6.1 AUTHENTICATION OF PLANT MATERIALshodhganga.inflibnet.ac.in/bitstream/10603/12880/12... · Figure 6.7: Effect of extraction time on extractive yields in microwave assisted extraction

P a g e | 194

CHAPTER 6 RESULTS AND DISCUSSION

6.1 AUTHENTICATION OF PLANT MATERIAL

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

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

was authenticated to be Anacardium occidentale Linn. belonging to family

Anacardiaceae.

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

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

6.2 STANDARDIZATION OF PLANT MATERIAL

6.2.1 Identification Tests

a) Organoleptic characters:

• Leaves: The powder of dried cashew leaves is green in color, with no

characteristic odour and the taste is slightly astringent.

• Testa: The powder of dried cashew testa is dark brown in color, with an aromatic

odour and slightly astringent taste.

b) Macroscopic characteristics:

In the macroscopic study of cashew leaves and testa, as depicted in Figure 6.1,

the following characteristics were observed:

• Leaves: The study of the macroscopic characters of fresh leaves reveal the

following characteristics:

Type: petiolated

Shape: elliptic obovate 4 to 22 cm long and 2 to 15 cm broad

Base: cuneate

Tip: obtuse

Venation: reticulate

Margin: entire and smooth

Arrangement: spiral

Texture: leathery

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• Testa: The study of the macroscopic characters of cashew nut testa reveal the

following characteristics:

Shape: kidney shaped

Size: 3-5 mm thick

Texture: irregular surface with fragile texture

Color: dark brown

(a) Leaves of cashew (b) Twig of cashew

(c) Stem of cashew (d) Testa of cashew

Figure 6.1: Macroscopic characters of cashew leaves and testa

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c) Microscopic analysis and powder characteristics:

• The results of microscopic analyses and powder characteristics study of leaves of

cashew are depicted in Figure 6.2 - Figure 6.6. Free hand sections of the leaves

were taken. A drop of phloroglucinol and hydrochloric acid each was used to detect

cellular arrangement in the sections of leaves and in the powdered drug.

Photomicrographs of the sections were also recorded with the help of Motic software.

(a) T.S. of lamina of cashew leaf

(b) T.S. of lamina of cashew leaf

Figure 6.2: T.S of lamina of cashew leaf

Parenchyma

Cuticle

Palisade

Cells

Epidermis

Collenchyma

Upper

Epidermis

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Figure 6.3: T.S. of cashew leaf through midrib

(a) Stomata of cashew leaf

(b) Stomata of cashew leaf

Figure 6.4: Microscopic images of stomata of cashew leaf

Upper

Epidermis

Mesophyll

Vascular

Bundle

(xylem and

phloem)

Stomata

Subsidiary

cells

Guard cells

Lower

Epidermis

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(a) Petiole of cashew leaf

(b) Petiole of cashew leaf

(c) Petiole of cashew leaf

Figure 6.5: T.S. of petiole of cashew leaf

Epidermis

Cortex

Fibre

Xylem

and

Phloem

Pith

Pericyclic

fibres

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(a) Trichomes of cashew leaf

(b) Epidermal cells of cashew leaf

(c) Palisade cells of cashew leaf

Figure 6.6: Powder characteristics of leaves of cashew

Trichomes

Epidermal

cells

Stomata

Polygonal

covering

trichomes

Lamina

portion with

palisade

cells,

parenchyma

and

epidermal

cells

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Study of transverse section of Leaf:

As observed in Figure 6.2 and Figure 6.3, the upper epidermis consisted of a

single layer of barrel-shaped cells. The epidermal cells were covered by a thick

cuticle and stomata were found along the epidermis. The mesophyll consisted of

two to three layers of compact cylindrical palisade cells and 4-5 layers of

parenchyma. In the midrib region; the upper epidermis was distinct, followed by

few layers of collenchymas and a wide region of different sizes of parenchyma

cells. The vascular bundle region was covered by endodermis and most of the part

of midrib was filled with corticle parenchyma and lignified xylem. Each vascular

bundle protected by an upper and a lower patch of sclerenchyma cells. A wide

nonlignified phloem region was found towards the lower epidermis protected by

thick sclerenchyma cells. The xylem was formed of vessels arranged into 5-8 rows

of vessels, in each row there were 2-6 vessels. The parenchyma cells below the

vascular bundle were formed of 3-5 layers varying sizes of cells. As seen in

Figure 6.4, the stomata were paracytic, rubiaceous celled with irregular subsidiary

cells.

Study of transverse section of petiole:

The general structure of the transverse section of the petiole appeared circular.

The outermost layer is formed of one layer of epidermis with no hairy structures.

The vascular bundles are arranged in a circle, and each vascular bundle is

preceded by pericyclic fibers. The phloem region is formed of primary and

secondary phloem and they are followed by the xylem. The pith is a wide region

of thickened parenchyma cells (Figure 6.5).

Study of diagnostic characters (powder characteristics) of leaves:

The diagnostic characters revealed in study of powder of cashew leaves were

epidermal cells, stomata, palisade cells and trichomes as seen in Figure 6.6.

The trichomes were single celled covering trichomes with sharp ends. Some

collapsed trichomes were also observed. Epidermal cells with ranunculaceous

stomata.

Stomata were surrounded by subsidiary cells, resembling other epidermal cells.

Epidermal cells are polygonal with irregular celled stomata. The palisade cells,

parenchyma with epidermal cells resemble the lamina portion of the leaves.

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6.2.2 Physicochemical analysis of cashew leaves and testa

The physicochemical analysis of cashew leaves and testa was carried out as per

the procedures and parameters mentioned in the Ayurvedic Pharmacopoeia of

India, and the results obtained are mentioned in Table 6.1 – 6.4.

Table 6.1: Determination of ash values

Type of ash Leaves of cashew

(%) ± SEM

Testa of cashew

(%) ± SEM

Total ash

Acid insoluble ash

Water soluble ash

Sulphated ash

10.5 ± 0.2

1.0 ± 0.5

4.5 ± 0.4

2.4 ± 0.3

8.3 ± 0.6

0.7 ± 0.7

3.3 ± 0.9

1.7 ± 0.5

n=3 determinations for values of each test mentioned above

Table 6.2: Determination of loss on drying

Loss on

drying

Leaves of cashew

(% w/w) ± SEM

Testa of cashew

(%w/w) ± SEM

7.5 ± 0.7

3.5 ± 0.3

n=3 determinations for values of the test mentioned above

Table 6.3: Determination of various extractive values

Extract

Leaves of cashew

(%) ± SEM

Testa of cashew

(%) ± SEM

Alcohol soluble extractive

Water soluble extractive

Ether soluble extractive

20.9 ± 0.9

7.6 ± 0.5

3.5 ± 0.3

38.3 ± 0.6

33.7 ± 0.4

5.6 ± 0.5

n=3 determinations for values mentioned above

Table 6.4: Determination of pH values

pH values

Extract of cashew

leaves ± SEM

Extract of cashew

testa ± SEM

5.5 ± 0.1

6.5 ± 0.3


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Determination of physicochemical parameters has been introduced in Ayurvedic

pharmacopoeia and in monographs of various herbal drugs. There are no reports

found for the determination of physicochemical parameters of cashew leaves and

testa and hence these investigations can serve as a reference for any further

determinations.

The total ash method measures the total amount of material remaining after

ignition and the amount of heavy metals and inorganic compounds and includes

“physiological and non-physiological” ash, which is the residue of the extraneous

matter (e.g. sand and soil) adhering to the plant surface. In the physiochemical

analysis, it was found that the determination of ash values showed a higher value

of ash present in cashew leaves as compared to testa (Table 6.1). The total ash

values, and water soluble ash values of both testa and leaves were found to be

higher than acid insoluble ash and sulphated ash values.

An excess of water in medicinal plant materials will encourage microbial growth,

the presence of fungi or insects, and deterioration of phytoconstituents following

hydrolysis. Limits for water content should therefore be set for every given plant

material. The test for loss on drying determines both water and volatile matter.

The results shown in Table 6.2, indicate that the leaves of cashew have a higher

moisture content as compared to testa.

Determination of extractive values reveals the amount of active constituents

extracted with solvents from a given amount of medicinal plant material. As

indicated in Table 6.3, it was observed that testa of cashew exhibited higher

extractive values as compared to leaves with alcohol, water and ether as extracting

solvents. The alcohol and water soluble extractive values were found to be higher

than ether soluble extractive values for leaves and testa of cashew. The alcohol

soluble extractive value was found to be greater, as being a relatively non-polar

solvent as compared to water, alcohol was able to extract polar as well as non

polar components.

In the determination of pH values of aqueous extracts of cashew leaves and testa

the leaves of cashew were found to have a more acidic pH than testa probably due

to the presence of higher amounts of constituents like anacardic acids (Table 6.4).

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6.3 EXTRACTION OF PLANT MATERIAL

The extraction of cashew leaves and testa were carried out by various techniques.

The results of extractive values obtained for each of the methods are as mentioned

in Table 6.5 – 6.9.

Table 6.5: Determination of extractive values (Soxhlet extraction)

Extract Leaves of cashew

(% w/w) ± SEM

Testa of cashew

(% w/w) ± SEM

Ethanol extract

Methanol extract

25.94 ± 0.70

8.60 ± 0.80

40.92 ± 0.50

46.40 ± 0.30


Table 6.6: Determination of extractive values (Decoction)

Extract Leaves of cashew

(% w/w) ± SEM

Testa of cashew nut

(% w/w) ± SEM

Aqueous extract 8.64 ± 0.50 36.5 ± 0.90


Table 6.7 (A): Determination of extractive values (Microwave assisted

extraction)

Time (sec.)

Microwave Extraction

(Low power 140 Watt)

% Yield ± (SEM)


(Low power-140 Watt)

% Yield ± (SEM)

Methanol extract of

cashew leaves

Aqueous extract of

cashew leaves

50 13.0 ± 0.40 15.0 ± 0.50

70 13.8 ± 0.10 17.4 ± 0.90

90 14.60 ± 1.0 17.8 ± 1.0

120 20.0 ± 1.20 29.0 ± 0.70

130 10.0 ± 0.20 19.2 ± 0.60

150 8.4 ± 0.50 19.0 ± 0.50

180 6.6 ± 0.40 16.0 ± 0.80

Soxhlet extraction at

(400-45

0C) for 18 hr

8.60 ± 0.80 8.9 ± 0.80

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Figure 6.7: Effect of extraction time on extractive yields in microwave

assisted extraction (cashew leaves)

Table 6.7 (B): Extractive yields of leaves with optimised conditions

for MAE

Extraction for 120 secs.

At Low Power

Leaves Of Cashew

(In Percentage W/W) ± SEM

Aqueous extract 29.0 ± 0.7

Methanol extract 20.2 ± 1.2


Table 6.8: Extractive yields of Testa for MAE

Extraction for 10 mins.

At Low Power

Testa of cashew

(In percentage w/w) ± SEM

Aqueous extract 35.0 ± 0.5

Methanol extract 44.2 ± 1.0


Table 6.9: Determination of various extractive values (polyphenol fractions)

Extract Extractive value

(In percentage w/w) ± SEM

Polyphenols of leaves 3.2 ± 1.0

Polyphenols of testa 5.4 ± 0.2


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� Extraction methods

• In literature, reports are found where Soxhlet has been used as control for

comparison with other extraction techniques. The extraction of cashew leaves and

testa with methanol and ethanol were carried out by Soxhlet extraction and with

water by decoction process. Since cashew is reported to be a rich source of

phenolic compounds and tannins, the extraction of testa and leaves was carried out

with solvents viz. ethanol and methanol. These solvents extract polar as well as

nonpolar phytoconstituents.

