbH2O2.

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E. Angiogenesis assay

a) Principle

Angiogenesis—the formation of new blood vessels from existing vasculature—is

an integral part of both normal and pathological processes. Endothelial cells are

the key cell type involved in this process. During angiogenesis, these cells:

1. Disrupt the surrounding basement membrane

2. Migrate toward an angiogenic stimulus

3. Proliferate to provide additional cells that make up a new vessel, and

4. Re-organize to form the necessary three-dimensional vessel structure.

One of the most well-established assays to model the formation of three-

dimensional vessels is known as the tube-formation assay. Endothelial Cell Tube

Formation provides an in vitro assay system that allows assessment of a number

of cellular events such as attachment, migration, invasion and differentiation in

the angiogenesis process as well as the modulation of these events by

antiangiogenic agents. A tube formation assay is performed in 96-well format and

a process of assaying for endothelial cell tube formation and its modulation in a

high throughput manner is carried out. Neovascularization is involved in

important pathological processes such as age-related macular degeneration,

arthritis, and solid tumor growth. Hypoxia and inflammation-mediated vascular

endothelial cell growth factor (VEGF-2) induction is generally accepted as the

driving force of new vessel growth. Angiogenesis is thus a target of therapy, and

there is an active search for agents capable of arresting both new vessel growth in

vivo and the proliferation of vessel endothelial cells (EC) in vitro.

Figure 5.11: Tube formation observed In HMEC’S

Source: American Association for Cancer Research ©2002

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b) Cell culture




c) Procedure

• Cultured HMEC cells were pre-incubated with varying conditions of plant extract

treatment or medium as control for 1 hr, trypsinized and added on to IBDI slides

previously filled with 10 µl of Matrigel.

• In total, 10.000 cells were added to each well in a 50 µl volume of culture medium

containing varying concentrations of hydrogen peroxide or vehicle.

• The angiogenic capacity of the cells subjected to various conditions mentioned

above were then evaluated using Angioquant software (Company) by measuring

tube length and junction formation after incubation period of 2, 4, and 24 hrs.

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F. Western blot analysis

a) Principle

It is an analytical method wherein a protein sample is electrophoresed on an SDS-

PAGE (SDS polyacrylamide gel electrophoresis) and electrotransferred onto

nitrocellulose membrane. The transferred protein is detected using specific

primary antibody and secondary enzyme labeled antibody and substrate.

A protein sample is subjected to polyacrylamide gel electrophoresis. After this the

gel is placed over a sheet of nitrocellulose and the protein in the gel is

electrophoretically transfered to the nitrocellulose. The nitrocellulose is then

soaked in blocking buffer (3% skimmed milk solution) to "block" the nonspecific

binding of proteins. The nitrocellulose is then incubated with the specific antibody

for the protein of interest. Then the nitrocellulose is incubated with a second

antibody, which is specific for the first antibody. The second antibody will

typically have a covalently attached enzyme which, when provided with a

chromogenic substrate, will cause a color reaction. Thus the molecular weight and

amount of the desired protein can be characterized from a complex mixture (e.g.

crude cell extract) of other proteins by western blotting.

b) Cell culture

HMEC cells were cultured in MCDB medium supplemented with 5% 200mM

L-glutamine, 10,000 Units/ml penicillin/ 10,000 µg/ml streptomycin and Fetal


c) Procedure

• Treated HMEC cells were washed with PBS and lysed in a lysis buffer containing

EDTA-free protease inhibitor mixture (Roche, USA).

• Following centrifugation at 23,000g for 15 min, the supernatant was collected and

stored at -20ºC until used. The protein concentration was determined by using the

Quant-iT protein assay kit (Invitrogen, UK ).

• After addition of sample loading buffer, proteins were resolved by 10.0% SDS–

bisacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene

difluoride membranes at 300 mA for 1.5 h.

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(a) (b)

(c) (d)

(e) (f)

Figure 5.12: Various steps of western blot analysis

(a) Stacking, (b) Loading of samples, (c) Electrophoresis, (d) Transfer of blot and

(e) Antibody detection (f) Analysis

Source: http://www.steve.gb.com/science/molecular_biology_methods.html

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• The membranes were blocked in 5% dry milk reconstituted in 0.1% Tween 20 in

PBS (PBST). For Nrf2 the blots were then incubated with primary antibodies

(1:100 dilution) in 5% dry milk/PBST, washed three times with PBST, and

incubated with horseradish peroxidase-conjugated secondary antibodies (1: 2000

dilution) in 5% dry milk/PBST for 1 h.

• For β-actin the blots were then incubated with primary antibodies (1:1000

dilution) in 5% dry milk/PBST, washed three times with PBST, and incubated

with secondary antibodies (1: 2000 dilution) in 5% dry milk/PBST for 30 minutes

each.

• The blots were washed again three times with PBST, and immunoreactive protein

complexes were visualized by ECL detection reagent (Sigma Aldrich). Reactive

antigens were visualized with ECL substrate and quantified by densitometric

analysis with ChemiDoc XRS (Bio-Rad).

• Protein expression data was quantified with Quantity One Software (Bio-Rad).

The intensity of Nrf2 bands was measured using densitometry, and the Nrf2

values were plotted after normalization with beta actin densitometric values.

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G. Quantitative Real-time PCR analysis (qPCR):

a) Principle

Polymerase chain reaction (PCR) is a method that allows exponential

amplification of short DNA sequences (usually 100 to 600 bases) within a longer

double stranded DNA molecule. PCR entails the use of a pair of primers, each

about 20 nucleotides in length, that are complementary to a defined sequence on

each of the two strands of the DNA. These primers are extended by a DNA

polymerase so that a copy is made of the designated sequence. After making this

copy, the same primers can be used again, not only to make another copy of the

input DNA strand but also of the short copy made in the first round of synthesis.

This leads to exponential amplification. Since it is necessary to raise the

temperature to separate the two strands of the double strand DNA in each round of

the amplification process, a major step forward was the discovery of a thermo-

stable DNA polymerase (Taq polymerase) that was isolated from Thermus

aquaticus, a bacterium that grows in hot pools; as a result it is not necessary to

add new polymerase in every round of amplification. After several (often about

40) rounds of amplification, the PCR product is analyzed on an agarose gel and is

abundant enough to be detected with an ethidium bromide stain.

Figure 5.13: BioRad RT-PCR system

Source: Nucleic Acids Research, 2001, 29(9):e45.

96 well plate

PCR

thermocycler

system

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Figure 5.14: Schematic representation of the PCR cycle

(1) Denaturing at 94-960C, (2) Annealing at 68

0C, (3) Elongation at 72

0C

(P= polymerase), (4) The first cycle is complete. Two resulting DNA strands

make up the template DNA for the next cycle, thus doubling the amount of DNA

duplicated for each new cycle.

Source: Nucleic Acids Research, 2001, 29(9):e45.

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b) Cell culture :




c) Procedure

• Total RNA was isolated using TRIzol Reagent (Cat. No. 15596-018, Invitrogen

Life Science Technologies), quantified by spectrophotometry (ND-1000,

Nanodrop technologies) and first-strand cDNA was synthesized using the

iScript™cDNA Synthesis Kit (Cat. No. 170-8891, Bio-Rad, Hercules, CA)

according to the manufacturer’s instructions.

• Specific primers for heme oxygenase-1 (decycling) (HO-1), NAD(P)H

dehydrogenase [quinone]-1 (NQO1), glutamate-cysteine ligase, catalytic subunit

(GCLC), glutamate-cysteine ligase, regulatory subunit (GCLM) and nuclear factor

(erythroid-derived 2)-like 2/NF-E2-related factor (Nrf2) were designed, as well as

primers for several housekeeping genes (B2MG, HPRT, PPIA, 18S, P0).

• Primers were designed to work at an annealing temperature of 60 degrees Celsius,

in cases where primers functioned sub-optimally, the optimal annealing

temperature was empirically established by setting a temperature gradient on the

thermocycler.

• The real-time PCR analysis was performed with iQ™ Sybr Green Supermix (Cat.

No. 170-8885, Bio-Rad, Hercules, CA), conducted according to the instructions of

the manufacturer. The final reaction volume was set at 15 uL.

• The samples were processed in MyIQ PCR system (Bio-Rad, Hercules, CA) and

analyzed using MyiQ System Software, Version 1.0.410 (Bio-Rad Laboratories

Inc.).

• After a hot start of 3 min, each cycle consisted of a denaturation step at 95 °C for

20 s, an annealing step specific for each set of primers for 30 s and an elongation

step at 72 °C for 30 s.

• After 45 cycles a melting curve was obtained by increasing the temperature with

0.5 °C increments from 65 °C to 95 °C. With every run, as internal calibration, a

10-fold dilution series of reference cDNA was included, attained by mixing equal

amounts of cDNA from each sample and subsequently diluting the mixture in

nuclease free water.

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• The reaction efficiency was calculated by using the formula 10–1/slope

(Rasmussen, 2001). Data were analyzed using the efficiency corrected Delta-

Delta-Ct method (Pfaffl, 2001).

• The Fold-change values of the genes of interest (GOIs) were normalized using the

geometric average of the Fold-change values of multiple housekeeping genes. The

best house-keeping genes were selected by implementing the pair-wise variance

algorithm introduced in (Vandesompele, 2002) using the geNorm applet

(http://medgen.ugent.be/~jvdesomp/genorm/).

