6.1 authentication of plant materialshodhganga.inflibnet.ac.in/bitstream/10603/12880/12... ·...
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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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CHAPTER 6 RESULTS AND DISCUSSION
• 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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
(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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CHAPTER 6 RESULTS AND DISCUSSION
(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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
n=3 determinations for values mentioned above
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
n=3 determinations for values mentioned above
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
n=3 determinations for values mentioned above
Table 6.7 (A): Determination of extractive values (Microwave assisted
extraction)
Time (sec.)
Microwave Extraction
(Low power 140 Watt)
% Yield ± (SEM)
Microwave Extraction
(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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CHAPTER 6 RESULTS AND DISCUSSION
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
n=3 determinations for values mentioned above
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
n=3 determinations for values mentioned above
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
n=3 determinations for values mentioned above
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CHAPTER 6 RESULTS AND DISCUSSION
� 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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds.
• Aqueous extract of testa: The aqueous extract of testa was found to contain
carbohydrates, proteins, flavonoids, alkaloids, tannins and phenolic compounds.
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
• 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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CHAPTER 6 RESULTS AND DISCUSSION
� 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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CHAPTER 6 RESULTS AND DISCUSSION
� 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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
� 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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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%
4 Ethanol extract 13.09%
5 Aqueous extract 13.65%
Polyphenol fraction of cashew testa
6 Aqueous extract 16.40%
7 Ethanol extract 15.44%
Polyphenol fraction of cashew leaves
8 Aqueous extract 7.0%
9 Ethanol extract 5.50%
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.17: HPTLC chromatogram of ethanol extract of leaves
Figure 6.18: HPTLC chromatogram of aqueous extract of leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.19: HPTLC chromatogram of ethanol extract of testa
Figure 6.20: HPTLC chromatogram of methanol extract of testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.21: HPTLC chromatogram of aqueous extract of testa
Figure 6.22: HPTLC chromatogram of polyphenol fraction of testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.23: HPTLC chromatogram of polyphenol fraction of leaves
Figure 6.24: HPTLC chromatogram of standard catechin
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CHAPTER 6 RESULTS AND DISCUSSION
� 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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CHAPTER 6 RESULTS AND DISCUSSION
� 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
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CHAPTER 6 RESULTS AND DISCUSSION
� 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
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 0.3 – 1.4µg / µl.
Figure 6.27: Calibration curve of catechin by HPLC
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CHAPTER 6 RESULTS AND DISCUSSION
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
4 Ethanol extract 13.20 ± 1.1
5 Aqueous extract 13.95 ± 0.5
Table 6.14: Catechin content in various extracts prepared by MAE
Sr.
No.
Time
(sec.)
Microwave Extraction
(Low power 140 Watt)
% Yield ± (SEM)
Microwave Extraction
(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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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.28: HPLC chromatogram of standard catechin
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.29: HPLC chromatogram of aqueous extract of cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.30: HPLC chromatogram of ethanol extract of cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.31: HPLC chromatogram of aqueous extract of cashew testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.32: HPLC chromatogram of ethanol extract of cashew testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.33: HPLC chromatogram of methanol extract of cashew testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.34: HPLC chromatogram of polyphenol fraction of cashew testa
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.35: HPLC chromatogram of polyphenol fraction of cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.36: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 50 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.37: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 70 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.38: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 90 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.39: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 120 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.40: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 130 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.41: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 150 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.42: HPLC chromatogram of aqueous extract prepared by
microwave extraction (for 180 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.43: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 50 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.44: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 70 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.45: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 90 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.46: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 120 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.47: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 130 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.48: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 150 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.49: HPLC chromatogram of methanol extract prepared by
microwave extraction (for 180 seconds) at low power
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CHAPTER 6 RESULTS AND DISCUSSION
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