• Several studies have been reported on the comparison of MAE with other

available conventional techniques. Recovery of phytoconstituents can be

enhanced by study of the behavior of three variables, namely irradiation time,

irradiation power and amount of extracting solvent. Extraction yields of plant

materials depend on the various extraction conditions. In order to study the effect

of Microwave assisted extraction on extractive yields of leaves of cashew, the

method of extraction was optimized. Water and methanol, were used as extracting

solvents. A threefold increase in the yield of aqueous extract was observed by use

of MAE within 120 seconds as compared to Soxhlet extraction for 18 hrs

extraction time. Microwaves give better extraction with polar solvents, especially

with water which has a high dielectric constant. Thus, extractive yield of leaves

with water was found to be higher than that of methanol.

• For MAE of testa, there was no significant change in the extractive yield even for

an extraction period of 10 mins. The yield obtained by MAE in 10 mins.

Extraction time was found similar to the yield obtained by soxhlet extraction

carried out for 18 hours. However, a reduction in extraction time and solvent

consumption was observed. Hence, it can be said that MAE can be a more

effective technique compared to conventional extraction methods for faster and

economical extraction of plant materials.

• Polyphenols are compounds of great interest to researchers worldwide for their

varying beneficial effects in various diseases (Na, 2008 and Larrauri, 1997). The

cashew nut testa is reported to be rich source of polyphenols and cashew nut shell

liquid is well known for its tannin content and is widely used by the tanning

industries (Subramanian, 1969). Hence an attempt was made to extract

polyphenols from leaves and testa of cashew.

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6.4 PRELIMINARY PHYTOCHEMICAL SCREENING OF EXTRACTS

The results of qualitative chemical tests for various extracts of cashew are

tabulated below

Table 6.10: Qualitative chemical tests

Sr.No

Chemical Tests EL AL ET MT AT

1

Test for Carbohydrates

+

+

+

+

+ Molisch's test .

Benedict's test

+

+

+

+

+

Test for Non-reducing sugars

+

+

+

+

+ Test for Gums

-

-

-

-

-

Test for mucilage

-

-

-

-

-

2

Tests for Proteins

+

+

+

+

+ Millons test

3

Tests for Amino Acids

-

-

-

-

-

Ninhydrin test

4

Test for Fats and Fixed Oil

-

-

+

+

+

Stain test

5

Saponification test

+

+

-

-

-

6 Test for Sterols and

Triterpenoids

-

-

-

-

-

Libermann- Buchard test

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7

Test for Glycosides

-

-

-

-

-

Legal’s test for Cardiac Glycosides

Keller Killiani test [for Deoxy sugars]

-

-

-

-

-

Froth Test for Saponin Glycosides

+ + - - -

Sodium picrate test (grignard reaction) for Cyanogenetic Glycosides

-

-

-

-

-

Tests for Coumarin Glycosides

- - - - -

8

Test for Flavonoids

+

+

+

+

+

Shinoda test (Magnesium Hydrochloride reduction)

Alkaline reagent test for Flavonoids

+

+

+

+

+

9 Tests for Alkaloids

+

+

+

+

+

Dragendorff’s test

10

Test for Tannins and Phenolic Compounds

Ferric chloride test

+

+

+

+

+

11

Tests for organic acids

-

-

-

-

- Calcium chloride test

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In the table, the following abbreviations were used:

EL: Ethanol extract of leaves

AL: Aqueous extract of leaves

ET: Ethanol extract of testa

MT: Methanol extract of testa

AT: Aqueous extract of testa

The symbol (+) denotes presence and (-) denotes absence of phytoconstituents.

From the qualitative chemical tests performed for estimation of various

phytoconstituents in extracts of cashew leaves and testa the following results were

obtained (Table 6.10):

• Ethanol extract of leaves: The ethanol extract of leaves was found to contain

carbohydrates, proteins, saponin glycosides, flavonoids, alkaloids, tannins and

phenolic compounds.

• Aqueous extract of leaves: The aqueous extract of leaves was found to contain

carbohydrates, proteins, saponin glycosides, flavonoids, alkaloids, tannins and

phenolic compounds.

• Ethanol extract of testa: The ethanol extract of testa was found to contain

carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds.

• Methanol extract of testa: The methanol extract of testa was found to contain


• Aqueous extract of testa: The aqueous extract of testa was found to contain


Gums, mucilage, amino acids, and organic acids were found to be absent in leaves

and testa of cashew.

The extraction for leaves and testa were carried out with solvents of similar

polarity. Polar solvents, viz. water, ethanol and methanol were used for extraction.

Hence, phytoconstituents of similar nature were found in extracts of testa and

leaves, except for saponin glycosides which were present in leaves and not in

testa.

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6.5 ISOLATION OF CATECHIN

• Isolation of catechin was carried out by Preparative Thin layer chromatography

(P-TLC).

• The presence of catechin in various extracts of cashew was confirmed by

co-chromatography with reference standard catechin. The spot with Rf value

identical to the marker catechin was isolated.

• The crude catechin thus obtained was recrystallised with hot water and the

percentage yield of pure catechin from crude catechin was calculated.

• The weights of catechin obtained before and after recrystallisation are as below:

Percentage yield of crude Catechin:

1g of the extract 90 mg of crude catechin

Thus, %Yield of crude catechin = 9.0% w/w

Percentage yield of catechin after recrystallisation:

1g of the extract 50 mg of Pure catechin

Thus, %Yield of Pure catechin = 5.0 w/w.

6.5.1 Identification of isolated catechin

The identity and purity of isolated catechin was further confirmed by chemical

spectral and chromatographic studies standard catechin was used for comparison

with the isolated catechin. Thus catechin was characterized for the following

chemical characteristics:

Physicochemical and spectral characteristics:

•••• Colour and shape:

Catechin appeared to be a buff white colored powder.

•••• Melting point: Melting point of isolated catechin was found to be 176 -1780C. It

was found to be identical to the melting point of reference catechin.

• pH: pH of the catechin solution was found to be 6.5.

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• Solubility:

Catechin was found to be soluble in methanol, water, and ethanol.

• Chemical test for catechin:

Catechin when added to vanillin and hydrochloric acid solution produced a pink

colour. The test was found to be positive for isolated catechin.

• The UV λmax (nm) : Isolated and marker catechin were found to exhibit a

similar λmax at 273nm.

Table 6.11: Physiochemical and spectral studies of isolated Catechin

Sr. No. Parameters Values for reference

catechin

Values for isolated

catechin

1 Colour and

crystal shape

Buff white colored

powder

Buff white colored

powder

2 Melting point(0C) 175

0C-177

0C 176 -178

0C

3 Derivatization

(Ethanolic FeCl3)

Bluish black color Bluish black color

4 HPLC Rt (min.) 2.6 2.6

5 HPTLC Rf 0.45 0.45

6 UV λmax (nm) 273 nm 273nm

7 pH of 1%

solution in water

6.5 6.5

8 Solubility Soluble in methanol,

water, chloroform

and insoluble

in benzene

Soluble in methanol,

water, chloroform

and insoluble in

benzene

Figure 6.8: Structure of catechin

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� UV spectral analysis of catechin

Figure 6.9: UV absorption spectrum of marker catechin

Figure 6.10: UV absorption spectrum of isolated catechin

Result: As indicated in Table 6.11 and Figure 6.8-6.10, it was observed that

isolated catechin the spectral, chemical and chromatographic characteristics

similar to reference catechin, thus indicating the identity of isolated catechin.

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� HPTLC profile of isolated catechin

In order to ascertain the purity of isolated catechin, HPTLC studies were

performed using marker catechin as the reference standard. Working solutions of

1mg/ml of isolated catechin and standard catechin were prepared in methanol and

the HPTLC analysis was carried out using the following optimised conditions.

Stationary phase : Silica gel 60 GF254 (Merck)

Mobile phase : Toluene: ethyl acetate: methanol: formic acid (6: 6:1:0.1)

Saturation time : 30 min.

Band width : 7 mm

Detection wavelength : 273 nm

Isolate Marker Isolate Marker Isolate Marker

In White Light @ 254 nm @ 366 nm

Figure 6.11: HPTLC video images of catechin

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Figure 6.12: HPTLC chromatogram of marker catechin

Figure 6.13: HPTLC chromatogram of isolated catechin

Result: As shown in Figure 6.11 - 6.13, the isolated catechin gave a single

isolated band at Rf of 0.45 similar to that of marker catechin. The percentage

purity of isolated catechin was found to be 99.82% when compared with standard

catechin.

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� HPLC profile of Isolated catechin

In order to ascertain the purity of isolated catechin, HPLC studies were carried out

using marker catechin as the reference standard. Standard catechin and isolated

catechin were analyzed by HPLC using the following conditions:

System : TOSOH-CCPM

HPLC method : Reverse Phase

Column : C18 (ODS)Phenomenex (250 x 4.60mm)-5µ

Mobile phase : Methanol (100%) HPLC Grade

Flow rate : 1ml/min

Wavelength : 273nm

Figure 6.14: HPLC chromatogram of reference catechin

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Figure 6.15: HPLC chromatogram of isolated catechin

Result: As seen in Figure 6.14 and Figure 6.15, isolated catechin gave a single,

sharp and well resolved peak at Rt of 2.6 min similar to that of reference catechin.

The HPLC profile of the standard and isolated catechin was found to be identical

at retention time (Rt) of 2.6 min. The HPLC profile of isolated catechin gave a,

well isolated peak with purity greater than 99.65%.

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6.6 CHROMATOGRAPHIC STUDIES

The HPTLC analysis of various extracts of cashew leaves and testa were carried

out by the optimized chromatographic conditions by HPLC and HPTLC

techniques and various components of the extracts were analysed

densitometrically. Catechin was used as a marker and the amount of it present in

various extracts was quantified.

� HPTLC Analysis

� Optimized Chromatographic parameters

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

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

Mobile phase : Toluene:ethylacetate:MeOH:formic acid (6:6:1:0.1v/v/v/v)

Saturation time : 15 mins.

Development distance: 80 mm

Derivatising agent: 5% alcoholic FeCl3 solution

Detection wavelength: 254 nm

� Calibration curve of catechin

In order to establish a calibration curve for estimation of catechin, the limit of

detection (LOD) and limit of quantitation (LOQ) were determined. The values

obtained for LOD and LOQ were 0.1 and 0.3 µg / µl respectively. The calibration

concentration range was between 0.4 - 2.0 µg / µl.

Figure 6.16: Calibration curve of catechin for HPTLC method

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The chromatograms obtained for various extracts of cashew leaves and testa and

polyphenol fractions are as depicted in Figure 6.17-6.24.

The amount of catechin in various extracts was estimated and is listed in Table

6.12. It was found that aqueous extract of leaves and testa contained the maximum

amount of catechin as compared to the other extracts.

In the polyphenol fraction of cashew leaves and testa the maximum amount of

catechin was found from the fraction prepared from aqueous extracts.

The images of fingerprints and spectra of various extracts and fractions at

different wavelengths are shown in Figure 6.25 and 6.26.

Table 6.12: Catechin content in various extracts of cashew by HPTLC

Sr.No Sample % of Catechin

Cashew leaf extracts

1 Ethanol extract 4.75%

2 Aqueous extract 5.70%

Cashew testa extracts

3 Methanol extract 12.75%



Polyphenol fraction of cashew testa



Polyphenol fraction of cashew leaves



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Figure 6.17: HPTLC chromatogram of ethanol extract of leaves

Figure 6.18: HPTLC chromatogram of aqueous extract of leaves

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Figure 6.19: HPTLC chromatogram of ethanol extract of testa

Figure 6.20: HPTLC chromatogram of methanol extract of testa

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Figure 6.21: HPTLC chromatogram of aqueous extract of testa

Figure 6.22: HPTLC chromatogram of polyphenol fraction of testa

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Figure 6.23: HPTLC chromatogram of polyphenol fraction of leaves

Figure 6.24: HPTLC chromatogram of standard catechin

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� HPTLC Fingerprints of extracts of leaves and testa of cashew

AL EL MT AT ET IC RC AL EL MT AT ET IC RC

Image @ 366nm Image @ 254nm

(a) (b)

AL EL MT AT ET IC RC EL ET RC

Image @ White Light Image @ white light after derivatization

(c) (d)

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� HPTLC Fingerprints of Polyphenol fractions of leaves and testa of cashew

RC PL PL PT RC PL PL PT RC PL PL PT

Image @366 nm Image @ 254 nm Image @ White Light

(a) (b) (c)

Figure 6.25: HPTLC video images (fingerprints of various extracts and

polyphenol fractions of cashew)

Figure 6.26: Spectra of catechin in various extracts

The abbreviations denoted on the tracks are as follows:

RC - Reference catechin IC - Isolated catechin

PL - Polyphenols of cashew leaves MT - Methanol extract of cashew testa

PT - Polyphenols of cashew testa AT - Aqueous extract of cashew testa

AL - Aqueous extract of cashew leaves ET - Ethanol extract of cashew testa

EL – Ethanol extract of cashew leaves

EL

RC

AT ET

MT

P a g e | 224


� HPLC Analysis

The HPLC analysis of various extracts of cashew leaves and testa were carried out

by the optimized chromatographic conditions. Catechin was used as a marker and

the amount of catechin present in various polyphenol fractions, extracts of cashew

prepared by conventional extraction process as well as by microwave extraction

was quantified.