• Additional house-keeping genes were included until an M-value of <0,15 was

achieved. To illustrate the behavior of the different house-keeping genes with

regard to each other (Pfaffl, 2004), a pair-wise non-parametric (spearman)

correlation matrix between the house-keeping genes was computed, using

GraphPad Prism, Version 5.0.

• Expression values were subsequently analyzed across biological replicates by

using an experiment-mean centered approach (Kubota, 1988), using a one-way

analysis of variance (ANOVA) with Tukey’s post test for determining the p-value.

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5.9 PHARMACOLOGICAL INVESTIGATIONS OF CASHEW EXTRACTS

FOR ANTIDIABETIC ACTVITY

5.9.1 Acute oral Toxicity Studies

The acute toxic class method set out in the OECD Guideline 423 is a stepwise

procedure with the use of 3 animals of a single sex per step. Depending on the

mortality and/or the mortality status of the animals, on average 2-4 steps may be

necessary to allow judgment on the acute toxicity of the test substance. This

procedure is reproducible, uses very few animals and is able to rank substances in

a similar manner to the other acute toxicity testing methods (Test Guidelines 420

and 425). The acute toxic class method is based on biometric evaluations with

fixed doses, adequately separated to enable a substance to be ranked for

classification purposes and hazard assessment. The method as adopted in 1996

was extensively validated in vivo against LD50 data obtained from the literature,

both nationally and internationally (OECD guidelines, 2001).

a) Background

The bioactive extracts of cashew leaves and testa were to be tested for antidiabetic

activity. Prior to any pharmacological activity it is essential to determine the LD50

of the candidate extracts, hence acute oral toxicity study was carried out.

b) Principle

The test is based on a stepwise procedure with the use of a minimum number of

animals per step, sufficient information is obtained on the acute toxicity of the test

substance to enable its classification. The test substance is administered orally to a

group of experimental animals at one of the defined doses. The substance is tested

using a stepwise procedure, each step using three animals of a single sex

(normally females). Absence or presence of compound-related mortality of the

animals dosed at one step will determine the next step, i.e.;

− no further testing is needed,

− dosing of three additional animals, with the same dose

− dosing of three additional animals at the next higher or the next lower dose

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level.

The method enables a judgement with respect to classifying the test substance to

one of a series of toxicity classes defined by fixed LD50 cut-off values.

A. Methodology

a) Selection of animal species

Female Albino mice (Wistar strain) were selected for the study. Healthy young

animals, 2 months old and 20-25g weight range of commonly used laboratory

strains were employed in the study. Females used were nulliparous and non-

pregnant. Normally females are used for the study, because literature surveys of

conventional LD50 tests show that, although there is little difference in sensitivity

between the sexes, in those cases where differences are observed females are

generally slightly more sensitive.

b) Housing and feeding conditions

The temperature in the experimental animal room was 22ºC (±3ºC) with the

relative humidity around 30%. Lighting was artificial, the sequence being 12

hours light, 12 hours dark. For feeding, conventional laboratory diet was used

with an unlimited supply of drinking water. Animals were caged based upon

various groups of plant extracts by dose, such that the number of animals per cage

must not interfere with clear observations of each animal.

c) Preparation of animals

The animals were randomly selected, marked to permit individual identification,

and kept in their cages for 5 days prior to dosing to allow for acclimatization to

the laboratory conditions. Animals were procured from Haffkine’s Research

Institute, Mumbai.

d) Preparation of doses

The extract was administered in a constant volume over the range of doses to be

tested by varying the concentration of the dosing preparation. Care was taken, not

to exceed 1.0 mL i.e. the maximum dose volume for administration. The extracts

were suspended in 0.05% CMC for administration. Doses were prepared freshly

prior to administration.

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e) Administration of doses

The test extracts were administered as a single dose by oral gavage using a

suitable intubation canula and observed for a period of 14 days.

a) Number of animals and dose levels

Three animals were used for each step for individual extracts in the study. Dose

limit at 2000 mg/kg (single dose) was administered to mice and observed for 14

days. Groups of 3 animals (control and test group), each were formed. The

animals in the control group received water.

B. Observations

Animals were observed individually after dosing at least once during the first 30

minutes, periodically during the first 24 hours, with special attention given during

the first 4 hours, and daily thereafter, for a total of 14 days, except where they

need to be removed from the study and humanely killed for animal welfare

reasons or were found dead.

All observations were systematically recorded with individual records being

maintained for each animal. Additional observations like that of tremors,

convulsions, salivation, diarrhea, lethargy, sleep and coma were observed when

toxic symptoms were seen in animals. Animals found in a mortality condition

were humanely killed. When animals were killed for humane reasons or found

dead, the time of death was recorded as precisely as possible.

• Body weight and food intake

Animals treated with extracts of cashew leaves and testa were observed for body

weight gain and food intake throughout the study. Individual weights of animals

were determined shortly before the test extracts were administered and daily

thereafter. Weight changes were calculated and recorded. At the end of the test

surviving animals were weighed and humanely killed.

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5.9.2 Evaluation of The Effect Of Cashew Leaves And Testa Extracts In

Streptozotocin-Nicotinamide Induced Type-II Diabetic Rats

A. Introduction

Although streptozotocin (STZ) is commonly used to produce an experimental

model of diabetes in laboratory animals, the destruction of the pancreatic islet cell

is an undesirable side effect when STZ is employed as a chemotherapeutic agent

in treatment of tumors. Therefore, suitable protective substances have been

sought, which will allow STZ to retain its complete anticancer activity but also

preserve normal pancreatic function in treated animals. Figure 5.15 illustrates the

mechanistic pathway of STZ showing the sites of action of protective agents,

several of which have nutritional importance. In reports published till date it is

observed that 2- deoxyglucose and 3-O-methyl glucose completely protected

against STZ action in rats which is probably due to structural similarity to STZ

and inhibition of the drug into the pancreatic cell. Nicotinamide, picolinamide,

theophylline and several benzamides all inhibit islet poly (ADP ribose) synthetase

hence do not deplete pancreatic NAD concentrations. The antioxidants vitamin E

and dimethyl urea also have protective effects in rodents. It has been postulated

that STZ can act as an oxidant and can initiate changes in the redox state of the

islet cell. Indeed, a fall in reduced glutathione and rise in the oxidized form occurs

in rat islet cells in vitro. As a result, lowered amounts of pyridine nucleotides are

produced by the hexose monophosphate shunt (HMPS). The role of STZ as a free

radical generator, however, remains controversial since neither superoxide

dismutase, nor vitamin C, both having antioxidant properties, demonstrated

significant protective effects (Ganda.1976).

It should also be mentioned that modification of STZ-induced diabetes by

nicotinamide and other protective agents may be of primary benefit over a limited

time span and over a longer period may actually potentiate the well known

oncologic action of STZ. To this point, researchers have demonstrated a dramatic

increase in β-cell tumors of surviving animals one year after administration using

poly (ADP ribose) synthetase inhibitors and STZ in rats (Okamoto, 1983). The

retardation of prompt DNA repair seems to be closely related to this phenomenon.

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Nicotinamide can exert a partial protection against the β cytotoxic effect of

streptozotocin. This model appears closer to human type 2 diabetes than other

available models (neonatally STZ- injected rats,GK rats),with regard to insulin

responsiveness to glucose and sulphonyl ureas (Okamoto, 1983).

Figure 5.15 Mechanistic pathway of STZ showing the sites of action of

protective agents

Source: Journal of Biological Chemistry, 257: 6084, 1982.

Figure 5.15 illustrates the protecting mechanism against action of streptozotocin

on pancreatic β-cells. As indicated in the figure, vitamin E and dimethyl urea may

protect against DNA strand breaks through antioxidant action, poly (ADP-ribose)

synthetase inhibitors such as nicotinamide, picolinamide, theophylline, etc. may

protect streptozotocin-induced depression of proinsulin synthesis by inhibiting

NAD degradation through poly (ADP-ribose) and 2 deoxyglucose and 3-O-methyl

glucose may protect by inhibiting cellular uptake of streptozotocin.

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B. Background

From the seven extracts prepared from leaves and testa of Cashew, three extracts

were selected for STZ - Nicotinamide Induced Type 2 Diabetes Mellitus model

based upon the acute toxicity study and antioxidant effects and IC50 values. The

doses of the extracts were selected based upon the literature available for the

cashew leaves and testa extracts.

C. Preparation of reagents

• Preparation of STZ and Nicotinamide solution

STZ is stable in citrate buffer (pH 4.5). A solution of STZ was prepared by

dissolving a weighed quantity of STZ in freshly prepared ice cold buffer pH (4.5)

solution and administered intra peritoneally. Nicotinamide was dissolved in

normal saline. The STZ in sodium citrate buffer was prepared freshly and injected

within 5 min so as to avoid degradation. Nicotinamide was administered 15 min

before STZ administration.

D. Experimental

• Animals groups

Healthy adult albino rats aged between 2 and 3 months of age, weighing 250–

300g were used for the pharmacological studies. The animals were housed in

polypropylene cages, maintained under standard conditions (12/12h light and

dark) at 25±3◦C and 35–60% humidity. They were fed with standard rat pellet diet

and water ad libitum. The study protocol was approved by Institutional animal

Ethical Committee (Approval No: CUSCP/IAEC/29&31/09-10). Animals were

procured from Haffkine’s Research Institute, Mumbai, and acclimatized with free

access to food and water for at least 1 week (Barik, 2008 and Shirwaikar, 2004).