Detection wavelength: 254 nm
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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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.50: HPLC Chromatogram of extract of cashew leaves subjected to
oven drying
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.51: HPLC chromatogram of extract of sun dried cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.52: HPLC chromatogram of extract of shade dried cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.53: HPLC chromatogram of extract of fresh cashew leaves
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
3 Aqueous Extract 7.23 ± 0.25
4 Ethanol Extract 6.68 ± 0.92
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
• 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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CHAPTER 6 RESULTS AND DISCUSSION
Table 6.17: Results of nitric oxide scavenging activity
Sr.No. Sample IC50 value (µµµµg/mL) ± SD
Extracts of Cashew Leaves
1 Aqueous Extract 1002.43 ± 1.2
2 Ethanol Extract 658.30 ± 0.98
Extracts of Cashew Testa
3 Aqueous Extract 518.6 ± 0.43
4 Ethanol Extract 331.84 ± 0.65
5 Methanol Extract 343.78 ± 0.47
Polyphenol Fractions of Cashew
6 Polyphenols of leaves 358.70 ± 0.79
7 Polyphenols of testa 330.64 ± 1.3
Extracts Prepared By Microwave Extraction
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
Extracts of Leaves Exposed to Various Drying Conditions
15 Sun dried leaves 356.70 ± 1.30
16 Oven dried leaves 380.90 ± 0.94
17 Shade dried leaves 375.8 ± 0.45
18 Fresh leaves 396.5 ± 0.17
19 Ascorbic acid (Control) 293.54 ± 0.83
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CHAPTER 6 RESULTS AND DISCUSSION
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
Extracts of Cashew Leaves
1 Ethanol extract 99.76 ± 1.1
2 Aqueous extract 104.38 ± 0.47
Extracts of Cashew Testa
3 Ethanol extract 83.3 ± 0.32
4 Methanol extract 113.3 ± 0.32
5 Aqueous extract 81.71 ± 0.64
Control
6 Ascorbic acid 23.60 ± 0.33
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CHAPTER 6 RESULTS AND DISCUSSION
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
Extracts of Cashew Leaves
1 Aqueous Extract 37.41 ± 0.12
2 Ethanol Extract 40.26 ± 0.98
Extracts of Cashew Testa
3 Aqueous Extract 54.36 ± 0.71
4 Ethanol Extract 58.31 ± 0.30
5 Methanol Extract 57.94 ± 0.56
Extracts Prepared By Microwave Extraction
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
Extracts of Leaves Exposed to Various Drying Conditions
13 Sun dried leaves 58.56 ± 0.28
14 Oven dried leaves 40.01 ± 0.49
15 Shade dried leaves 37.41 ± 0.53
16 Fresh leaves 49.041 ± 0.89
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
Figure 6.58: Viability assay of ethanolic extract of cashew testa, 3 hrs after
stimulation with tbH2O2
Figure 6.59: Viability assay of polyphenols of cashew testa, 3 hrs after
stimulation with tbH2O2
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CHAPTER 6 RESULTS AND DISCUSSION
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
stimulation with tbH2O2
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
• 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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CHAPTER 6 RESULTS AND DISCUSSION
• 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
On day 0 Day 7 Day 15
Normal control 85.33 ± 3.18 85.50 ± 2.16 85.50 ± 2.75
Diabetic control 263.33 ± 9.05 268.83 ± 8.74 279.83 ± 8.09
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
Values are expressed as mean ± SEM, n = 6
* 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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CHAPTER 6 RESULTS AND DISCUSSION
• 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
On day 0 Day 15 Day 30
Normal control 78.67 ± 0.84 76.5 ± 1.38 75.83 ± 1.82
Diabetic control 150.33 ± 6.92 149.33 ± 5.86 154.50 ± 5.40
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*
Values are expressed as mean ± SEM, n = 6;
* Significantly different from Control, p<0.05
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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CHAPTER 6 RESULTS AND DISCUSSION
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*
Values are expressed as mean ± SEM, n = 6;
* Significantly different from Control, p<0.05
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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CHAPTER 6 RESULTS AND DISCUSSION
Table 6.28: Effect of extracts on serum triglyceride levels in rats
Treatment Serum triglyceride( mg/dl) ± SEM
On day 0 Day 15 Day 30
Normal control 58.55 ± 3.65 61.21 ± 3.99 60.06 ± 4.13
Diabetic control 97.33 ± 8.12 98.71 ± 7.97 104.89 ± 7.55
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 *
Values are expressed as mean ± SEM, n = 6;
* Significantly different from Control, p<0.05
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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CHAPTER 6 RESULTS AND DISCUSSION
Table 6.29: Effect of extracts on serum total cholesterol levels in rats
Treatment Serum total cholesterol (mg/dl) ± SEM
On day 0 Day 15 Day 30
Normal control 78.89 ± 3.22 77.82 ± 2.78 78.09 ± 3.87
Diabetic control 99.49 ± 2.82 102.42 ± 3.38 104.37 ± 4.95
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 *
Values are expressed as mean ± SEM, n = 6;
* 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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CHAPTER 6 RESULTS AND DISCUSSION
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
Diabetic control 34.01 ± 2.01 49.38 ± 4.94 20.98 ± 1.51
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 *
Values are expressed as mean ± SEM, n = 6
* 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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CHAPTER 6 RESULTS AND DISCUSSION
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*
Values are expressed as mean ± SEM, n = 6
* 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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CHAPTER 6 RESULTS AND DISCUSSION
• 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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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.
(Sections treated with hematoxylin and eosin x 100)
a b
c d
e
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CHAPTER 6 RESULTS AND DISCUSSION
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.
(Sections treated with hematoxylin and eosin x 100)
a b
c d
e
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CHAPTER 6 RESULTS AND DISCUSSION
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
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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
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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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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CHAPTER 6 RESULTS AND DISCUSSION
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.