� Optimized Chromatographic parameters

The optimized parameters for HPLC analysis were:

Solvent system: Methanol:Water (90:10 v/v)

Flow rate: 1ml/min

Column: C18 column


� Calibration curve of catechin

In order to establish a calibration curve for estimation of catechin, the limit of

detection (LOD) and limit of quantitation (LOQ) were determined. The values

obtained for LOD and LOQ were 0.1 and 0.3 µg / µl respectively. The calibration

concentration range was between 0.3 – 1.4µg / µl.

Figure 6.27: Calibration curve of catechin by HPLC

P a g e | 225


The peaks in HPLC fingerprints were identified by comparing the retention times

in the chromatograms of extracts with those of reference standard catechin peak.

The Chromatograms obtained for various extracts of cashew leaves and testa and

polyphenol fractions are as depicted below in Figure 6.28- 6.49.

The catechin content in various extracts of testa and leaves prepared by

conventional techniques was quantified and is listed in Table 6.13. Aqueous

extract of testa and leaves were found to contain maximum amount of catechin.

The catechin content in methanol and aqueous extracts of leaves prepared by

microwave assisted extraction (MAE) was quantified and is listed in Table 6.14.

It was observed that the extraction time of 120 secs. yielded the maximum amount

of catechin in methanol and aqueous extract of leaves prepared by MAE.

Table 6.13: Catechin content in various extracts of estimated by HPLC

Sr.No. Extract % Catechin content

Cashew leaf extracts

1 Ethanol extract 4.95 ± 1.0

2 Aqueous extract 5.83 ± 0.9

Cashew testa extracts

3 Methanol extract 12.95 ± 0.7



Table 6.14: Catechin content in various extracts prepared by MAE

Sr.

No.

Time

(sec.)


(Low power 140 Watt)

% Yield ± (SEM)


(Low power-140 Watt)

% Yield ± (SEM)

Methanol extract of

cashew leaves

Aqueous extract of

cashew leaves

1 50 2.1.± 0.2 2.5 ± 0.3

2 70 2.5 ± 0.1 3.4 ± 0.6

3 90 3.50 ± 1.0 6.8 ± 1.2

4 120 8.0 ± 1.7 8.8 ± 0.5

5 130 7.8 ± 0.7 8.2 ± 0.2

6 150 6.4 ± 0.3 7.9 ± 0.3

7 180 4.6 ± 0.5 6.0 ± 0.7

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Figure 6.28: HPLC chromatogram of standard catechin

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Figure 6.29: HPLC chromatogram of aqueous extract of cashew leaves

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Figure 6.30: HPLC chromatogram of ethanol extract of cashew leaves

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Figure 6.31: HPLC chromatogram of aqueous extract of cashew testa

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Figure 6.32: HPLC chromatogram of ethanol extract of cashew testa

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Figure 6.33: HPLC chromatogram of methanol extract of cashew testa

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Figure 6.34: HPLC chromatogram of polyphenol fraction of cashew testa

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Figure 6.35: HPLC chromatogram of polyphenol fraction of cashew leaves

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Figure 6.36: HPLC chromatogram of aqueous extract prepared by

microwave extraction (for 50 seconds) at low power

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Figure 6.43: HPLC chromatogram of methanol extract prepared by


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6.7 EFFECT OF VARIOUS DRYING METHODS ON THE POLYPHENOL

CONTENT AND ANTIOXIDANT ACTIVITY OF CASHEW LEAVES

The leaves of cashew were subjected to various drying conditions in order to

study the effect of varying temperatures on the polyphenol content and antioxidant

activity of the extracts. The results of the antioxidant activity and total phenolic

content estimation are detailed in section 6.8.

6.7.1 Quantitation of catechin content of various extracts of cashew

The catechin content in extracts of leaves subjected to drying conditions was

carried out by HPLC method. The conditions used for analysis are mentioned

below:

The optimized parameters for HPLC analysis were:

Solvent system: Methanol:Water (90:10 v/v)

Flow rate: 1ml/min

Column: C18 column


Table 6.15: Comparison of various drying techniques of cashew leaves based

on catechin content

Sr.No Extracts % of Catechin ± SEM

1 Oven dried leaves 6.11% ± 0.35

2 Sun dried leaves 7.94 % ± 0.18

3 Fresh leaves 5.70% ± 0.16

4 Shade dried leaves 7.50 % ± 0.53

n=3 determinations for each of the values mentioned above

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Figure 6.50: HPLC Chromatogram of extract of cashew leaves subjected to

oven drying

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Figure 6.51: HPLC chromatogram of extract of sun dried cashew leaves

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Figure 6.52: HPLC chromatogram of extract of shade dried cashew leaves

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Figure 6.53: HPLC chromatogram of extract of fresh cashew leaves

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As observed in the Table 6.15 and Figure 6.50 - 6.53 it is observed that the

extracts prepared from cashew leaves exposed to sunlight for drying contained the

maximum amount of catechin.

Various temperatures significantly influence the extraction yield of phenolics

from plants and the antioxidant activity of the phenolic compounds. In this study

the cashew leaves were exposed to various drying conditions and upon

chromatographic quantitation, a considerable difference was observed in the

catechin content of various extracts. The results suggest that the order of catechin

content in various extracts prepared was as Sun dried > Shade dried > Oven dried

> Fresh leaves.

The leaves of cashew are taken as food accompaniments in Malaysia (Abas, 2006)

and finding a suitable method to preserve them along with maintaining their

antioxidant effect would be of help to the consumers. The results showed that only

oven drying brought about significant reduction in catechin content with fresh

leaves as the comparison group. This might be due to some chemical

transformations during the process of drying. Thermal processing of food is

primarily intent to inactivate pathogens and other deteriorative microorganisms

capable of making it unsuitable for human consumption. However, it is believed

that thermal treatments are the main cause of the depletion in natural antioxidants

(Mokbel, 2005; Mohan, 2008). In this study, increasing the temperature to about

80°C seemed to cause depletion in the antioxidant content. Since many

plants/fruits have antioxidants, it is important to maintain this nutrient content for

its benefit by controlling the extraction temperature or exposure of leaves to

temperatures higher than 400

C. Sun dried leaves were found to contain the

maximum amount of catechin, which suggests that if leaves dried in sunlight are

consumed in some form instead of fresh leaves, the availability of antioxidants

would be more as compared to fresh or shade dried leaves.

In recent years polyphenols have received increasing attention from chemists and

food technologists. Another phenomenon that might affect the Polyphenol content

or antioxidant activity of the leaves subjected to various drying conditions is the

browning effect. Such compounds present in food have been found to take part in

both enzymic and nonenzymic browning reactions. The specific enzymes that take

part in browning reactions involving polyphenols have been known by different

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names but in general can be referred to as polyphenoloxidases. Enzyme chemists

have been able to isolate, purify, and characterize polyphenoloxidase enzymes

from several sources. The oxidation of polyphenolic substrates, secondary

reactions, inhibition, and activation have also been investigated thoroughly during

the last few decades. During the processing and storage of food products,

especially fruits and vegetables, several nonenzymic changes leading to browning

involving polyphenols have been noted. The common cause of darkening of many

products is attributable to the interaction between polyphenols and heavy metals,

especially iron. Formation of colored anthocyanidin pigments has been suspected

in others. The inhibition of enzymic discoloration involving polyphenols is best

effected by application of heat or by influence of certain chemicals. In addition to

these the effect of other agents such as freezing (Negishi, 1964), moisture content

(Draudt, 1966) and so on have also been investigated to a limited extent. Since

browning of this nature involves an enzymic step, factors such as concentration of

the substrate, pH of the medium, and availability of oxygen have an influence on

the rate of the reaction. In practice, however, some of these factors are difficult to

control during storage and processing of food materials. In a comparative study

between fruits and vegetables, it was noted that polyphenoloxidases of fruits are

more stable than those of vegetables (Yankov, 1963).

Thus, a thorough detailed investigation of the enzymic reactions occurring in

leaves of cashew would be required in order to arrive to mechanisms that lead to

increase in catechin content of leaves exposed to sunlight.

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6.8 EVALUATION OF ANTIOXIDANT ACTIVITY

The extracts and fractions of cashew leaves and testa were subjected to various in

vitro and cell line based antioxidant assays, in order to ascertain their antioxidant

effect. The results obtained from various assays and tests are detailed below.

6.8.1 Assessment of Free Radical Scavenging Capacity in vitro

A. DPPH. radical scavenging assay

The radical scavenging activities of the extracts and fractions of leaves and Testa

of cashew were estimated by comparing the percentage inhibition of formation of

DPPH. radicals by the extracts and those of Ascorbic acid. The results of the assay

are as indicated in Table 6.16.

• Extracts of Cashew Leaves

A steady increase in the percentage inhibitions of the absorbance of the DPPH

radicals by the extracts up to a concentration of 16.0 µg/mL, and 18.0 µg/mL for

aqueous and ethanolic extracts respectively, after which there was a leveling off

with much slower increase in inhibition. This pattern of DPPH inhibition is

commonly observed with plant extracts. Overall, the Aqueous and Ethanol

extracts of leaves of cashew were able to inhibit the formation of DPPH. radicals.

The aqueous extract and ethanol extracts had IC50 values of 12.76 µg/mL and

9.41µg/mL respectively which is inversely related to its antioxidant ability. The

IC50 value of Ascorbic acid (standard) was found to be 5.30µg. Based upon the

IC50 values of the extracts it can be concluded that, ethanol extract is more potent

as an antioxidant than aqueous extract.

• Extracts of Cashew Testa

Radical scavenging activities of the extracts of cashew testa, were estimated by

comparing the percentage inhibition of formation of DPPH radicals by the extracts

and those of Ascorbic acid depicting a steady increase in the percentage

inhibitions of the absorbance of the DPPH radicals by the extracts up to a

concentration of 14.0 µg/mL for aqueous and methanol extracts and 20.0 µg/mL

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for ethanol extract, after which there was a slower increase in inhibition. Overall,

the aqueous, ethanol and methanol extracts of cashew were able to inhibit the

formation of DPPH. radicals and had IC50 values of 7.62, 6.68 and 7.23µg/mL of

dried extract which is inversely related to its antioxidant ability. Among the three

extracts of testa that were tested for antioxidant activity, ethanol extract was found

to be most potent with the least IC50 value comparable with Ascorbic acid used as

standard. This suggests that ethanol extract of testa can exhibit significant

antioxidant activity at a much lower concentration.

• Polyphenol Fractions of Cashew

Polyphenolic compounds are well known in literature for their antioxidant effects

and hence an attempt was made to evaluate the efficacy of polyphenol rich

fraction from cashew leaves and testa. The IC50 values for Polyphenol fraction of

test and leaves were found to be 7.51 and 7.42µg/mL respectively which is

inversely related to its antioxidant ability. Thus, the results indicate that the

polyphenol fractions may serve as potential antioxidant candidates.