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• Two study groups for standardization of STZ-Nicotinamide dose were as follows:

Group 1: Streptozotocin: 60 mg/kg (i.p) and nicotinamide: 120 mg/kg (ip)

Group 2: Streptozotocin: 60 mg/kg (i.p) and nicotinamide: 100 mg/kg (ip)

• The study groups for interventional study of STZ-nicotinamide model containing

six animals each were as follows:

Group 1: Normal control [treated with saline]

Group 2: Positive control [treated with Glibenclamide 0.45 mg/kg]

Group 3: Diabetic control [treated with streptozotocin (60 mg/kg i.p) 15 min after

the administration of (100 mg/kg i.p) nicotinamide]

Group 4: Treatment group [treated with ethanol extract of Cashew testa

175 mg/kg]

Group 5: Treatment group [treated with polyphenol fraction of Cashew testa

50 mg/kg]

Group 6: Treatment group [treated with ethanol extract of Cashew leaves

100 mg/kg]

Group 7: Treatment group [treated with ethanol extract of Cashew testa

350 mg/kg in divided doses]

E. Methodology - Induction of Experimental diabetes

a) Standardization of Streptozotocin–Nicotinamide (STZ-NA) dose to induce

Type II Diabetes condition– NIDDM in rats

• All the animals had free access to water and food. A rat model of type 2 diabetes

mellitus (non-insulin dependent diabetes mellitus, NIDDM) was induced in

overnight-fasted rats by a single intraperitoneal injection of streptozotocin (60

mg/kg) 15 min after the intraperitoneal administration of nicotinamide (100 mg/kg

i.p)

• Blood samples were obtained from the retro-orbital plexus in both streptozotocin-

injected and control animals at 72 hours and on day 7 after an overnight fast.

Hyperglycemia was confirmed by elevated blood glucose levels determined at

72 h and then on day 7 after injection. Only rats confirmed to have permanent

NIDDM were used for the antidiabetic study.

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• The rats were supplied with 5% glucose water and ad libitum basal diet during the

next 24 hours to avoid sudden hypoglycemia post-injection. On day 2, water was

replaced with drinking water. Fasting blood glucose levels were determined by

glucose oxidase method. Rats with fasting blood glucose levels above 200 mg/dL

were considered diabetic.

b) Intervention study

• A rat model of type 2 diabetes mellitus (non-insulin dependent diabetes mellitus,

NIDDM) was induced in overnight-fasted rats by a single injection of

streptozotocin (60 mg/kg i.p) 15 min after the administration of nicotinamide (100

mg/kg i.p).

• The rats were supplied with 5% glucose water and ad libitum basal diet during the

next 24 hours to avoid sudden hypoglycemia post-injection. On day 2, water was

replaced with drinking water.

• Blood samples were obtained from the retroorbital plexus in both streptozotocin-

injected and control animals at 72 hours and on day 7 after an overnight fast. Rats

with stable elevated fasting blood glucose levels on day 7 and above 200 mg/dL

were considered diabetic and chosen for the interventional study. Fasting blood

glucose levels were determined by glucose oxidase method.

• The animals were grouped randomly based on their blood glucose levels, each

having six animals. The control group received 0.05 % suspension of CMC and in

the treatment group the drug/extracts were suspended in 0.05% CMC

administered orally for 15 days.

• At the end of the experimental period, the rats were fasted overnight and blood

samples were withdrawn from the retro orbital plexus. Serum samples were used

for the various biochemical estimations.

c) Statistical analysis

All statistical analyses were made using the software InStat for windows. All

results were expressed as mean ± SEM. Post hoc Dunnett’s test was used to

determine statistical significance. The values were considered statistically

significant when p<0.05.

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5.9.3 Evaluation of the effect of cashew leaves and testa extracts in neonatal

streptozotocin induced (n- STZ) rat model of Type 2 Diabetes Mellitus

A. Introduction

In this model STZ is used within 5 days from birth in rats in dose ranging from

80-100 mg/kg to develop symptoms similar to type II diabetes, known as (n0-n5

model). Most of the beta cells are destroyed by STZ in neonates, but they

gradually regenerate to half the original mass. The animals exhibiting blood

glucose level above 100 mg/dL are considered to be diabetic. By varying the

administration time of STZ one can obtain hyperglycemia of differing severity

depending upon the extent of beta cell damage and regeneration. Susceptibility of

rats to STZ varies with species, strain, sex, age and nutritional state of the animals,

and all the STZ treated animals do not develop hyperglycemia. By 6 weeks of age

neonates showed basal hyperglycemia and abnormal glucose tolerance. Variations

in blood glucose levels, high mortality (30-50%) due to STZ toxicity and lack of

response to oral hypoglycemic drugs are the drawbacks of n0-n5. A study

showed that a spilt dose regimen of STZ injected over two consecutive days (day

0 and 1) after birth induces hyperglycemia and decreases pancreatic insulin stores

by day 5 as compared to a single dose (Portha, 2003; and Portha, 2007 b).

NIDDM or type II diabetes is the most common form of the disease which is

caused by impaired insulin secretion paralleled by a progressive decline in β-cell

function and chronic insulin resistance. Insulin resistance is a main reason in

pathogenesis of type II diabetes and occurs when the cellular mechanisms fail to

respond to insulin effects. The neonatal rats treated with streptozotocin (STZ) on

the first day of birth showed hyperglycemia and reduction in pancreatic insulin

amount during neonatal period and which could be maintained up to adulthood.

This diabetic rat’s model resembles the human NIDDM. This model is the result

of the spontaneous evolution of the administration of streptozotocin to 2-day-old

neonates. Adult diabetic rats are mildly hyperglycemic, mildly hypoinsulinemic

and strongly intolerant to glucose. Neonatal streptozotocin administration has

been used widely to mimic the physiopathology of the gestational diabetic state.

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a) The β-cell function in n-STZ models

• Quantitative and qualitative defects in insulin secretion in Type 2 diabetes

mellitus:

Type 2 diabetic subjects display more subtle changes in the dynamics of insulin

secretion, such as blunting of the first phase insulin secretion and disruption of the

insulin secretory pulses. The first phase is a very brief surge of insulin that follows

an acute secretagogue challenge such as an intravenous glucose bolus. It peaks

after 2-4 min and dissipates within 6-10 min. If the challenge is sustained, the

prolonged second phase starts; the second phase supervenes and lasts until the

glucose is cleared. The first phase is a more efficient signal than the second, both

in enhancing glucose clearance and priming the liver to shut down glucose

production. Glucose regulates β-cell function in two ways. It produces a direct

release of insulin as a result of enhancement of the concentrations and its pre-

stimulus level modulates the response to the islet secretagogues. It is suggested

that the first phase response to glucose is specifically abolished in Type 2 diabetes

mellitus and the second phase insulin secretion is attenuated. It is not clear which

defect has a greater impact on glycemia. Insulin, like other hormones, is secreted

in pulses and this appears to be a fundamental signal for hormone signaling. In

normal subjects under fasting conditions, insulin release occurs in regular pulses

with a periodicity of about 13 min. But in subjects with Type 2 diabetes mellitus

insulin secretory profiles are more chaotic, and also the regular 13 min pulses are

absent. As indicated in figure 5.15, streptozotocin liberates toxic amounts of nitric

oxide that inhibits aconitase activity and participates in DNA damage. As a result

of the streptozotocin action, β-cells undergo the destruction by necrosis

(Arulmozhi, 2004).

• Defects in exocrine pancreas in n-STZ rats:

The acute STZ administration at birth to neonates affects subsequent exocrine

pancreas development, particularly that of amylase while exogenous insulin

attenuated the effect.

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Figure 5.16: Probable mechanism of action of STZ

Source: Indian J Pharmacol, 2004, 36(4), 217-221.

• β-cell regeneration in n-STZ rats:

It has been reported that after the n0-STZ injection, from the postnatal day 4

onwards, signs of regeneration are apparent, in that numerous insulin positive

cells are found throughout the acinar parenchyma and within the duct epithelium,

but in 4-month-old animals the regeneration process remains incomplete. The

timing of the STZ injection is the critical factor for the efficiency of the

regeneration process, which coincides with the normal development of islet cell

mass in the rat. It has been proved that there is some capacity of β cell

regeneration in the neonatal rat pancreas (which is lacking in the adult rodents)

and the capacity of the β cell regeneration in the Wistar strain decreases quickly

during the first postnatal week and thereafter it is no longer significant. It is also

explained that the regeneration of the β cells in the Sprague Dawley neonates is

less efficient than in the Wistar strain. The recovery from diabetes mellitus in the

Sprague-Dawley n2-STZ model is due to the partial replenishment of the β-cell

mass from the replication of the existing β-cells, rather than neogenesis from

undifferentiated precursors (Arulmozhi, 2004).

• Insulin resistance in n-STZ models:

There are evidences that a severe reduction in the β cells obtained from subjects

with Type 2 diabetes mellitus or animals after STZ injection is associated with no

severe insulin resistance. It is found that the induction of insulin resistance in an

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individual with reasonably normal islet function leads to modest elevation of the

plasma glucose level, whereas in an individual with impaired islet cell functions it

leads to hyperglycemia.

In contrast to the above findings, in 8-week-old n0-STZ female rats, it was shown

that hepatic glucose production measured in the basal state was higher in the

diabetes mellitus models than in the controls, despite similar peripheral insulin

levels in both groups. It is found that in white and brown adipose tissues, an

increased responsiveness to insulin action is detected when comparing diabetic

females to control females and insulin action was found normal in the skeletal

muscles and diaphragm of the same adult females. The observations of the

hormonal insulin action in the liver and white and brown adipose tissues indicated

that glucose is preferentially channeled towards the liver and adipose tissue in

n0-STZ females. The studies in the n5-STZ model showed that the glucose

utilization by the whole body mass induced by hyperinsulinemia was significantly

reduced and the hepatic glucose production rate was less efficiently suppressed by

submaximal or maximal insulin levels, which indicated that the insulin resistance

is present in vivo at the level of the peripheral tissues and the liver. When β-cell

insult is the primary factor responsible for the emergence of moderate to severe

hyperglycemia in rats, insulin resistance can develop secondarily and a certain

degree of insulin deficiency is necessary to induce insulin resistance.

b) Merits of n-STZ rat model of Type 2 diabetes mellitus

By altering the dose and the day of the STZ injection, the n-STZ models exhibit

various stages of Type 2 diabetes mellitus, such as impaired glucose tolerance,

mild, moderate and severe hyperglycemia. The n-STZ rats exhibit slightly lowered

plasma insulin levels, slightly elevated plasma glucose levels and lowered

pancreatic insulin content. As indicated in figure 5.2, the β-cells in the n-STZ rats

bear a resemblance to the insulin secretory characteristics found in Type 2 diabetic

patients. As indicated in table 5.2, the pattern of insulin release found in the n0-

STZ and n2- STZ rats is qualitatively similar to that of the rat, which is a

genetically diabetic non-obese model of human diabetes mellitus.

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c) Defects in n-STZ models

The impairment of glucose-induced insulin release in n-STZ rat is clearly related

to a defect in oxidative glycolysis. This leads to a severe decrease in the

mitochondrial oxidative catabolism of glucose-derived pyruvate. It coincides with

a lower ATP/ADP ratio in simulated islets and their subsequent alteration of ionic

events rightly coupled to the fuel function of the hexose in the islet cells. It has

been found that the n-STZ rats exhibited an increased amylin-insulin molar ratio.

Table 5.2: Comparison of human type 2 diabetes and n-STZ diabetic animals

Parameters

Human Type 2 diabetes n-STZ diabetes

Pancreatic insulin ++ + / -

Basal plasma glucose ++ ++

Basal plasma insulin ++ +

Glucose tolerance - -

Insulin tolerance ++ + / -

Obesity - + / -

Diabetic complications + +

‘ + ‘ = Present; ‘ - ‘ = Absent

� n-STZ rat model of Type 2 diabetes mellitus

A. Background

From the seven extracts prepared from leaves and testa of cashew, three extracts

were selected for STZ - nicotinamide induced Type 2 Diabetes Mellitus model

based upon the acute toxicity study and antioxidant effects and IC50 values. The

doses of the extracts were selected based upon the literature available for the

cashew leaves and testa extracts. The extracts which showed significant activity in

the STZ - nicotinamide induced Type 2 Diabetes Mellitus model, were selected

for neonatal streptozotocin-induced rat model of Type 2 diabetes mellitus (Barik,

2008 and Shirwaikar, 2004).

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B. Preparation of reagents

• Preparation of STZ solution

STZ is stable in citrate buffer (pH 4.5). A solution of STZ was prepared by

dissolving accurately weighed amount of STZ in freshly prepared ice cold citrate

buffer (pH 4.5) solution and administered intra peritoneally. The STZ in sodium

citrate buffer was prepared freshly and injected within 5 min so as to avoid

degradation. The buffer was prepared freshly before injection as the drug degrades

after 15-20 minutes in the citrate buffer.

C. Experimental design

• Animal groups

Animals were procured from Haffkine’s Research Institute, Mumbai, and

acclimatized with free access to food and water for at least 1 week. Healthy adult

albino rats aged between 2 and 3 months of age, weighing 250–300g were used

for the pharmacological studies. The animals were housed in polypropylene cages,

maintained under standard conditions (12/12h light and dark) at 25±3◦C and 35–

60% humidity. They were fed with standard rat pellet diet and water ad libitum.

The study protocol was approved by Institutional animal Ethical Committee

(Approval No: CUSCP/IAEC/29&31/09-10).

Three study groups for standardization of STZ dose were as follows:

Group 1: Control: Citrate buffer (i.p) on day 2

Group 2: Streptozotocin: 70 mg/kg (i.p) on day 5 {n5-STZ}



• The study groups for interventional study of neonatal streptozotocin-induced

(n-STZ) model containing six animals each were as follows:

Group 1: Normal control [treated with saline]

Group 2: Positive control [treated with standard Pioglitazone 2 mg/kg]

Group 3: Diabetic control [treated with streptozotocin (100 mg/kg i.p)]

Group 4: Treatment group [treated with ethanol extract of cashew testa

175 mg/kg]

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D. Methodology - Induction of experimental diabetes

a) Standardization of streptozotocin dose to induce Type II Diabetes condition in

neonates

• All the animals had free access to water and food. A type 2 diabetes mellitus (non-

insulin dependent diabetes mellitus, NIDDM) was induced in neonates after 2 and

5 days of birth. The neonates were injected by a single intraperitoneal injection of

streptozotocin (100 mg/kg i.p)

• The pups were weaned after 30 days, and the animals were checked for fasting

glucose levels after 3 months of STZ injection.

• When animals were 12 weeks old, an oral glucose tolerance test was performed

(2g glucose/kg b.w was fed). Blood samples were withdrawn at 0,30,60,90 and

120 min. Glucose was administered after 0 min reading. All the animals were

fasted overnight before experiments. Animals which were intolerant to glucose by

OGTT were selected for the experiments.

b) Intervention study

• Two day old neonates were injected with 100 mg/kg of streptozotocin. When

animals were 12 weeks old, an oral glucose tolerance test was performed.

• All the animals were fasted overnight before experiments. Animals which were

intolerant to glucose by OGTT and fasting blood glucose levels >120 mg/dL were

selected for the interventional study with test compounds. Fasting blood glucose

levels were determined by glucose oxidase method.

• The animals were grouped randomly based on their blood glucose levels, each

having six animals. The control group received 0.05 % suspension of CMC. The

treatment group received the drug/extracts suspended in 0.05% CMC. The

administration of the drugs was done orally for 30 days.

• At the end of the experimental period, the rats were fasted overnight and blood

samples were withdrawn from the retro orbital plexus. Serum samples were used

for the various biochemical estimations. The whole blood was collected in EDTA

coated vials and used for the estimation of glycated haemoglobin.

• Serum was stored at –20 0 C for insulin estimation and analyzed by Radio immuno

assay. Histopathology was conducted on selected organs which were preserved in

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10% formalin solution. Tissues were processed, sectioned and stained with

hematoxylin and eosin.

c) Statistical analysis

• All statistical analyses were made using the software InStat for windows. All

results were expressed as mean ± SEM. Post hoc Dunnett’s test was used to

determine statistical significance. The values were considered statistically

significant when p<0.05.

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F. Details of various procedures used for biochemical estimations

a) Determination of blood glucose level (Glucose Oxidase Method)

• Principle of the method

Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid. The

formed hydrogen peroxide (H2O2), is detected by a chromogenic oxygen acceptor,

phenol-aminophenazone in the presence of peroxidase (POD):

β-D-Glucose + O2 + H2O GOD

Gluconic acid + H2O2

H2O2 + Phenol + Aminophenazone POD

Quinone + H2O

The intensity of the color formed is proportional to the glucose concentration in

the sample (Kaplan, 1984 a).

• Clinical significance

Glucose is a major source of energy for most cells of the body; insulin facilitates

glucose entry into the cells. Diabetes is a disease manifested by hyperglycemia;

patients with diabetes demonstrate an inability to produce insulin.

• Procedure

1. Assay conditions:

Wavelength of detection: . . . . . . . . 505 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path

Temperature. . . . . . . . . . . . . . . . . . 37ºC

2. The instrument was adjusted to zero with distilled water.

3. Into a cuvette the following solutions were pippeted:

Blank Standard Sample

Working Reagent (mL) 1.0 1.0 1.0

Standard (µL) -- 10 --

Sample (µL) -- -- 10

4. The samples were mixed and incubated for 10 min at 37ºC

5. The absorbance (A) of the samples and standard, were measured against the

blank. The colour was stable for at least 30 minutes.

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• Calculations

(A)Standard -

--------------------- x 100 (Standard conc.) = mg/dL glucose in the sample

(A) Sample

• Conversion factor: mg/dL x 0.0555= mmol/L.