• Extracts Prepared By Microwave Extraction

Microwave assisted extraction has been known to increase the extractive yields of

substances. In case of materials containing polyphenols, anthocyanins and

flavanoids this behaviour of increase in yields can occur for two reasons: (i) at a

high temperature, new compounds can be generated as a result of non-enzymatic

browning or the Maillard reaction. These compounds, referred to as melanodins or

Maillard reaction products (MRPs), possess antioxidant activity and function as an

antioxidant via a chain-breaking mechanism. Several authors have noted that the

antioxidant activity afforded by the generation of MRPs does not compensate for

that lost by the phenolic compounds (Morales, 2001; Yilmaz, 2005), and (ii)

during oxidation of polyphenolics, the oxidation products formed during the

intermediate stages have shown to posses greater antioxidant activity than the

endogenous polyphenolics; however, these intermediate compounds are only

temporary (Manzocco, 2000). At the same time, constituents with moieties

possessing antioxidant behaviour and bound to different components of the

food/plant matrix can be released / cleaved from cell walls during thermal

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operations thereby allowing them to exhibit antioxidant activity. With an increase

in extraction time in microwave extraction an increase in the antioxidant behavior

was observed. The extraction time of 180 seconds exhibited the least IC50 values.

The results thus indicate that microwave assisted extraction can effectively extract

antioxidant compounds from cashew leaves with water as the extracting solvent.

• Extracts of Leaves Exposed to Various Drying Conditions

Drying of plant material at high temperatures can result in significant degradation

of the polyphenolics and also affect antioxidant and free-radical scavenging

capacities (Larrauri, 1997). In the experiments carried out, it was observed that the

order of antioxidant activity for extracts of leaves exposed to various drying

conditions was Sun dried > Shade dried > Oven dried > Fresh leaves. Thus, it can

be interpreted that sunlight temperature and browning of leaves in sun light might

lead to some reactions that lead to increase in the antioxidant activity of the

compounds.

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Table 6.16: Results of DPPH. radical scavenging assay

Sr.No. Sample IC50 value (µµµµg/mL) ± SD

Extracts of Cashew Leaves

1 Aqueous Extract 12.76 ± 0.35

2 Ethanol Extract 9.41 ± 0.73

Extracts of Cashew Testa



5 Methanol Extract 7.62 ± 0.45

Polyphenol Fractions of Cashew

6 Polyphenols of leaves 7.51 ± 0.51

7 Polyphenols of testa 7.42 ± 0.36

Extracts Prepared By Microwave Extraction

8 50 seconds 8.90 ± 0.44

9 70 seconds 8.63 ± 0.56

10 90 seconds 8.30 ± 0.61

11 120 seconds 7.40 ± 0.99

12 130 seconds 7.59 ± 0.83

13 150 seconds 7.20 ± 0.72

14 180 seconds 6.90 ± 0.67

Extracts of Leaves Exposed to Various Drying Conditions

15 Sun dried leaves 11.2 ± 0.70

16 Oven dried leaves 13.5 ± 0.81

17 Shade dried leaves 12.6 ± 0.78

18 Fresh leaves 14.3 ± 0.51

19 Ascorbic acid (Control) 5.3 ± 0.96

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B. Nitric oxide scavenging activity

Sodium nitro-prusside in aqueous solution at physiological pH spontaneously

generates nitric oxide which interacts with oxygen to produce nitrite ions that can

be estimated using Griess reagent. Scavengers of nitric oxide compete with

oxygen, leading to reduced production of nitrite ions.

• Extracts of Cashew Leaves

Overall, the ethanol extract of cashew leaves showed higher nitric oxide

scavenging ability compared to the aqueous extract as indicated in Table 6.17.

The IC50 values of ethanol and aqueous extracts were found to be 658.3 µg and

1002.3 µg of dry extract respectively. The presence of high levels of phenolic

compounds in the ethanol extract may have partly contributed to the observed

antioxidant activities. This study provided evidence on the potential health

benefits of cashew leaves. However, a detailed investigation of the molecular

mechanisms responsible for this activity is further required to understand the

mechanism of action of cashew leaves as antioxidant.

• Extracts of Cashew Testa

Sodium nitroprusside in aqueous solution at physiological pH spontaneously

generates nitric oxide which interacts with oxygen to produce nitrite ions that can

be estimated using Griess reagent. Scavengers of nitric oxide compete with

oxygen, leading to reduced production of nitrite ions. Overall, the ethanol extract

of cashew testa showed higher nitric oxide scavenging ability compared to the

aqueous extract and methanol extract. The IC50 values of aqueous, ethanol and

methanol extracts were found to be 518.6, 331.48 and 343.78µg/mL respectively.

All three extracts exhibited comparable antioxidant activity. Ethanol extract was

found to be most potent antioxidant with the least IC50 value when compared with

Ascorbic acid used as standard.

• Polyphenol Fractions of Cashew

Based upon the results of Griess assay it was observed that polyphenols of cashew

testa and leaves had considerable antioxidant activity as compared to Ascorbic

acid used as a control. However, a considerable difference between the

antioxidant activity of both the fractions was not observed.

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• Extracts Prepared By Microwave Extraction

Microwave assisted extraction led to an increase in the extractive yield as

observed in the previous experiments. The effect of this extraction process on the

antioxidant compounds in cashew leaves was ascertained by evaluation of

antioxidant activity. It was observed that the antioxidant effect increased with

increase in extraction time period. However, after 130 seconds there was no

considerable difference in the antioxidant activity of the extracts.

• Extracts of Leaves Exposed to Various Drying Conditions

Among the extracts of leaves exposed to varying drying conditions, it was

observed that leaves dried in sunlight exhibited the maximum antioxidant effect as

compared to shade dried, fresh and oven dried leaves. Thus, it can be inferred that

Sun drying may be the optimal drying condition amongst other conditions selected

in the study which leads to increase in the antioxidant phytoconstituents.

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Table 6.17: Results of nitric oxide scavenging activity

Sr.No. Sample IC50 value (µµµµg/mL) ± SD








Polyphenol Fractions of Cashew

6 Polyphenols of leaves 358.70 ± 0.79

7 Polyphenols of testa 330.64 ± 1.3


8 50 seconds 376.20 ± 0.53

9 70 seconds 365.50 ± 0.29

10 90 seconds 357.89 ± 0.63

11 120 seconds 350.79 ± 0.38

12 130 seconds 348.90 ± 0.35

13 150 seconds 347.50 ± 0.46

14 180 seconds 345.60 ± 0.40





18 Fresh leaves 396.5 ± 0.17

19 Ascorbic acid (Control) 293.54 ± 0.83

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6.8.2 Determination of Antioxidant Capacity against Lipid Peroxidation

A. Thiobarbituric acid Reacting substances (TBARS) test

Lipid peroxidation (LPO) can inactivate cellular components and plays an

important role in oxidative stress in biological systems. Furthermore, several toxic

byproducts from the peroxidation can damage other bio-molecules (Box, 1997;

Esterbauer, 1996). It is well established that transition of metal ions such as iron

and copper stimulate lipid peroxidation through various mechanisms (Halliwell,

1984). These may either generate hydroxyl radicals to initiate the lipid

peroxidation process or propagate the chain process via decomposition of lipid

hydroperoxides (Braughler, 1987). In this study, the extracts inhibited the lipid

peroxidation to a considerable extent as compared to standard i.e. Ascorbic acid.

The effect of extracts against lipid peroxidation could be attributed to presence of

phenolics, flavonoids, and glycosides. Ethanol extract of cashew testa and leaves

exhibited good peroxidation inhibitory activity than methanol and aqueous

extracts.

As observed in the results shown in Table 6.18, the ethanol extract of leaves and

aqueous extract of testa showed better anti-lipid Peroxidation activity as compared

to the other extracts.

Table 6.18: Results of anti-lipid peroxidation activity

Sr.No Extract IC50 Value (µg/mL) ± SD






4 Methanol extract 113.3 ± 0.32


Control

6 Ascorbic acid 23.60 ± 0.33

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6.8.3 Determination of Total Phenolics Content

A. Folin - Ciocalteu method

Phenolic compounds have been proved to be responsible for the antioxidant

activity in plants. The amounts of total phenolics in cashew extracts were

measured in this study. These extracts were found to have various phenolic levels

as indicated in Table 6.19. The ethanol extract of cashew test and leaves, Sun

dried leaves extract and Microwave extracted leaves for 180 seconds had the

highest content of total phenolics. The various levels of phenolics in these

extracts could be partly due to the differences in growing conditions. Under field

conditions, the phenolic compositions of plant tissues vary considerably with

seasonal, genetic, and agronomic factors (Hilton, 1973). In addition, a large

variability at various stages of maturation and growing conditions such as

temperature and extraction conditions affect the contents of phenolic compounds

(Zheng, 2001).

Table 6.19: Results of total phenolics content estimation

Sr.No. Sample Total Phenolic content

(mgGAE/g of extract) ± SD









6 50 seconds 32.90 ± 0.36

7 70 seconds 38.28 ± 0.69

8 90 seconds 39.02 ± 0.61

9 120 seconds 39.64 ± 0.42

10 130 seconds 40.01 ± 0.84

11 150 seconds 47.37 ± 1.4

12 180 seconds 49.43 ± 0.63





16 Fresh leaves 49.041 ± 0.89

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6.8.4 Antioxidant Capacity in Cultured Cells

NF-E2-related factor (Nrf2) is responsible for regulation of antioxidant response

element (ARE)–driven expression of genes encoding the majority of phase II

detoxification and antioxidant enzymes, such as NAD(P)H:quinone

oxidoreductase-1 (NQO1), glutathione S-transferases, glutamate–cysteine ligase,

and heme oxygenase-1 (HO-1). Basal and inducible antioxidant/phase II

detoxifying enzyme expression was found to be abrogated in the Nrf2-deficient

mice (Ramos-Gomez, 2001; Xu, 2006). The association of Nrf2 with ARE in the

promotor regions of antioxidant genes is a key regulatory step in stress protein

expression. Keap1 has been identified as a cytosolic binding protein for Nrf2

which associates with the Kelch domain of Keap1, and is sequestered in

association with the actin cytoskeleton under normal physiological conditions,

which in turn allows proteasomal degradation of Nrf2. Under oxidative stress or

treatment with electrophilic reagents, Nrf2 is released through the oxidation of the

cysteine residues on Keap1, allowing Nrf2 to translocate into the nucleus

(Nguyen, 2004).

A. ROS Assay :

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. Catechin, polyphenols of cashew testa, and aqueous extract of cashew testa

were found to inhibit ROS production. The results of ROS assay for some selected

extracts, showed a concentration dependent decrease in production ROS by

oxidation of H2DCFDA dye after 3 hrs incubation period are shown in Figure

6.54 - 6.56. Varying concentrations of tbH2O2 (0, 75 and 150 µM) were used to

induce oxidative stress conditions. With 0 µM tbH2O2, pre-incubation with plant

extracts exhibited slight variations in already low ROS levels. But with oxidative

stress conditions induced by 75 or 150 µM tbH2O2, increasing concentration of the

extracts decreased the amount of ROS levels formed, as indicated by a decrease in

fluorescence signal.

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Figure 6.54: ROS assay of catechin, 3 hours after tbH2O2 stimulation

Figure 6.55: ROS assay of polyphenols of testa, 3 hours after tbH2O2

stimulation

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Figure 6.56: ROS assay of aqueous extract of cashew testa, 3 hours after

tbH2O2 stimulation

B. Viability Assay:

The Cell Proliferation Reagent WST-1 provides a colorimetric assay for the

quantification of cell viability and proliferation. WST-1 is a tetrazolium salt that

when in contact with metabolically active cells gets cleaved to formazan by

mitochondrial dehydrogenases. The formazan dye is then measured using a

scanning spectrophotometer at wavelengths 420-480 nm. The effect of plant

extracts on cell viability under oxidative stress conditions induced by tbH2O2

after 3 hrs incubation period is shown in Figure 6.57-6.61. In the graphs indicated

below EBM2 is used to represent the culture medium used as a control to ascertain

whether the media components or plant extracts affect WST-1 dye processing in

the absence of cells. First of all, incubation pre-treated cells with vehicle (o µM

tbH2O2) showed that the WST-1 dye was processed to formazan and thus that the

cells were viable. Incubation with tbH2O2 decreased cell viability as indicated by

decreased formazan production. Pre-incubation with increasing concentrations of

catechin, ethanol extract of cashew leaves, ethanol extract of cashew testa and

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polyphenols of cashew testa and leaves were found to rescue cell viability after

H2O2 treatment to some extent, however, a complete rescue of cell viability was

not observed for any of the extracts. Unexpectedly, the presence of formazan was

detected in all EBM-2 wells, suggesting that cells were mistakenly added to the

wells.