• Reference Values

Serum or plasma: 60 – 110 mg/dL 3.33 – 6.10 mmol/L CSF: 60 – 80% of the

blood value These values are for orientation purpose; each laboratory should

establish.

b) Determination of blood glucose level (Hand-held accu Check Glucose meter)


The Measurement of the blood glucose level by ACCu check glucose meter

occurs through the following electrochemical and enzymatic reactions described

below:

Incubation period: After drop detect, glucose dehydrogenase catalyzes a selective

electron-transfer reaction between glucose in the sample and M (potassium

ferricyanide) in the reagent layer as indicated in figure 5.17:

Figure 5.17: Enzyme reactions occuring in glucose measurement strips

during measurement

Source: Current separations, 21 (2),45-48; 2005.

Each molecule of glucose reduces two molecules of ferricyanide, creating two

molecules of ferrocyanide. The final ferrocyanide concentration is directly

correlated to the sample glucose concentration.

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Measurement period: During the measurement period,the meter applies a potential

difference between the working and counter electrodes. The counter electrode

potential is defined by the ratio of ferricyanide and ferrocyanide at the electrode

surface:

Since the amount of ferrocyanide is small relative to the amount of ferricyanide,

the concentration ratio (and hence the counter electrode potential) is effectively

constant. This applied potential difference is sufficient to provide a diffusion-

limited current at the working electrode, so the ferrocyanide concentration may be

determined by biamperometry.

As shown in figure 5.18, the meter measures working electrode current, which is

linked to ferrocyanide concentration. Because the ferrocyanide concentration is

coupled to glucose concentration, the current measurement permits calculation of

blood glucose.

Figure 5.18: Measurement of blood glucose level by glucometer

Source: Current separations, 21 (2),45-48; 2005.

• Procedure:

The measurement sequence itself consists of five time segments:

1. When biosensor is inserted, the meter automatically turns on and performs a series

of tests.

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2. A small drop of blood from the rat tail is placed on the strip. After the blood drop

is placed on the biosensor strip, the meter applies a potential difference to detect

sample (Drop detect).

3. Following sample application (Drop detect), electrode potential difference is

removed and enzymatic reaction is permitted to proceed (Incubation period ).

4. After the meter applies a potential difference and measures current (Measurement

period).

5. Current data are analyzed, the result is recorded and displayed.

c) Quantitative determination of alanine aminotransferase (ALT)

• Principle of The Method

Alanine aminotranferase (ALT) catalyses the reversible transfer of an amino

group from alanine to - ketoglutarate forming glutamate and pyruvate.

The piruvate produced is reduced to lactate by lactate dehydrogenase (LDH) and

NADH:

L-Alanine + α-Ketoglutarate ALT

Glutamate + Pyruvate

Pyruvate + NADH + H+

LDH Lactate + NAD+ LDH

The rate of decrease in concentration of NADH, measured photometrically, is

proportional to the catalytic concentration of ALT present in the sample (Murray,

1984).

• Clinical Significance

The ALT is a cellular enzyme, found in highest concentration in liver and kidney.

High levels are observed in hepatic disease like hepatitis, diseases of muscles and

traumatisms, its better application is in the diagnosis of the diseases of the liver.

When they are used in conjunction with AST aid in the diagnosis of infarcts in the

myocardium, since the value of the ALT stays within the normal limits in the

presence of elevated levels of AST.

• Procedure


Wavelength of detection: . . . . . . . . . . . 340 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . 1 cm light path

Constant temperature . . . . . . . . .. . . . . .25ºC

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3. The solutions were pipetted into a cuvette as mentioned below:

Working Reagent (mL) 1.0

Sample (µL) 100

4. The solutions were mixed, and incubated for 1 minute. The initial absorbance

(A) of the sample, was measured at 1 minute intervals thereafter for 3 minutes.

6. The difference between absorbances and the average absorbance differences per

minute ( A/min) were calculated as follows:

∆ A/min x 1750 = U/L of ALT

A: Absorbance

U/L: Units/ Litre

• Reference values

25ºC 30ºC 37ºC

Men up to 22 U/L 29 U/L 40 U/L

Women up to 18 U/L 22 U/L 32 U/L

Normal newborns have been reported to show a reference range of up to double

the adult, attributed to the neonate’s hepatocytes. These values decline to adult

levels by approximately 3 months of age.

d) Quantitative determination of aspartate aminotransferase (AST)


Aspartate aminotransferase (AST) formerly called glutamate oxaloacetate (GOT)

catalyses the reversible transfer of an amino group from aspartate to ketoglutarate

forming glutamate and oxalacetate (Murray, 1984). The oxalacetate produced is

reduced to malate by malate dehydrogenase (MDH) and NADH:

L-Aspartate + α -Ketoglutarate AST

Glutamate + Oxaloacetate

Oxaloacetate + NADH + H+

MDH Malate + NAD

+

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The rate of decrease in concentration of NADH, measured photometrically, is

proportional to the catalytic concentration of AST present in the sample.


The AST is a cellular enzyme, is found in highest concentration in heart muscle,

the cells of the liver, the cells of the skeletal muscle and in smaller amounts in

other weaves.

Although an elevated level of AST in the serum is not specific of the hepatic

disease, is used mainly to diagnostic and to verify the course of this disease with

other enzymes like ALT and ALP. Also it is used to control the patients after

myocardial infarction, in skeletal muscle disease and other.

• Procedure


Wavelength of detection: . . . . . . . . . . ..340 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . 1 cm. light path

Constant temperature . . . . . . . . . . . . . . .25ºC


3. The solutions were pipetted out into a cuvette as follows:

Working Reagent (mL) 1.0

Sample (µL) 100

4. The solutions were mixed and incubated for 1 minute.

5. The initial absorbance (A) of the sample, was measured and the absorbances

absorbances at 1 minute intervals thereafter for 3 minutes were also recorded.

6. The difference between absorbances and the average absorbance differences per

minute ( A/min) were calculated as follows:

∆ A/min x 1750 = U/L of AST


25ºC 30ºC 37ºC

Men up to 19 U/L 26 U/L 38 U/L

Women up to 16 U/L 22 U/L 31 U/L

P a g e | 172


e) Quantitative determination of cholesterol

• Principle of the method The cholesterol present in the sample originates a

coloured complex, according to the following reaction:

Cholesterol esters + H2O Cholesterol esterase

Cholesterol + fatty acids

Cholesterol + O2 Cholesterol oxidase

4-Cholestenona + H2O2

2 H2O2+ Phenol + 4-Aminophenazone peroxidase

Quinonimine + 4H2O

The intensity of the color formed is proportional to the cholesterol concentration

in the sample (Naito, 1984).


Cholesterol is found in all body cells. The liver makes all of the cholesterol the

body needs to form cell membranes and to make certain hormones. The

determination of serum cholesterol is one of the important tools in the diagnosis

an classification of lipemia. High blood cholesterol is one of the major risk factors

for heart diseases.

• Procedure


Wavelength of detection: . . . . . . . .. 505 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path

Temperature . . . . . . . . . . . . . . . .. . .37ºC


3. The solution was pipetted out into a cuvette as follows:

Blank Standard Sample

WR (mL) 1.0 1.0 1.0


Sample (µL) -- -- 10

4. The solutions were mixed and incubated for 5 min. at 37º C or 10 min. at room

temperature.

P a g e | 173


5. The absorbance (A) of the samples and Standard, were measured against the blank.

The colour is stable for at least 60 minutes.

• Calculations

(A) Sample -

-------------------- x 200 (Standard conc.) = mg/dL Cholesterol in the sample

(A) Standard



Risk evaluation is done based on the following values:

Less than 200 mg/dL Normal

200-239 mg/dL Borderline

240 mg/dL and above High

f) Determination of HDL cholesterol (HDL-C)


The very low density (VLDL) and low density (LDL) lipoproteins from serum or

plasma are precipitated by phosphotungstate in the presence of magnesium ions.

After removed by centrifugation the clear supernatant containing high density

lipoproteins (HDL) is used for the determination of HDL cholesterol (Naito,1984).


HDL particles carry cholesterol from the cells back to the liver. HDL is known as

“good cholesterol” because high levels are thought to lower the risk of heart

disease. A low HDL cholesterol levels, is considered a greater risk for heart

disease.

• Procedure

Precipitation process:

1. The solutions were pipetted into a centrifuge tube as mentioned below:

Working reagent (µL) 100

Sample (mL) 1.0

P a g e | 174


2. The solution mixture was allowed to stand for 10 min at room temperature.

3. The solution was then centrifuged at 4000 r.p.m. for 20 min

4. The supernatant was collected and HDLc was tested.

• Calculations (Naito,1984)

With calibrator

(A) Sample -

-------------------- x 200 (calibrator conc.) = mg/dL HDL-C in the sample

(A) Calibrator

With Factor:

A505 nm Sample x 320 = mg/Dl HDL-C in the sample.

A546 nm Sample x 475 = mg/Dl HDL-C in the sample

• Calculation of LDL-cholesterol

According to the Friedewald Formula (Naito, 1984).:

LDL cholesterol = Total cholesterol - Triglycerides x HDL cholesterol

-------------------

5

• Reference Values

HDL-cholesterol:

Men Women

Lower risk > 55 mg/dL > 65 mg/dL

Standard risk 35-55 mg/dL 45-65 mg/dL

Increased risk < 35 mg/dL < 45 mg/dL

LDL-cholesterol:

Suspected above : 150 mg/dL

Increased above : 190 mg/dL

g) Quantitative determination of creatinine


The assay is based on the reaction of creatinine with sodium picrate (Jaffé’s fluid).

Creatinine reacts with alkaline picrate forming a red complex. The time interval

chosen for measurements avoids interferences from other serum constituents. The

P a g e | 175


intensity of the color formed is proportional to the creatinine concentration in the

sample (Murray, 1984).


Creatinine is the result of the degradation of the creatine, component of muscles, it

can be transformed into ATP, that is a source of high energy for the cells. The

creatinine production depends on the modification of the muscular mass, and it

varies little and the levels usually are very stable. It is excreted by the kidneys.

With progressive renal insufficiency there is retention in blood of urea, creatinine

and uric acid. Elevated creatinine level may be indicative of renal insufficiency

(Murray, 1984).

• Procedure


Wavelength: . . . . . . . . . . . . . . . . . 492 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .1 cm. light path

Temperature. . . . . . . . . . . . . . . . . . 37ºC


3. The solutions were pipetted into a cuvette as follows:

Blank Standard Samples

WR (mL) 1.0 1.0 1.0


Sample ( µL) -- -- 100

4. The solutions were mixed and the absorbance (A1) after 30 seconds and after 90

seconds (A2) of the sample addition was recorded.

6. ∆A was calculated as, ∆A= A2 – A1

• Calculations

∆A Sample – ∆A Blank

------------------------------ x 2 (Standard conc.) = mg/dL of creatinine in sample

∆A Standard – ∆A Blank

• Conversion factor: mg/dL x 88.4 = mol/L.

P a g e | 176



Serum levels

Male 0,7 - 1,4 mg/dL

Female 0,6 - 1,1 mg/dL

Urine: 15-25 mg/Kg/24 h

Male 10 - 20 mg/Kg/24 h

Female 8 – 18 mg/Kg/24 h

h) Quantitative determination of total protein by Biurett method


Proteins give an intensive violet-blue complex with copper salts in an alkaline

medium. Iodide is included as an antioxidant.

The intensity of the color formed is proportional to the total protein concentration

in the sample (Koller, 1984).


The proteins are macromolecular organic compounds, widely distributed in the

cells. They are structural and transport elements. The serum proteins are divided

in two fractions, albumin and globulins. The determination of total proteins is

useful in the detection of:

-- High protein levels caused by hemoconcentration like in the dehydrations or

increase in the concentration of specific proteins.

-- Low protein level caused by hemodilution by an impared synthesis or loss (as

by hemorrhage) or excessive protein catabolism.

• Procedure


Wavelength of detection: . . . . . . . . . . . . . . ..540 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 1 cm. light path

Temperature . . . . . . . . . . . . . . . . . . . . . . . . . .37ºC


P a g e | 177




Working Reagent

(mL)

1.0 1.0 1.0

Standard (µ L) -- 25 --

Sample ( µL) -- -- 25

4. The solutions were mixed and incubated for 5 min at 37ºC .

5. The absorbance (A) of the samples and Standard, against the Blank were measured.

The colour is stable for at least 30 minutes.

• Calculations

(A) Sample

------------------------------ x 7 (Standard conc.) = g/dL of total protein in sample

(A) Standard


Adults: 6.6 – 8.3 g/dL

Newborn: 5.2 – 9.1 g/dL

i) Quantitative determination of triglycerides


Sample triglycerides incubated with lipoproteinlipase (LPL), liberate glycerol and

free fatty acids. Glycerol is converted to glycerol-3-phosphate (G3P) and

adenosine-5-diphosphate (ADP) by glycerol kinase and ATP. Glycerol-3-

phosphate (G3P) is then converted by glycerol phosphate dehydrogenase (GPO) to

dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2). In the last

reaction, hydrogen peroxide (H2O2) reacts with 4-aminophenazone (4-AP) and p-

chlorophenol in presence of peroxidase (POD) to give a red colored dye:

Triglycerides + H2O LPL

Glycerol + free fatty acids

Glycerol + ATP Glycerol Kinase

G3P+ ADP

G3P + O2 GPO

DAP + H2O2

H2O2 + 4-AP + p-Chlorophenol POD

Quinone + H2O

P a g e | 178


The intensity of the color formed is proportional to the triglycerides concentration

in the sample (Buccolo, 1973).


Triglycerides are fats that provide energy for the cell. Like cholesterol, they are

delivered to the body’s cells by lipoproteins in the blood. A diet with a lot of

saturated fats or carbohydrates will raise the triglyceride levels. The increase in

serum triglyceride levels are relatively non-specific. For example liver

dysfunction resulting from hepatitis, extra hepatic biliary obstruction or cirrhosis,

diabetes mellitus is associated with the increase.

• Procedure

a) Assay conditions:

Wavelength of detection: . . . . . . . . . . .505 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . . . . .1 cm light path

Temperature . . . . . . . . . . . . . . . . . . . . 37ºC




Working reagent (mL) 1.0 1.0 1.0


Sample ( µL) -- -- 10

4. The solutions were mixed and incubated for 5 min at 37ºC .

5. The absorbance (A) of the samples and Standard, against the Blank were

measured. The colour is stable for at least 30 minutes.

• Calculations

(A) Sample

------------------------- x 200 (Standard conc.) = mg/dL of triglycerides in sample

(A) Standard



Men 40 – 160 mg/dL

Women 35 – 135 mg/dL

P a g e | 179


j) Quantitative determination of urea


Urea in the sample is hydrolyzed enzymatically into ammonia (NH3) and carbon

dioxide (CO2). Ammonia ions formed react with -ketoglutarate in a reaction

catalysed by glutamate dehydrogenase (GLDH) with simultaneous oxidation of

NADH to NAD+:

Urea + H2O + 2 H+

Urease 2 NH3 + CO2

2 NH3 + α - Ketoglutarate + NADH GLDH

H2O + NAD+ + L-Glutamate

The decrease in concentration of NADH, is proportional to urea concentration in

the sample (Kaplan, 1984).


Urea is the final result of the metabolism of proteins; it is formed in the liver from

its destruction. It can appear elevated urea in blood (uremia) in: diets with excess

of proteins, renal diseases, heart failure, gastrointestinal hemorrhage, dehydration

or renal obstruction.

• Procedure


Wavelength of detection: . . . . . . . . . 340 nm

Cuvette: . . . . . . . . . . . . . . . . . . . . .. . 1 cm light path

Temperature. . . . . . . . . . . . . . . . . .. . .37ºC




WR (mL) 1.0 1.0 1.0


Sample ( µL) -- -- 10

4. The solutions were mixed and the absorbance were measured after 30 sec. (A1) and

90 sec (A2).

6. ∆A was calculated as, ∆A= A2 – A1.

P a g e | 180


• Calculations

∆A Sample

------------------------------ x 50 (Standard conc.) = mg/dL of urea in sample

∆A Standard

10 mg/L urea blood urea nitrogen (BUN) divided by 0.466 = 21 mg/L urea = 0.36

mmol/L urea1.

• Conversion factor: mg/dL x 0.1665 = mmol/L.

• Reference values Serum : 15- 45 mg/dL (2.49-7.49 mmol/L)

Urine : 20 - 35 gr/24 h.

k) Quantitative determination of glycohemoglobin In Blood

• Principle

Glycosylated hemoglobin (GHb) has been defined operationally as the fast

fraction hemoglobins HbA1 (Hb A1a, A1b,A1c ) which elute first during column

chromatography. The non - glycosylated hemoglobin, which consists of the bulk

of hemoglobin, has been designated HbAo. A hemolysed preparation of whole

blood is mixed continuously for 5 minutes with a weakly binding cation-exchange

resin. The labile fraction is eliminated during the hemolysate preparation and

during the binding. During this mixing, HbAo binds to the ion exchange resin

leaving GHb free in the supernatant. After the mixing period, a filter separator is

used to remove the resin from the supernatant. The percent glycosylated

hemoglobin is determined by measuring absorbances of the ratio of the

absorbances of the glycosylated hemoglobin (GHb) and the total hemoglobin

fraction of the control and the test is used to calculate the % GHb of the sample

(Trivelli, 1971).

• Normal Reference Values

Normal : < 8.0 %

Good control : 8.0 - 9.0 %

Fair control : 9.0 - 10.0 %

Poor control : > 10.0 %

P a g e | 181


• Procedure

Wavelength of detection : 415 nm

Temperature : R.T.

Light path : 1 cm

• Hemolysate Preparation

1. About 0.5 ml of Lysing Reagent was added into tubes labeled as Control (C) and

Test (T).

2. About 0.1ml of the reconstituted control was added and mixed well with blood

sample into the appropriately labeled tubes until complete lysis was evident. The

mixture was allowed to stand for 5 minutes.

• Glycosylated hemoglobin (GHb) Separation

1. The Ion-Exchange Resin tubes were labelled as Control and Test.

2. About 0.1 ml of the hemolysate from Step A was added into the appropriately

labeled Ion Exchange Resin tubes.

3. A resin Separator was inserted into each tube, approximately 1 cm above the liquid

level of the resin suspension.

4. The tubes were mixed on a vortex mixer continuously for 5 minutes.

5. The resin was allowed to settle, and the resin separator was pushed into the tubes

until the resin was firmly packed.

6. Each supernatant was aspirated directly into a cuvette and absorbance of each was

measured against distilled water.