Figure 6.57: Viability assay of ethanolic extract of cashew leaves, 3 hrs after

stimulation with tbH2O2

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Figure 6.58: Viability assay of ethanolic extract of cashew testa, 3 hrs after


Figure 6.59: Viability assay of polyphenols of cashew testa, 3 hrs after


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Figure 6.60: Viability assay of catechin, 3 hrs after stimulation with tbH2O2

Figure 6.61: Viability assay of polyphenols of cashew leaves, 3 hrs after


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C. Angiogenesis assay:

The method is based on the differentiation of ECs on a basement membrane

matrix, Matrigel, derived from the Engelbreth-Holm-Swarm tumor. ECs from

human umbilical cords as well as from other sources differentiate and form

capillary-like structures on Matrigel in the presence of 10% bovine calf serum

(BCS) and 0.1 mg/mL of endothelial cell growth supplement (ECGS), which is a

mixture of both acidic and basic fibroblast growth factor (Croix, 2000). An

inhibition of angiogenesis was observed by tbH2O2. The ability of the extracts to

rescue this inhibition of angiogenesis caused by tbH2O2 was assessed.

HMEC cells were seeded on matrigel. Measurement of angiogenic capacity was

based on the mean tube length observed after 24 hrs. Photographs of the tubes

formed were taken with the help of Olympus DP71 Microscope and the mean

tubule length was quantified with Angioquant software. The deleterious effect of

oxidative stress on angiogenic capacity of HMEC cells was observed on untreated

cells as well as cells pretreated with catechin. Catechin, showed no significant

antiangiogenic effect nor any statistically significant inhibitory effect on the

angiogenesis inhibiting activity of tbH2O2.

As observed in Figure 6.62, in-vitro investigations have indicated that catechin

was not able to inhibit several key events of the angiogenic process. In our

experiments we were unable to derive conclusions about the effect of extracts on

the angiogenesis inhibiting activity of tbH2O2. The data obtained from Matrigel

assays needs to be reanalyzed. Reports have indicated that certain polyphenolic

compounds inhibit certain angiogenesis processes such as proliferation and

migration of endothelial cells and vascular smooth muscle cells and the expression

of two major proangiogenic factors, vascular endothelial growth factor (VEGF)

and matrix metalloproteinase-2, by both redox-sensitive and redox-insensitive

mechanisms (Kondo, 2002).

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EGM2 H2O2

2.5 Catechin 2.5 Catechin + H2O2

Figure 6.62: Representative matrigel assay of catechin (2.5 micrg) after

24 hrs incubation

D. Western blot analysis:

The proteins were isolated from cells treated with varying concentrations of

catechin, control, tbHQ and tbH2O2 for 3 hrs. The expression of Nrf2 and beta-

actin by proteins extracted from pretreated HMEC cells was measured by Western

blot analysis with indicated specific antibodies. The experiments were repeated

three times a representative blot is shown below in Figure 6.63.

Upon activation, Nrf2 protein is stabilized and translocates to the nucleus to

heterodimerize with other leucine zipper transcription factors such as Nrf1, mafK,

junD, and c-fos, and bind to ARE in target gene promoters. To study the effects of

pretreatment with plant extracts on Nrf2 protein expression, Western blot analysis

was performed.

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The image depicted in Figure 6.63 represents the blot prepared to evaluate NrF2

expression. Multiple bands of low signals at varying positions were observed in

the lane of standard NrF2 lysate. This casts a doubt on location of the exact band

for NrF2 in the controls as well as in the lysates of cells treated with catechin. The

protocol adopted needs to be optimized to obtain a higher signal of the protein of

interest, so that the expression of the protein can be better visualized and

quantified. Thus the experiments of Western Blot need to be repeated, in order to

conclude about the effect of catechin on NrF2 protein expression in HMECs.

Where, 1o Antibody- Nrf2 H-300 and 2

o- Swine antibody – Anti rabbit

immunolglobulin/HRP. L – Ladder, M- medium, CL-Control lysate, N- Nrf2

lysate, t- tbH2O2. 2.5 C, 25 C and 125 c are varying concentrations of catechin in

micrograms used for treatment.

Figure 6.63 Western Blot for Nrf2 expression of HMEC cells treated with

varying concentrations of catechin, tBHQ and hydrogen peroxide.

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E. RT-PCR analysis:

Exposure of HMEC cells to catechin increased the NrF2 protein levels as

observed in the western blot assay, whereas an increase was not found upon tbHQ

and tbH2O2 stimulation of cells. tbHQ was used as a positive control for NrF2

activation, however an optimal increase in the induction of NrF2 target genes was

not observed even with tbHQ treatment. Expression of phase II enzymes is

important in protecting the cells against stress conditions. We evaluated mRNA

expression profiles of phase 2 enzymes in catechin, tbHQ (positive control) and

tbH2O2 treated cells using real-time PCR. Treatment of HMEC cells with 2.5 and

25µM concentration of catechin resulted in an upregulation of the Nrf2 target gene

HMOX. Upregulation of the Nrf2 target gene HMOX was observed compared to

tbHQ (positive control) and vehicle as the control. At a 25µM concentration of

catechin for GCLC and NQO-1 a decrease was observed upto 1.5 and 0.6 fold

respectively.

The treatment with tbH2O2 also exhibited a decrease in the responses for HMOX,

GCLC, and GCLM, and NQO-1 upto 1.8, 0.9, 0.5 and 0.9 fold respectively.

Hence, we may infer that a down regulation of HMOX, and GCLM, and NQO-1

genes was observed with tbH2O2. The expression of Nrf2 genes GCLC and

GCLM was decreased upto 1.3 and 0.8 fold by tbH2O2. Hence, we observe a

down regulation of 3 out of 4 Nrf2 genes by catechin as indicated in the results of

our experiments depicted in Figure 6.64.

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Figure 6.64: Effect of catechin, tbHQ and tbH2O2 stimulation on expression of

NrF2 genes in HMEC’s

***p<0.0001 and **p≤ 0.001 to 0.01. Significant p - values were obtained for

HMOX and GCLM gene expression by catechin.

The objective of the present study was to evaluate the effect of oxidative stress on

cultured endothelial cells (treated with plant extracts), specifically with reference

to the intracellular protective mechanism that is governed by Nrf2. Several

compounds, including known Nrf2 activators, bioactive plant extracts, phenolic

and catechin fractions from cashew were tested for their potential to reduce

oxidative stress and its detrimental effects on HMECs.

Growing evidence indicates an important role for ROS in diabetes, hypertension,

restenosis after balloon angioplasty and atherosclerosis. However, little and

contradictory information exists about the mechanisms by which ROS elicit their

effect on the structure and function of the cells of the blood vessels. Fewer data

exist on the correlation between the ROS status and endothelial cell death. In the

present work, plant extracts which exhibited direct antioxidant effect in in-vitro

assays like DPPH radical Scavenging assay and Greiss assay were used as

candidates for various indirect in-vitro assays on HMECs. ROS assay was

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performed to establish the antioxidant potential of the plant extracts. For

mimicking oxidative stress, we selected tert-butyl hydrogen peroxide (tbH2O2).

The cells pretreated with plant extracts showed a decrease in ROS levels after

tbH2O2 stimulation, indicated by a decrease in dye flurorescence. The effect may

be attributed to the activity of plant extracts to reduce ROS formation, especially

in the concentration range from 2.5 to 25µg.

We questioned whether the oxygen radicals affect directly the HMECs, whether

their effect is dependent on concentration and whether this insult may lead to cell

death. To this purpose, cultured HMECs were exposed to oxidative stress for

different time intervals and concentrations. The ability of plant extracts to reverse

the effect of oxidative stress on cell viability was estimated by use of WST-1 dye

assay. The results showed that exposure of cultured HMECs to tbH2O2 led to an

expected decrease in cell viability. The cell viability decreased with increasing

concentrations of tbH2O2, even in the cells treated with cashew extracts. Thus we

may infer that, although the plant extracts exhibit significant antioxidant activity

in the ROS assay, their mode of reduction of ROS species does not improve cell

viability as measured by mitochondrial dehydrogenase activity.

Several recent studies have indicated that polyphenols, flavanols and anthocyanins

have in vitro and in vivo antiangiogenic properties by inhibiting the expression of

two strong proangiogenic factors, VEGF and matrix metalloproteinase (MMP-2),

and also by preventing the proliferation and migration of endothelial cells (ECs)

and vascular smooth muscle cells (VSMCs) (Diaz, 1997).

The antiangiogenic properties of polyphenolic compounds could contribute to

explain the reduced risk of coronary heart diseases (viz. Arteriosclerosis) and

cancer mortality following chronic consumption of moderate amounts of red wine

and green tea.

Angiogenesis is a key process in the development of several pathologies,

including cancer, and the inhibition of angiogenesis is regarded as a promising

tool to combat cancer. To develop novel angiogenesis inhibiting agents, the

phenotype of tumor endothelial cells is subject to intensive investigation to

identify putative target molecules for interference (Croix, 2000). Proper validation

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of target molecules and inhibitors requires reproducible experimental in vitro

approaches. Tissue-specific micro-environmental factors have a pronounced

influence on the phenotype of the endothelial cells within the tissue.

HMEC have been extensively used in angiogenesis research, and we used HMEC

cells to evaluate the effect of plant extracts on angiogenesis. In vitro investigations

have indicated that polyphenolic compounds are able to inhibit several key events

of the angiogenic process such as proliferation and migration of endothelial cells

and vascular smooth muscle cells and the expression of two major proangiogenic

factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-

2, by both redox-sensitive and redox-insensitive mechanisms (Kondo, 2002).

However, in our experiments we were unable to derive conclusions about the

effect of extracts on the angiogenesis inhibiting activity of tbH2O2. The data

obtained from Matrigel assays needs to be reanalyzed.

Nrf2 levels in cells are regulated by further phosphorylation, nuclear export, and

degradation, which may be enhanced by ARE-linked expression of Keap1 (Jain,

2007). Nrf2 may also exhibit ARE-linked expression. We investigated the

activation status of Nrf2 in human microvascular endothelial cells by assessing

nuclear translocation of Nrf2 by immunoblotting in protein fractions.

Quantitative Western blotting for Nrf2 revealed increased expression of a protein

at 57-kDa in fractions of cells treated with 25 microgram concentration of

catechin. The lysates from tbHQ and tbH2O2 stimulated cells did not increase the

expression to a greater extent as compared to catechin. This may be due to

inability of tbHQ and tbH2O2 to translocate Nrf2 to Antioxidant Response

enzyme (ARE) binding sites in nucleus with stimulation period of 3 hours. The

experiments need to be designed more appropriately in order to conclude about

the Nrf2 expression activity of catechin, tbHQ and tbH2O2.

A sharp distinct band was observed at 57-kDa in all the lanes. But as this band

also appears to be in the negative control lanes of the lysates of cells treated with

lysates alone, we are uncertain about locating the right band.

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We next focused on the effect of the plant extracts on the expression of Nrf2 target

genes. The results from q-PCR experiments suggest that in Human microvascular

endothelial cells, catechin activates Nrf2 in a concentration dependent manner. This

effect was observed as an increase in HMOX1 expression, a target gene of Nrf2. A

down regulation of most of the selected NrF2 genes was observed in our experiments

with tbHQ and tbH2O2, and catechin. Hence the experiments should be still

investigated further with proper positive controls in order to infer about the effect of

catechin on expression of the target genes of Nrf2.