• Total Hemoglobin (THb) fraction

1. About 5.0 ml of distilled water was dispensed into tubes labeled as Control and

Test. To the above mentioned tubes 0.02 ml of hemolysate from Step A was added

into the appropriately labeled tube and mixed.

3. Each absorbance was measured against distilled water.

• Calculations

Ratio of Control (RC) = Abs. Control GHb / Abs. Control THb

Ratio of Test (RT) = Abs. Test GHb / Abs. Test THb

% GHb = [Ratio of Test (RT) / Ratio of Control (RC ) ] x 10 (Value of Control)

P a g e | 182


l) Radio immuno assay of Insulin using rat serum samples

• Principle

Human insulin is a polypeptide hormone that originates in the ß-cells of the

pancreas and serves as a principal regulator for the storage and production of

carbohydrates. Its secretion is normally stimulated by increases in the amount of

glucose in the circulation.

Insulin radioimmunoassay (RIA) is a double-antibody batch method. Insulin in the

specimen competes with a fixed amount of 125

I-labelled insulin for the binding

sites of the specific insulin antibodies. Bound and free insulin are separated by

adding a second antibody, centrifuging, and decanting. The radioactivity in the

pellet is then measured. The radioactivity is inversely proportional to the quantity

of insulin in the specimen.

This test is used to measure insulin levels in the bloodstream and is also useful in

determining pancreatic ß-cell activity. Conditions such as obesity, a high-

carbohydrate diet, and inactivity tend to increase expected normal values. Values

are found to be elevated shortly after food intake and in cases of acromegaly,

Cushing's syndrome, and thyrotoxicosis.

• Procedure

There is an antigen - antibody reaction between the antigen (Hormone) of interest

and its specific antibodies raised from laboratory animals. The quantum of

antigen-antibody binding is monitored using a radiolabelled antigen or antibody.

The RIA kit contains test tubes which have been coated with the antibody. To

these tubes, standards and samples were added along with the tracer. The contents

were incubated for 2 hours and the contents of the tubes were decanted. The tubes

were washed with the wash solution provided in the kit and the counting was

carried out in a gamma counter. The counts obtained for known standards used in

the assay were utilised for generating a dose - response curve and the unknown

concentration in serum samples were extrapolated from the data (Hales,

1963). The schematic representation of the RIA assay procedure is given in figure

5.19.

P a g e | 183


Figure 5.19: Assay procedure for RIA

• Preparation of synthetic serum:

γ Globulin (2%)+Bovine serum albumin(4%), diluted with double distilled

water. Sufficient volume was prepared based on the assay requirement.

• Preparation of working standard for constructing standard curve:

* Prepared from 200 µU/mL insulin stock

Insulin

Standard

Standard A

(mL)

Assay buffer

(mL)

Insulin concentration

(µIU/mL)

B 1 1 100

C 0.5 1.5 50

D 0.5 3.5 25

E 0.5 7.5 12.5

F 0.3 7.7 7.5

G 0.5 0.5 of F

(7.5 µIU/mL)

3.75

Contents of the tubes were mixed using vortex mixer, covered with aluminium

foil and incubated overnight at 2-4 0 C in refrigerator. After adding tracer, the

samples were incubated for 3 hrs. At the final step after centrifugation,

supernatant was decanted. The radio activity of precipitate antigen antibody

complex was measured using multiwell radio activity counter.

P a g e | 184


5.10 DEVELOPMENT OF FORMULATION OF ETHANOL EXTRACT OF

CASHEW TESTA

5.10.1 Introduction

Herbal medicines are widely used in conventional as well as alternative medical

practices in many countries, both developed and developing. Although herbal

medicines have been used by the people for centuries, they have not yet been

developed to a level as to serve as a substitute or complementary to synthetic

drugs.

WHO has issued general guidelines to assist in ensuring the safety and efficacy of

Complementary and Alternative Medicines (CAM) products. These include

suitable plant identification tests (physical and/or chemical) and if possible,

chromatograms of the purported active/s or marker compound/s present in

products should be provided. Alternatively, characteristic fingerprints of the plant

material used in the products are essential to prove botanical authenticity.

The USP (2005) has included some plant products in a section devoted to dietary

supplements. Although a monograph for solid oral dosage forms has not been

included, the USP does specify that some formulated dietary supplements such as

Glucosamine tablets should undergo dissolution and weight variation testing or in

some cases, only weight variation and disintegration testing are required such as

for the specifications for American Ginseng tablets. The general test scheme for

dietary supplements and pharmaceutical medicines is shown in Table 5.3

(Löbenburg, 2005). Hence, whilst several of the tests for the QC of dietary

supplements in the USA have recently been described in the USP as shown in the

table below, mandatory testing for those criteria has not yet been implemented.

This situation is similar in other countries as well making some of those tests

optional rather than mandatory.

P a g e | 185


Table 5.3: Pharmaceutical test scheme for pharmaceuticals and dietary

supplements (Löbenburg,2005)

Pharmaceuticals Dietary supplements

<301> Acid-neutralizing capacity <1216> Tablet friability

<701> Disintegration <2040> Disintegration and

dissolution of dietary

supplements

<711> Dissolution <2091> Weight variation

<724> Drug release <2750> Manufacturing practices

of dietary

supplements

<785> Osmolarity <2090> Weight variations of

dietary supplements

<905> Uniformity of dosage forms

----

<1087> Intrinsic dissolution

----

<1088> In vitro and in vivo

dissolution evaluation

of dosage forms

----

<1090> In vivo bioequivalence guide

----

<1216> Tablet friability ----

The numbers in < > refer to the specific sections in the USP 28 (2005)

5.10.2 Classification of herbal preparations

The International Pharmaceutical Federation (FIP) has published guidelines for

the classification of herbal preparations according to the amount of information

available on the efficacy and chemical composition of the product.

As per these guidelines, herbal preparations can be broadly classified into the

following categories:

1. Extracts in which the known/accepted pharmacological activity is assigned

solely to a single or group of constituents. Standardization may be achieved by

adjusting the level of actives by addition of inert excipients or extracts which

have higher or lower levels of the desired active compounds.

2. Extracts where the pharmacological effects are associated with constituents or

groups of constituents which synergistically contribute to the desired effect of

P a g e | 186


which the mechanism is largely unknown i.e. active marker/s have been identified.

Standardization can be achieved by blending batches of either raw botanical

materials or herbal preparations of higher and lower quality but the addition of

inert excipients is not permitted.

3. Extracts where there is no documented evidence of an identified active constituent

which is responsible for the therapeutic effect. Chemical compounds which may

not contribute to any pharmacological activity are then selected as markers for

GMP purposes (Westerhoff, 2002; Lang, 2001).

According to the European Agency for the Evaluation of Medicinal products

(EMEA), preparations which fall under categories 2 and 3 as well as immediate-

release formulations are exonerated from dissolution testing. In addition, in

preparations where the active constituent is highly soluble in aqueous media

which have pH values consistent with that of the GIT, disintegration testing is

then a sufficient indicator of GMP.

5.10.3 Background for the development of immediate release Tablet

Ethanol extract of cashew testa exhibited significant antioxidant and antidiabetic

activity in various in vivo and in vitro models including the studies performed on

cell lines in the research work reported in the present compilation in section 5.8-

5.93 and 6.8-6.93. Thus, an attempt was made to design and develop an economic,

effective formulation from the bioactive extract.

Moreover, tablets are the most common solid oral dosage forms for many reasons

including ease of manufacturing, convenience for the patient, accurate dose

administration, and better stability than liquids and parenteral dosage forms.

Direct compression is the simplest and most economical method for the

manufacturing of tablets because it requires less processing steps than other

techniques such as wet granulation and roller compaction.

5.10.4 Methodology

• Equipments used

UV- Visible spectrophotometer, Jasco V-530; Roche tablet friabilator, Dissolution

P a g e | 187


tester USP (XXIII) - Electrolab TDT-06T, Monsanto tablet Hardness tester -

Campbell electronics, Mumbai. Vernier Caliper-Mitutoyo, Japan.

• Preparation of the dry powder extract (DPE) for tablet formulation

Dry plant extracts usually lack good flow properties to be processed by direct

compression. In addition, because the active components of the extracts are

diluted by coextracted substances, high dosages are required. This is in conflict

with the limited proportion in which the extracts can be incorporated into the final

mixture for tablet compression (Vennat, 1993). Numerous reports have addressed

techniques used to solve these problems, such as wet granulation with non-

aqueous solvents (Diaz, 1996), direct compression of spray-dried extracts

(Plazier-Vercamen, 1986) and selection of suitable excipients for the formulation

of dry plant extracts in direct compression tablets (Renoux, 1996).

The crude ethanol extract of testa of cashew was defatted three times with n-

hexane. The extract was then dried. The dried ethanol extract was mixed with

various excipients to obtain a non-adherent and free-flowing dry extract powder.

The amount of dibasic calcium phosphate (DCP) was optimized to 10%, to obtain

a free flowing powder blend based upon the water sorption properties of 6, 8, 10,

12 and 14 % w/w of DCP.