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6.9 PHARMACOLOGICAL INVESTIGATIONS OF CASHEW EXTRACTS

FOR ANTIDIABETIC ACTVITY

6.9.1 Acute Oral Toxicity Studies – Acute Toxic Class Method

Acute Oral Toxicity Studies were carried out in Albino mice following OECD 423

Guidelines, for extracts which showed a better antioxidant activity in vitro and

the results obtained are indicated in Table 6.20. Dose limit at 2000 mg/kg (single

dose) was administered to mice and observed for 14 days.

The crude extract/s and polyphenol fractions of leaves and testa of cashew did not

produce toxic symptoms or changes in behavior or death and found to be safer in

mice upto the dose of 2000 mg/kg body weight, except for Polyphenol fraction of

leaves.

Animals treated with ethanol extracts of leaves and testa of cashew exhibited

normal body weight gain and food intake throughout the study. Animals treated

with polyphenol fraction of leaves showed a slight abnormal contractions in the

abdominal region, possibly due to high polyphenolic content which might have

caused gastric irritation.

Acute toxicity tests have shown that the LD50 of the extract in mice was higher

than 2000 mg/kg except for Polyphenol fraction of leaves for which the limit was

2000 mg/kg and it’s categorized under category 5 and category 4 of GSH as per

OECD guidelines 423 respectively.

The data describing the toxicity of ethanol extracts and polyphenol fractions of

ethanol extract of cashew leaves and testa indicates a moderate toxicity of

polyphenol fraction of cashew leaves. Nevertheless, the folk medicine generally

uses aqueous extracts of the cashew leaves (Konan, 2007). We may therefore

conclude that the long history of the cashew leaves used in folk medicine without

toxicity reports seems to be largely supported by the data shown here.

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Table 6.20: Acute toxicity studies of extracts of cashew

Test Substance

Dose

Level

LD50 Cut

off value

Mortality

at selected

doses

LD50 cutoff

Ethanol Extract

of Testa

2000

mg/kg

b.w.

5000

mg/kg

b.w

0/6

> 2000 mg/kg

Category 5 of

GSH

Ethanol Extract

of Leaves

2000

mg/kg

b.w.

5000

mg/kg

b.w

0/6

> 2000 mg/kg

Category 5 of

GSH

Polyphenols of

ethanol extract

of Testa

2000

mg/kg

b.w.

5000

mg/kg

b.w

0/6

> 2000 mg/kg

Category 5 of

GSH

Polyphenols of

ethanol extract

of leaves

300

mg/kg

b.w.

1000

mg/kg

b.w

1/6

> 300 mg/kg

Category 4 of

GSH

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6.9.2 Evaluation of The Effect Of Cashew Leaves And Testa Extracts In

Streptozotocin-Nicotinamide Induced Type-II Diabetic Rats

From the eight extracts prepared from leaves and testa of cashew, three extracts

were selected for checking the hypoglycemic activity 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 screened by STZ - Nicotinamide Induced Type 2 Diabetes

Mellitus were then selected for evaluation of antidiabetic activity by Neonatal

streptozotocin model.

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]

Extracts, fractions and standard were administered in the form of oral solution and

suspension once daily for 15 consecutive days to diabetic animals. Control

animals received only vehicle. For blood glucose levels, the blood was withdrawn

by tail snipping on day 0,7,15 and estimated using glucose strips (Accu check

active, Roche diagnostics, Germany).On day 15,blood was collected and

estimated for various biochemical parameters. The results obtained for each of the

parameters are given in Table 6.21 – 6.24.

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• Determination of Physical end points

Table 6.21: Effect of extracts on body weight on rats

Treatment Body weight (g)

On day 1 Day 7 Day 14

Normal control 237.17 ± 6.26 245.17 ± 5.56 254.83 ± 5.33

Diabetic control 200.67 ± 5.24 193.33 ± 4.27 184.33 ± 4.90

Glibenclamide

0.45 mg/kg 203.17 ± 7.04 202.83 ± 6.85 206.83 ± 5.43

Ethanol extract

of cashew testa

(175 mg/kg)

201.5 ± 6.28 203.33 ± 6.63 200.33 ± 4.60

Polyphenols of

cashew Testa

(50 mg/kg)

208.17 ± 6.26 207.33±7.31 203.5 ± 6.01

Ethanol Extract

of cashew

leaves (100

mg/kg)

207.50 ± 8.06 205.50 ± 8.44 199.33 ± 7.32

Ethanol extract

of cashew testa

(350 mg/kg) in

divided doses

210 ± 5.06 207 ± 3.90 200 ± 4.16

Values are expressed as mean ± SEM, n = 6

* Significantly different from diabetic control, p<0.05

As observed in Table 6.21, ethanol extract of leaves, ethanol extract of testa and

polyphenols of cashew testa and ethanol extract of leaves (double dose i.e. 350

mg/kg) evaluated for their antidiabetic effects on STZ-Nicotinamide induced

model. The results showed no significant difference in the body weights of

diabetic animals treated with extracts as compared with diabetic control at p<0.05.

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• Determination of Biochemical End points

Table 6.22: Effect of extracts on fasting blood glucose levels in rats

Treatment Fasting blood glucose mg/dl ± SEM


Normal control 85.33 ± 3.18 85.50 ± 2.16 85.50 ± 2.75


Glibenclamide 0.45

mg/kg 259.33 ± 10.16 198.67 ± 9.15* 121.17 ± 8.10*

Ethanol extract of

cashew testa (175

mg/kg)

260.83 ± 8.86 221.83 ± 7.45* 161.17 ± 5.61*

Polyphenols of cashew

Testa (50 mg/kg) 269.52 ± 3.6 228.24 ± 6.2* 144.21 ± 2.3*

Ethanol Extract Of

cashew leaves (100

mg/kg)

259.13 ± 2.5 218.13 ± 7.1* 154.14 ± 3.61*

Ethanol extract of

cashew testa

(350 mg/kg) in divided

doses

259.13 ± 2.5 191.34 ± 5.9* 150.54 ± 8.6*

Values are expressed as mean ± SEM, n = 6;

* Significantly different from Control, p<0.05

As observed in Table 6.22, ethanol extract of leaves, ethanol extract of testa and

polyphenols of cashew testa and ethanol extract of leaves (double dose i.e. 350

mg/kg) tested for their antidiabetic effects on STZ-Nicotinamide induced model.

The diabetic animals treated with extracts showed a significant decrease in the

fasting blood glucose levels as compared with diabetic control at p<0.05.

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Table 6.23: Effect of extracts on percent reduction in fasting blood glucose

levels in rats

Treatment % Reduction in FBG

Day 7 Day 15

Normal control 0.73 ± 1.5 -0.32 ± 1.0

Diabetic control -2.13 ± 0.6 -6.40 ± 1.3

Glibenclamide 0.45 mg/kg 23.49 ± 1.0 53.45 ± 1.6

Ethanol extract of cashew testa (175 mg/kg) 14.9 ± 0.6 38.2 ± 2.5

Polyphenols of cashew Testa (50 mg/kg) 15.31 ± 3.2 46.49 ± 1.8

Ethanol Extract Of cashew leaves

(100 mg/kg) 15.82 ± 3.0 40.51 ± 3.5

Ethanol extract of cashew testa

(350 mg/kg) in divided doses 26.14 ± 2.0 42.16 ± 1.3

Percent reduction in glycemia was calculated with respect to the zero day level

according to the following formula:

Percent reduction in glycemia = [(Gi-Gt)/Gi] x 100

Where Gi is initial glycemia values and Gt is the glycemia value at 7 and 15 days.

As seen in the results in Table 6.23, ethanol extract of testa, polyphenols of

cashew testa and ethanol extract of leaves, at single dose levels and ethanol extract

of testa at double dose caused a 14.9%, 15.31%, 15.82 % and 26.14% reduction in

the fasting blood glucose levels of diabetic animals treated with extracts on day 7.

On day 15 a reduction of 38.02%, 46.49%, 40.51% and 42.16 % in the fasting

blood glucose levels of diabetic animals treated with extracts was observed.

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Table 6.24: Effect of extracts on serum biochemical parameters in rats

Treatment

Serum parameters on day 15

TG

(mg/dl) TC (mg/dl)

HDL-C

(mg/dl)

LDL-C

(mg/dl)

VLDL-C

(mg/dl)

Normal

control 72.81±3.27* 75.21±1.7* 72.3 ± 2.2 11.3 ± 2.2 14.1±0.6*

Diabetic

control 203.43±3.36 122.81±3.5 43.8 ± 3.6 37.6 ± 5.8 40.6 ± 0.6

Glibenclamide

(0.45 mg/kg) 73.07 ± 1.2* 108.8 ± 3.8* 38.2 ± 2.8 55.6 ± 4.4 14.6±0.2*

Ethanol

extract of

cashew testa

(175 mg/kg)

78.44±3.87* 111.51±4.05* 39.7 ± 2.0 56.0±4.2* 15.6±0.7*

Polyphenols

of cashew

Testa (50

mg/kg)

86.85 ± 2.7* 95.0 ± 3.0* 39.2 ± 0.9 38.6 ± 3.4 17.1±0.4*

Ethanol

Extract Of

cashew leaves

(100 mg/kg)

99.62 ± 2.4* 90.3 ± 2.4* 45.1±5.8* 25.2 ± 6.9 19.9±0.9*

Ethanol

extract of

cashew testa

(350 mg/kg)

in divided

doses

74.82 ± 1.4* 93.3 ± 7.4* 42.1 ± 3.6 27.2 ± 6.2 17.9±0.6*

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Values are expressed as mean ± SEM, n = 6,*Significantly different from diabetic

control,p<0.01

TG – Triglyceride; TC - Total cholesterol; HDL-C – High density lipoprotein

cholesterol

LDL-C - Low density lipoprotein cholesterol

VLDL-C – Very low density lipoprotein cholesterol

LDL-C, VLDL- C calculated using friedwald formula

VLDL = TG/5; LDL=TC-(HDL+TG/5)

As observed in the results stated in Table 6.24, ethanol extract of testa,

polyphenols of cashew testa and ethanol extract of leaves, at single dose levels

and ethanol extract of testa at double dose levels tested for their antidiabetic

effects on STZ-Nicotinamide induced model showed statistically significant

results as compared with diabetic control at p<0.01 for the lipid profile Viz,

triglyceride, total cholesterol, and VLDL-c levels. The total cholesterol showed

statistically significant results as compared with diabetic control at p<0.01.

The fundamental mechanism underlying hyperglycemia involves over-production

(excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization

of glucose by the tissues (Latner, 1958). Persistent hyperglycemia, the common

characteristic of diabetes can cause most diabetic complications. In all patients,

treatment should aim to lower blood glucose to near-normal levels.

The diabetic syndrome in rats administered STZ and partially protected with

suitable dosages of nicotinamide is characterized by stable moderate

hyperglycemia, glucose intolerance and altered but significant glucose stimulated

insulin secretion (Masiello, 1998).

In our investigation, the blood glucose level estimation studies revealed that the

ethanolic extracts and Polyphenol fractions of cashew leaves and testa have the

capacity to lower blood glucose levels.

The marked increase in serum triglycerides and cholesterol observed in diabetic

rats is in agreement with the findings of Nikkila and Kekki, 1973. The most

common lipid abnormalities in diabetes are hypertriglyceridemia and

hypercholesterolemia (Khan, 1995; Mitra, 1995). Hypertriglyceridemia is also

associated with metabolic consequences of hypercoagulability, hyperinsulinemia,

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insulin resistance and insulin intolerance (Gingsberg, 1994). In our study,

administration of the extract to the STZ induced diabetic rats significantly (p <

0.05) improved these parameters. The observed hypolipidaemic effect may be

because of decreased cholesterogenesis and fatty acid synthesis.