A. Pre-compression Parameters (Micrometric evaluation) (Renoux, 1996)

1. Determination of Water uptake characteristics (moisture sorption study in

desiccators)

Many drug substances, particularly plant extracts, have a tendency to adsorb

moisture. The adsorption and equilibrium moisture content can depend upon

different factors such as the atmospheric humidity, temperature, surface area,

exposure time and the mechanism for moisture uptake. With most hygroscopic

materials, changes in moisture level can greatly influence many important

parameters, including chemical stability, flow properties and compatibility. The

effects of moisture on various physical properties such as angle of repose, bulk

and true densities, porosity and compression properties of various plant extracts

have been reported with different authors (Mishra, 1996).

P a g e | 188


The addition of DCP to the crude extract decreases the hygroscopicity. Powder

beds of 1.0 g of the dry extract preparations containing 2, 4, 6, 8, and 10 % w/w of

DCP were prepared in petri dishes and stored at 25° C in desiccators. The

moisture content of the various extract preparations was measured at 24 hrs

intervals for 15 days.

Based on the results of the moisture content studies, the dry extract preparation

containing 10% DCP was selected for tablet formulation. Hence, density, flow

property and compressibility of this dry extract blend were further investigated.

2. Density

• Bulk (poured) density

Bulk density was measured according to the European Pharmacopoeia (European

Pharmacopoeia, 2005). 150 g of dry powder blend was filled without compacting

into a 250 mL graduated cylinder using a powder funnel and was weighed to ± 0.1

g (m). The unsettled volume (V0) was read to ± 1 mL and the bulk density was

calculated as the quotient m/V0. Measurements were performed in triplicate and

the mean value was calculated

• Tapped density

A settling apparatus (model STAV 2003, J. Engelsmann AG) was used to measure

the tapped density. The settled volume was read after 500 (V500) and 1250 taps

(V1250). Another 1250 taps were carried out when the difference between V500

and V1250 was greater than 2 mL. The tapped density (g/mL) was expressed as

the quotient of m/V1250 or m/V2500. Measurements were performed in triplicate

and the mean value was calculated (Rai, 1995).

Carr compressibility index, CC % = [(tapped density – bulk density)/bulk

density]*100

and Hausner ratio, HR = [tapped density/bulk density] were calculated from the

bulk and tapped density results.

P a g e | 189


Table 5.4: Grading of powders

Consolidation index (Carr %) Flow

5-15 Excellent

12-16 Good

18-21 Fair to passable

23-35 Poor

33-38 Very poor

>40 Very very poor

A. Powder flow properties

• Angle of repose

Angle of repose was determined by pouring 150 ml of the dry extract preparation

though a funnel of 10 mm pore size, adjusted at 75 mm height from the base, into

10 cm diameter plate placed below the tip of the funnel. The height (h) of the

powder cone was measured. The grading of powders as per their flow properties

and its relation to angle of repose is indicated in table 5.4 and 5.5.

The angle of repose (θ) was calculated using the following equation:

tan θ = h/r

Where, θ is angle of repose,

h is height of the cone

r is radius of the pile formed

The mean value of three measurements was taken.

• Flow rate

The flow rate of the dry extract preparation was determined by pouring 100 gm of

the dry extract preparation though a funnel of pore size 10 mm with closing end.

The amount of the dry extract powder passing per unit of time under gravitational

force was recorded.

P a g e | 190


Table 5.5: Relationship between angle of repose and flow properties

Angle of repose (θ) In degrees Flow

<25 Excellent

25-30 Good

30-40 Passable

>40 Very poor

B. Determination of the dose for the formulation

Based upon the effective dose reported for the extract and the results of

antidiabetic activity in rats, the dose of the extract in formulation was extrapolated

to 350 mg twice a day. In each tablet the amount of extract incorporated was 187

mg.

C. Formulation of Tablet

Various ingredients were added to the dry extract powder prepared previously and

proportions of various excipients added were optimized. All ingredients were

passed through mesh 60 # before use. Before the compression process the

hardness was adjusted by altering the pressure applied for compression. The dry

powder blend equivalent to 187 mg of extract (i.e 50mg of blend) was used for

compression in single punch direct compression machine with 10.5 mm punch.

As indicated in Table 5.6, the codes F1- F6 represent the formulations prepared

for optimization of various ingredients in the tablet.

D. Post Compression parameters (Vennat, 1993; European Pharmacopoeia 2005)

• Shape and color of tablets

Tablets were observed for color and shape under a lens by placing the tablets in

light.

• Uniformity of thickness

Three tablets were selected randomly from each batch of formulation and

thickness was measured individually with dial caliper (Mitutoyo, Japan). The

thickness was measured in mm and standard deviation was also calculated.

P a g e | 191


Table 5.6: Various tablet formulations of ethanol extract of cashew testa

Ingredients Amount of ingredients in (mgs)

F1 F2 F3 F4 F5 F6

Extract 187.0 187.0 187.0 187.0 187.0 187.0

Avicel -102

(directly

compressible

Micro crystalline

Cellulose- MCC)

65.0 75.0 85.0 95.0 105.0 115.0

Dibasic Calcium

Phosphate (DCP)

35.0 35.0 35.0 35.0 35.0 35.0

Croscarmellose 56.0 46.0 36.0 26.0 16.0 6.0

Talc 7.0 7.0 7.0 7.0 7.0 7.0

• Hardness testing

Hardness of a tablet represents its ability to withstand mechanical shocks during

handling. Hardness of the tablets was tested using ‘Monsanto’ Hardness tester.

The USP does not specify tablet hardness parameters for dietary supplements. It is

expressed as kg/cm2. Three tablets were randomly picked and hardness of each

was determined. The mean and standard values were calculated.

• Friability testing

Friability was determined using Roche Friabilator. It is expressed in percentage

(%). Ten tablets were initially weighed (W initial) and transferred to the friabilator.

The friabilator was operated at 25 rpm for 4 minutes. The final weight of tablets

(W final) was recorded and the friability % was calculated as :

F = (W initial) - (W final) x 100

------------------------------------

(W initial)

Compressed tablets that lose less than 1.0% of their weight are considered

acceptable.

P a g e | 192


• Weight Variation

The weight variation tolerance for uncoated and film-coated botanical dosage

forms as specified in the USP 28 (<2091>) [240] was used to compare various

batches of tablets used in the validation of previously described methods.

According to the USP, 20 tablets of each dosage form must be weighed

individually and not more than 2 of the tablets are permitted to deviate from the

mean by more than 7.5% (United States Pharmacopeia, 2005).

• Content uniformity testing

Two tablets were weighed, powdered and powder equivalent to 187 mg of extract

was transferred to 100.0 ml volumetric flask. To it, 0.1M HCl (pH 1.2) was added

upto the mark. The flask was kept in a sonicator for 10 min to facilitate effective

extraction of drug. The solution was then filtered and 10.0 ml of the filtrate was

taken and diluted with 0.1M HCl to prepare appropriate working solutions. The

absorbance of the solution was determined spectrophotometrically at 273 nm

(United States Pharmacopeia, 2005).

• Disintegration Test

Tablet disintegration was determined according the specifications for uncoated

and film-coated tablets in the USP 28 (<2040>) [240], using Apparatus A.

Distilled water, maintained at 37 ± 0.5°C was used as the immersion fluid. One

tablet was placed in each of the six baskets and then raised and lowered vertically

at a constant frequency within the disintegration medium for the allocated time

period. According to the USP, 6 tablets should disintegrate completely within 20

minutes. If 1 or 2 of these tablets fail to disintegrate, the same test should be

repeated on an additional 12 tablets. Of the 18 tablets tested, 16 must disintegrate

completely within 20 minutes in order to meet the requirements. The time in

seconds taken for complete disintegration of the tablet with no palpable mass

remaining in the apparatus was measured and recorded (United States

Pharmacopeia, 2005).

• Dissolution conditions

Dissolution tests were performed using the Type 2 (Paddle) apparatus of the USP

28 (<711>). Each of the 8 vessels contained 900 ml of the appropriate dissolution

P a g e | 193


media and the temperature of the vessel contents was maintained at 37 ± 0.5°C.

The rotation speed of the paddles was 100 rpm. Volume adjustments of the media

were made by replacement of the withdrawn sample volume with fresh buffer at

the same pH.

A 0.1 M HCl solution was used for the dissolution medium at pH 1.2. The

reference standard, catechin representing typical amounts the tablets were added

to investigate the solubility of these components under the conditions of the

dissolution tests. The conditions of Dissolution were as follows:

Dissolution medium: 0.1 N HCl

Temperature: 370

C ± 10C

RPM: 100

Volume withdrawn for every time point: 10 ml

λmax : 273 nm

• Stability testing

The stability of a formulation is defined as its ability to remain within its physical,

chemical, therapeutic and toxicological specifications within a particular

container.

The purpose of stability determination is to provide evidence on how the equality

of a drug substance or a drug product varies with time under the influence of a

variety of environmental conditions such as humidity, temperature, and light and

enables recommended storage conditions, re-test periods and shelf lives to be

established.

ICH guidelines mention the length of study and storage condition:

Long term testing 25 ± 20 C / 60

0 % ± 5% RH for 12 months

Accelerated testing 40 ± 20 C / 75

0 % ± 5% RH for 6 months

In the present study, stability studies were carried out at 25 ± 20 C / 60

0 % ± 5%

RH and 40 ± 20 C / 75

0 % ± 5% RH for a period of 3 months.

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