Various studies on medicinal plants have reported a similar lipid lowering activity

(Ram, 1997; Sharma, 1997; Jouad, 2003). The characteristic loss of body weight

associated with STZ induced diabetes is due to increased muscle wasting in

diabetes (Swanston-Flat, 1990). The animals treated with extracts of cashew testa

and leaves showed a weight loss in our studies, which may be directly due to the

lipid lowering activity of the extract or indirectly to the influence on various lipid

regulation systems. The significant antidiabetic activity of the cashew extracts in

our study may be attributed to its principle antioxidant constituents. Longer

duration studies on chronic models may contribute towards the development of a

potent antidiabetic drugs and help gain insights into molecular mechanisms of

action of herbal drugs.

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6.9.3 Evaluation of the effect of cashew leaves and testa extracts in neonatal

Streptozotocin induced (n- STZ) rat model of Type 2 Diabetes Mellitus

The aim of our study was to investigate the effects of bioactive extracts of leaves

and testa of cashew in streptozotocin induced neonates. Various biochemical and

physical parameters were estimated and the results are indicated in

Table 6.25 – 6.31.

• Determination of physical endpoints

Table 6.25: Effect of extracts on body weight in rats

Treatment Body weight (g)

On day 1 Day 10 Day 20 Day 30

Normal control 229.17±7.4 238.17±6.9 245.83±6.1 249.50±5.1

Diabetic

control 206.83±11.7 218.67±12.2 210.83±12.1 209.33±13.3

Pioglitazone 2

mg/kg 222.67±8.8 230.33±10.1 236.17±8.82 239.67±8.42

Ethanol extract

Of leaves

(100 mg/kg) 213.67±13.5 215.33±18.0 223.67±14.0 229.83±15.0

Ethanol extract

of testa (175

mg/kg) 266.33±12.8 218.33±12.9 220.83±15.0 222.67±15.3


* Significantly different from diabetic control, p<0.05

As observed from the results shown in Table 6.25, ethanol extract and

polyphenols of cashew testa were evaluated for their antidiabetic effect on

neonatal STZ (n2-STZ) model of type 2 diabetes in rats at single dose levels. Upto

a period of 15 days and 30 days, there was no significant difference observed in

the body weights of the treatment group and the diabetic control group at p<0.05.

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• Determination of Biochemical End points

Table 6.26: Effect of extracts on fasting blood glucose levels in rats

Treatment Fasting blood glucose (mg/dl) ± SEM


Normal control 78.67 ± 0.84 76.5 ± 1.38 75.83 ± 1.82


Pioglitazone 2 mg/kg 144.83 ± 7.24 129.50 ± 5.47* 113.33 ±3.30*

Ethanol extract of

cashew leaves(100

mg/kg)

147.67 ± 6.09 135.83 ± 4.40 123.83 ±2.87*

Ethanol extract of testa

(175 mg/kg) 145.83 ± 6.17 138.67 ± 5.33 128.50 ±4.36*



As indicated in the results shown in Table 6.26, ethanol extract and polyphenols

of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-

STZ) model of type 2 diabetes in rats at single dose levels.

The extracts showed statistically significant reduction in the reduction of blood

glucose levels at p<0.05 on day 30.

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Table 6.27: Effect of extracts on serum insulin and glycated haemoglobin in rats

Treatment

Serum insulin

on day 30

(Uiu/ml)

Glycated

haemoglobin

on day 30

(%)

FIRI

on day 30

Normal control 16.99 ± 1.5 3.92 ± 0.08 51.71 ±5.1

Diabetic control 16.52 ± 1.9 5.6 ± 0.27 103.37 ±14.8

Pioglitazone 5 mg/kg 13.43 ± 1.4 4.25 ± 0.18* 60.44 ±5.86*

Ethanol extract Of

cashew leaves (100

mg/kg)

11.69 ± 0.93 4.92 ± 0.17* 57.90 ±4.69 *

Ethanol extract of

testa (175

mg/kg)

12.99 ± 1.4 4.98 ± 0.14 66.76 ± 4.10*



As observed from the results shown in Table 6.27, ethanol extract and

polyphenols of cashew testa were evaluated for their antidiabetic effect on

neonatal STZ (n2-STZ) model of type 2 diabetes in rats at single dose levels. A

statistically significant decrease in the glycated haemoglobin levels was observed

at p<0.05. Ethanol extract of leaves and ethanol extract of testa decreased the

fasting insulin resistance index (FIRI) and it was comparable to standard –

pioglitazone.

A decrease in serum insulin levels was also observed as compared with diabetic

control but it was not found to be statistically significant at p<0.05.

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Table 6.28: Effect of extracts on serum triglyceride levels in rats

Treatment Serum triglyceride( mg/dl) ± SEM


Normal control 58.55 ± 3.65 61.21 ± 3.99 60.06 ± 4.13


Pioglitazone 2

mg/kg 94.80 ± 3.05 84.59 ± 2.67 75.93 ± 2.52*

Ethanol extract

Of cashew

leaves(100

mg/kg)

96.63 ± 4.22 88.84 ± 4.16 81.69 ± 4.47*

Ethanol extract

of testa (175

mg/kg)

98.95 ± 3.40 91.51 ± 4.32 83.49 ± 4.55 *



Ethanol extract and polyphenols of cashew testa were evaluated for their

antidiabetic effect on neonatal STZ (n2-STZ) model of type 2 diabetes in rats at

single dose levels.

The triglycerides levels on day 30 were found to be decreased and statistically

significant as compared to diabetic control at p<0.05.

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Table 6.29: Effect of extracts on serum total cholesterol levels in rats

Treatment Serum total cholesterol (mg/dl) ± SEM


Normal control 78.89 ± 3.22 77.82 ± 2.78 78.09 ± 3.87


Pioglitazone 2

mg/kg 95.11 ± 2.72 88.90 ± 2.64 * 84.21 ± 3.18 *

Ethanol extract Of

cashew leaves(100

mg/kg)

96.32 ± 2.59 91.93 ± 2.51 87.91 ± 3.21 *

Ethanol extract of

testa (175 mg/kg) 98.12 ± 2.29 94.98 ± 2.53 90.77 ± 2.81 *


* Significantly different from Control, p>0.05

As shown in Table 6.29, ethanol extract and polyphenols of cashew testa were

evaluated for their antidiabetic effect on neonatal STZ (n2-STZ) model of type 2

diabetes in rats at single dose levels.

Decrease in total cholesterol levels were found as compared with diabetic control

and the values were significant at p<0.05.

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Table 6.30: Effect of extracts on lipid parameters in rats

Treatment Serum Parameter on day 30

HDL-C (mg/dl) LDL-C (mg/dl) VLDL-C (mg/dl)

Normal control 37.35 ± 1.81 28.73 ± 3.95 12.01 ± 0.82


Pioglitazone 5

mg/kg 39.74 ± 2.28 29.31 ± 3.80 * 15.15 ± 0.78 *

Ethanol extract of

cashew leaves

(100 mg/kg)

36.19 ± 1.80 35.39 ± 3.03 16.34 ± 0.89 *

Ethanol extract of

testa (175

mg/kg)

35.77 ± 1.56 38.30 ± 3.84 16.69 ± 0.91 *


* Significantly different from diabetic control, p>0.05

TG – Triglyceride; TC - Total cholesterol; HDL-c – High density lipoprotein

cholesterol

LDL-C - Low density lipoprotein cholesterol

VLDL-C – Very low density lipoprotein cholesterol

LDL-C, VLDL- c calculated using friedwald formula

VLDL = TG/5;LDL=TC-(HDL+TG/5)

As observed in the results shown in Table 6.30, ethanol extract and polyphenols

of cashew testa were evaluated for their antidiabetic effect on neonatal STZ (n2-

STZ) model of type 2 diabetes in rats at single dose levels.

The lipid profiles for VLDL-C levels showed statistically significant results as

compared with diabetic control at p<0.05. However a significant reduction in

LDL-C and HDL-C were not observed at p<0.05.

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Table 6.31: Effect of extracts on renal function biomarkers in rats

Treatment Serum biomarkers of Liver and Kidney on day 30

SGOT SGPT Urea Creatinine

Normal

control 273.14 ± 5.47 71.88 ± 4.91 61.18 ± 1.64 0.67 ± 0.02

Diabetic

control 286.71± 28.46 85.34± 10.85 77.83 ± 2.12 0.81 ± 0.06

Pioglitazone

2 mg/kg 291.83± 20.61 91.16 ± 7.28 67.60 ± 3.88 0.75 ± 0.02

Ethanol

extract Of

cashew

leaves(100

mg/kg)

222.19± 23.23 66.29 ± 4.27 66.93 ± 3.96 0.66±0.01*

Ethanol

extract of

testa (175

mg/kg)

260.94± 32.86 74.97 ± 9.90 70.80 ± 3.74 0.71± 0.02*


* Significantly different from diabetic control, p>0.05

As indicated in Table 6.31, Ethanol extract and polyphenols of cashew testa were

evaluated for their antidiabetic effect on neonatal STZ (n2-STZ) model of type 2

diabetes in rats at single dose levels.

The renal markers were accessed to ascertain the effect of drug treatment in

diabetic rats. However, there was no significant difference observed between the

treatment group and diabetic control at p<0.05.

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• The Histological and Histochemical Studies

After blood sampling for the biochemical analysis, the animals were sacrificed,

quickly dissected, and small slices of the liver and kidney were taken and fixed in

10% formalin. The specimens were dehydrated in ascending grades of ethanol,

cleared in xylene, and embedded in paraffin wax. Sections of 6 µm in thickness

were prepared and stained with Haematoxylin and Eosin to examine under

microscopy at 100x magnification.

Histopathological Results

Liver: The liver of control rats appears to be divided into the classical hepatic

lobules; each is formed of cords of hepatocytes radiating from the central vein to

the periphery of the lobule. The cell cords were separated by narrow blood

sinusoids (Figure 6.65 - a). The histopathological examination of diabetic rats

showed periportal necrosis of the hepatocytes near the portal areas. The liver also,

showed dilated and congested portal vessels as well as areas of inflammatory cell

infiltration (Figure 6.65 - b). In diabetic rats treated with standard Pioglitazone

liver of control rats appears to be divided into the classical hepatic lobules; each is

formed of cords of hepatocytes radiating from the central vein to the periphery of

the lobule. The cell cords were separated by narrow blood sinusoids as in normal

control rat (Figure 6.65 - c). In diabetic rats treated with extracts of testa and

leaves, the liver architecture appears more or less like control (Figure 6.65 - d

and e).

Kidney: Examination of the kidney of the normal control rats revealed normal

glomeruli with thin glomerular basement membranes, normal cellularity and

patent capsular space surrounding by proximal and distal were normal (Figure

6.66 - a). Light microscopy of the kidney sections from diabetic rats showed an

increase in the mesangial cell and matrix of the glomeruli and hyalinization of the

arterioles (Figure 6.66 - b). In diabetic rats treated with pioglitazone, the kidney

architecture appears more or less like normal control (Figure 6.66 – c). In diabetic

rats treated with leaves and testa extract, the kidney architecture appears more or

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less like normal control with the exception of some inflammatory infiltration that

appeared in the interstitum (Figure 6.66 – d and e).

Pancreas: In the pancreas of normal control rats many round (Figure 6.67 - a)

and elongated islets were evenly distributed throughout the cytoplasm, with their

nucleus lightly stained than the surrounding acinar cells. In diabetic rats, (Figure

6.67 - b) the islets were damaged, shrunken in size and infiltration of

lymphocytes was observed. In rats treated with plant extracts and standard

Pioglitazone, islets were comparable to normal rats and there was not much

shrinkage in size of the islet although slight damage was observed. (Figure 6.67 –

c, d and e).

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Figure 6.65: Photomicrographs of liver

a) normal rat b) diabetic rat c) diabetic rats treated with standard drug

d) diabetic rat treated with ethanol extract of leaves e) diabetic rats treated

with ethanol extract of testa.

(Sections treated with hematoxylin and eosin x 100)

a b

c d

e

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Figure 6.66: Photomicrographs of kidney a) normal rat b) diabetic rat c)

diabetic rats treated with standard drug d) diabetic rat treated with ethanol

extract of leaves e) diabetic rats treated with ethanol extract of testa.


a b

c d

e

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Figure 6.67: Photomicrographs of pancreas

a) normal rat b) diabetic rat c) diabetic rats treated with standard drug

d) diabetic rat treated with ethanol extract of leaves e) diabetic rats treated

with ethanol extract of testa.


a b

c d

e

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In further agreement with Portha et al. (2002),it can be suggested that this animal

model is also suitable for measuring insulin secretion in comparison with a (non-

glucose-dependent) insulin-secreting drug like tolbutamide. The data obtained

from the experiments provided a clear evidence that an oral single dose of this

plant extracts stimulates insulin secretion and this may partially explain the

mechanism of the efficacy of cashew leaves and testa.

A model of type 2 diabetes can be induced in rats by either i.v. (tail vein) or i.p.

treatment with STZ in the first days of life. At 8–10 weeks of age and thereafter,

rats neonatally treated with STZ manifest mild basal hyperglycemia, an impaired

response to the glucose tolerance test, and a loss of pancreatic β-cell sensitivity to

glucose (Pascoe and Storlien, 1990). The (n-STZ) rat model exhibits a clear basal

hyperglycemia with glucose intolerance, high HbA1c values, a strong reduction of

pancreatic insulin stores, a decreased (50%) basal plasma insulin level, and a lack

of plasma insulin response to glucose (Portha et al., 2002).

It has been observed that STZ at first abolished the pancreatic β-cell response to

glucose, but a temporary return of responsiveness then appears which is followed

by its permanent loss (Mythili et al., 2004). It is necessary to reemphasize that

natural products display several effects besides lowering blood glucose in these

experimental models. In view of the lack of parallel studies of their toxicity, these

models of diabetes induced by either alloxan or STZ are considered a screening

step in the search for drugs for the treatment of diabetes.

Experimental diabetes in animals has provided considerable insight into the

physiologic and biochemical derangement of the diabetic state. Many of this

derangement were in the form of significant changes in lipid metabolism and

structure (Sochar, 1985). These structural changes are clearly oxidative in nature

and are associated with development of vascular disease (Baynes, 1999). In

diabetic rats, increased lipid peroxidation was also associated with

hyperlipidaemia (Morel, 1989). During diabetes, a profound alteration in the

concentration and composition of lipids occurs. Liver and kidney are important for

glucose and lipid homeostasis, they participates in the uptake, oxidation and

metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids

P a g e | 300


and triglycerides. Thus it is expected to have changes in liver and kidney during

diabetes (Seifter, 1982).

The results obtained indicate that the n-STZ diabetic animal group developed a

moderate type 2 diabetes; however the animals were in better conditions during

the experiments (with lower blood-sugar concentrations); it confirms that the (n-

STZ) model is suitable for investigations on type 2 diabetes.

Thus, the results presented here suggest that that these extracts of cashew testa and

leaves could be developed as a phytomedicine.

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6.10 DEVELOPMENT OF FORMULATION OF ETHANOL EXTRACT OF

CASHEW TESTA

6.10.1 Pre-compression Parameters (Micrometric evaluation)

1. Determination of Water uptake characteristics (moisture sorption study in

desiccators)

Figure 6.68: Physical appearance of the DEP’s containing various

percentages of DCP after 15 days in desiccators

2 % of DCP

4 % of DCP

10 % of DCP 8 % of DCP

6% of DCP

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Based on the results of the moisture content studies, the dry extract preparation

containing 10% DCP was selected for tablet formulation (Table 6.32). Hence,

density, flow property and compressibility of this dry extract blend were further

investigated.

Table 6.32: Water content of the dry extract preparations (DEP)

Quantity of DCP in Dry

extract powder (DEP)

Water content (%) ± SD

2.0 % 5.5 ± 0.17

4.0 % 5.1 ± 0.12

6.0 % 4.7 ± 0.23

8.0 % 4.3 ± 0.20

10.0 % 3.5 ± 0.10

2. Density

Compressibility indices less than 15% are indicative of free-flowing powders;

indices greater than 40% usually correspond to very poor flow (Carr, 1965). As

shown in the Table 6.33, the compression index had decreased form 29.8 to 19.9

indicating the improvement of the flow properties due to the formulation of the

crude extract in to dry extract preparation using DCP.

Table 6.33: Porosity, compression index and Hausner ratio of the dry extract

preparation (DEP) as well as the crude extract

Sample Porosity (%) Compression

(Carr’s)

index (%)

Hausner

ratio

DEP 55.0 19.9 1.25

Ethanol extract of

testa

56.0 29.8 1.42

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The HR is a simple test usually used to evaluate fluidity, where values less than

1.25 indicate good flow properties and values greater than 1.5 indicate poor flow

properties (Fonner et al.,1966). As shown in the table, the dry extract preparation

decreased the HR from 1.42 to 1.25, indicating the improvement of flowability

after mixing with DCP.

A. Powder flow properties

• Angle of repose

The dry extract preparation showed an angle of repose 36.2° which is classified as

passable (fair) flow according to Wells and Aulton (1988) and the flow rate was

found to be 7.9 gm/sec.

B. Formulation of Tablet

Based on the preliminary investigation, the tablet formulation containing dry

extract preparation was formulated with various proportions of different additives

as mentioned below in Table 6.34.

Table 6.34: Optimised formula of tablet formulation

Sr.No Ingredients Quantity for each tablet

of 350 mg

1 Extract 187.0 mg

2 Avicel -102 (directly compressible

Micro crystalline Cellulose- MCC)

85.0 mg

3 Dibasic Calcium Phosphate (DCP) 35.0 mg

4 Croscarmellose 36.0 mg

5 Talc 7.0 mg

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C. Post Compression parameters

The results of post compression parameters and results of various pharmacopoeial

tests of the tablets prepared from the optimized formula are shown in

Table 6.35 – 6.38 and in Figure 6.69 - 6.72.

• Shape and color of tablets

Tablets were brown in color and flat circular shape when observed under a lens by

placing the tablets in light.

• Uniformity of thickness

Three tablets were picked randomly from each batch of formulation and thickness

was measured individually with dial caliper (Mitutoyo, Japan). The thickness of

the tablet ranged from 4.9 ± 0.01 to 4.10 ± 0.05 mm. Uniformity in values

indicates that the tablets were compressed without sticking to dies or punches.

• Hardness testing

The hardness of the tablets was between 3.5 kg/cm2- 4.0 kg/cm

2. The lower

standard deviation value indicated that hardness of the tablets were almost

uniform and possess good mechanical strength and sufficient hardness.

• Friability testing

The friability of compressed tablets was within approved range (<1%) in the

tablets. This indicates that that the tablets possess good mechanical strength.

• Weight Variation

All the tablets passed the weight variation tests as the % weight variation was

within the pharmacopoeial limits of ± 10%. The weight of all the tablets were

found to be almost uniform. This can be attributed due to the good flow property

and good compressibility of the tablets.

• Content uniformity testing

The drug content of the tablets was ascertained spectrophotometrically for five

times. The catechin content of the tablets were between 8.88 ± 0.12 to 8.10 ± 0.13

mg when determined spectrophotometrically at 273 nm. The Limit of detection

(LOD) and Limit of quantitation (LOQ) were found to be 10 and 30 micrograms

P a g e | 305


respectively. The calibration range was established between 30-110 micrograms.

• Disintegration Test

The internal structure of tablet, i.e. pore size distribution, water penetration into

tablets and swelling of disintegration substance are suggested to be the mechanism

of disintegration. The disintegration time increases with increase in DCP and

MCC content. The disintegration time for tablets were found to be less than 2.5

mins.

• Dissolution conditions

The cumulative drug release was calculated based upon the amount of catechin

present in each tablet. The drug releases at 10 mins, 15 mins and 30 mins were

28.9 %, 72.6% and 93.50% respectively. The rapid dissolution might be due to

rapid breakdown of the tablet and faster absorption of the drug.

• Stability testing

In the stability studies of the formulation the tablets were analysed for 6 months

period for drug content uniformity, hardness, in vitro disintegration time and

friability. From the results obtained we could conclude that the tablets were stable

and they retained their original properties.

The results of the post compression parameters are listed in Table 6.35 – 6.38.

Figure 6.69: Tablets prepared from ethanol extract of testa

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Table 6.35: Results of post compression parameters for tablets

Parameters Values

Uniformity of thickness (mm) (n=3) 4.8 ± 0.01 mm

Hardness (n=3) kg/cm2 3.7 kg/cm

2

Friability (%) (n=10) 0.45 ± 0.50

Uniformity of weight (n=20) (mg) 349.79 ± 0.32

Drug content (n=3) (mg) 186.7 ± 0.45

In vitro disintegration time (n=6) 2.3 ± 0.12

Table 6.36: Results of in vitro dissolution profile of tablets

Time period (min) Cumulative % drug release

5 28.90

10 72.60

15 86.50

20 88.20

30 90.89

40 91.99

50 93.42

60 95.43

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Figure 6.70: UV absorption spectra of catechin

Figure 6.71: UV absorption spectra of ethanol extract of testa in tablet

formulation

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Figure 6.72: Calibration curve for catechin by UV spectroscopic analysis

Table 6.37: Results of stability studies (25 ± 20 C / 60

0 % ±5% RH)

Parameters 1 month 2 months 3 months 6 months

Uniformity of

thickness

(mm) (n=3)

4.7 ± 0.01

mm

4.6 ± 0.09

mm

4.8 ± 0.01

mm

4.8 ± 0.03

mm

Hardness

(n=3) kg/cm2

3.5 kg/cm2 3.8 kg/cm

2 3.4 kg/cm

2 3.3 kg/cm

2

Friability (%)

(n=10)

0.43 ± 0.5 0.42 ± 0.2 0.43 ± 0.60 0.42 ± 0.60

Uniformity of

weight (n=20)

(mg)

348.99 ± 0.3 349.79 ± 0.1 347.59 ± 0.5 346.0 ± 0.3

Drug content

(n=3) (mg)

186.9 ± 0.15 187.5 ±0.1 187.0 ± 0.2 186.0 ± 0.2

In vitro

disintegration

time (n=6)

2 mins. and

20 secs. ±

0.13

2 mins. and

10 secs. ±

0.43

2 mins. and

30 secs. ±

0.23

2 mins. and

15 secs. ±

0.33

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Table 6.38: Results of stability studies (40 ± 20 C / 75

0 % ± 5% RH)

Parameters 1 month 2 months 3 months 6 months

Uniformity

of thickness

(mm) (n=3)

4.6 ± 0.3 mm 4.7 ± 0.3

mm

4.8 ± 0.3 mm 4.7±0.3 mm

Hardness

(n=3) kg/cm2

3.55 kg/cm2 3.45 kg/cm

2 3.40 kg/cm

2 3.41 kg/cm

2

Friability (%)

(n=10)

0.41 ± 0.25 0.43±0.65 0.44 ± 0.55 0.45 ± 0.5

Uniformity

of weight

(n=20) (mg)

350.17 ± 0.4 349.97 ± 0.5 349.77 ± 0.50 348.77 ± 0.5

Drug content

(n=3) (mg)

187.2 ± 0.13 187.0 ± 0.1 186.99 ± 0.14 186.50 ± 0.1

In vitro

disintegration

time (n=6)

2min ± 0.11 2 min and

20 secs. ±

0.12

2 min and 10

secs. ± 0.10

2 min and

30 secs. ±

0.30

Thus, an attempt to develop a oral dosage form from the bioactive extract of

cashew testa was satisfactorily successful and it was evaluated for various

parameters and stability studies were carried out. This dosage form can prove to

be as a health supplement with antioxidant and anti-diabetic properties, the

efficacy of which have been evaluated in animal models.

Documents

6.1 AUTHENTICATION OF PLANT MATERIALshodhganga.inflibnet.ac.in/bitstream/10603/12880/12... · Figure 6.7: Effect of extraction time on extractive yields in microwave assisted extraction