Research J. Pharmacognosy and Phytochemistry 2010; 2(5): 372-376
Isolation and Quantitative Analysis of a
ISSN 0975- 2331
Research Journal of Pharmacognosy
and Phytochemistry. 2(5): Sept.-Oct.
2010, 372-376
Research Article
*Corresponding Author:
Dr. Pratima Tatke,
Associate Professor in Pharm. Chem.,
C. U. Shah College of Pharmacy,
S.N.D.T. Women's University, Juhu
Campus, Juhu Tara Road, Santacruz
West,
Mumbai-400 049, M.S.
E. Mail.- patatke@yahoo.com
Received on 29.05.2010
Accepted on 07.07.2010
© A&V Publication all right reserved
Pratima A. Tatke et.al.
Bioactive Polyphenol - Catechin in Anacardium
occidentale Linn. (Leaves and Testa) by HPLC
Analysis
Yogini S. Jaiswal1, Pratima A. Tatke1*, Satish Y.Gabhe1 and
Ashok B. Vaidya2
1
C.U.Shah College of Pharmacy, S.N.D.T Women’s University, Mumbai-
400049, India.
2
ICMR Advanced Centre of Reverse Pharmacology in Traditional Medicine,
Kasturba, Health Society, Vile Parle-(W), Mumbai- 400 056, India.
ABSTRACT:
The present paper reports a method developed for isolation, characterization,
structural elucidation and quantitative analysis of bioactive polyphenol –
Catechin, from extracts of leaves and testa of Anacardium occidentale Linn
(Cashew). Isolation of Catechin was carried out by Preparative TLC. The
isolated Catechin was characterized by P-NMR, MS, IR spectroscopy and
various chemical and chromatographic analysis. The method was established
by using HPLC analysis and quantitation of isolated Catechin with marker
Catechin. The purity of isolated Catechin was found to be 99.30% based
upon the comparison of peak purity and peak areas of isolated Catechin and
marker Catechin.
The HPLC separation system consisted of a C18 reversed-phase column, an
isocratic elution system of methanol-water (9:1 v/v), and a UV-Visible
detector with 254nm as the detection wavelength. The limit of detection
(LOD) and limit of quantitation (LOQ) were found to be 0.2 µg and 0.6 µg
respectively with an Rt value of 2.5 min.
The maximum amount of Catechin was found in aqueous extract of leaves
(5.70%) and testa (13.65%).
Thus the developed HPLC method is rapid and simple technique for
separation and determination of Catechin from extracts of A.occidentale
leaves and testa. The method can be suitably applied for determination of
Catechin from various plant extracts.
KEYWORDS: Catechin; Anacardium occidentale Linn.; Polyphenol;
HPLC
1. INTRODUCTION:
The polyphenols like Catechin comprise of a range of substances that play a
role in protecting biological systems against the deleterious effects of
oxidative processes on macromolecules, such as proteins, lipids,
carbohydrates and DNA1. Malaysian population consumes traditional
vegetables and herbs, raw or cooked as accompaniments with their main
meal.
Many of these vegetables are claimed to possess medicinal properties
although there are no scientific evidences to support the claims. One of the
commonly consumed vegetables is the leaves of A. occidentale (commonly
known as Cashew).
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Research J. Pharmacognosy and Phytochemistry 2010; 2(5): 372-376 Pratima A. Tatke et.al.

A. occidentale has been used in the treatment of various 2.2 Preparation of standard solutions:
diseases including malaria, yellow fever and diarrhea2,3. Standard stock solutions (1 mg/ml) of reference Catechin
Cashew is a tropical tree indigenous to Brazil, and is a were prepared in methanol. Working solutions of Catechin
member of the family Anacardiaceae. The biological were prepared by appropriate dilutions of the stock
activities of this plant are widely reported and it has been solutions with methanol. All solutions were prepared
reported to possess antidiabetic, anti-bacterial, anti-fungal, freshly prior to analysis.
anti-oxidant and anti-inflammatory activities4-7. The
2.3 Preparation of plant extracts:
antioxidant activities and phenolic content of this plant have
been reported mainly in the nuts, leaves and stem barks8-12. Fully matured shade dried leaves, and dried testa of
The kernel of cashew nut is covered with a thin reddish- A.occidentale were collected and ground to coarse powder
brown skin or testa that has been reported as a good source form. The samples were extracted by using Soxhlet
of hydrolysable tannins with Catechin and epiCatechin as extractor, with ethanol and methanol, for 18 h with a mass
the major polyphenols13,14. to volume ratio of 1:6 (g/mL). The methanol and ethanol
extracts were evaporated to dryness on the rotary
Plants contain a large amount of structurally and evaporator. Aqueous extract was prepared by refluxing for
functionally diverse components. Medicinal plants serve as 18h and mass to volume ratio of 1:6 (g/mL). The aqueous
an important source to invent potential drugs and safe extract was freeze dried and used for analysis.
antioxidant compounds. Numerous novel bioactive
compounds have been isolated and identified from plants. 2.4 Apparatus and operating conditions:
The HPLC analysis was done on a TOSOH-CCPM system
However, isolation and purification of pure compounds using UV-Visible detector. Melting point was determined
from plants is usually difficult, tedious and expensive using Remik melting point apparatus, India. The UV
process. Reports on the identification of novel compounds spectrum was recorded using JASCO UV Visible
from plants are available in significant numbers; however spectrophotometer. The IR spectrum was recorded using
research publications on the quantitative analysis of novel JASCO FTIR 5300 using the KBr pellet method. The NMR
bioactive compounds are relatively few, due to the lack of spectrum was recorded using Bruker 400 Ultra shield TM
standard compounds. Recently, chromatographic fingerprint spectrophotometer in Dimethyl Sulphoxide (DMSO). The
technique has been accepted by WHO as a strategy for the MASS spectrum was recorded using micro TOF-Q
quality assessment of herbal medicines15-22. instrument carried out in the negative mode (M+ -1).
Chromatographic techniques like HPLC and HPTLC have
recently gained increasing importance due to their 2.5 Isolation and purification of Catechin:
emphases on the characterization of the complete sample Isolation of Catechin was carried out by Preparative TLC
composition. The methods developed by use of these technique, from methanol extract of cashew leaves. The
techniques can also be applied to determination of standard methanol extract was defatted with n-hexane (AR grade).
compounds as markers, bioactive components and Hexane layer was discarded and methanol layer was
enhancement of herbal medicinal product quality23. concentrated and applied on Silica gel GF254 precoated
plates for preparative TLC. The mobile phase used for
The aim of the present research work was to isolate and chromatography was Toluene: Ethyl acetate: Methanol:
characterize the bioactive polyphenol – Catechin, from Formic acid (6:6:1:0.1v/v/v/v) with saturation time of 20
extracts of Anacardium occidentale Linn. and thereby min. The Catechin spot was identified by co-
quantitate the amount of Catechin present in the selected chromatography with reference Catechin, extracted and
extracts of testa and leaves of Cashew by optimised HPLC purified by recrystallisation. The recrystallisation process
method. was carried out with hot water.

2. EXPERIMENTAL: 2.6 Identification of Catechin:

2.1 Materials:
Cashew Leaves were collected from Tungareshwar forests
of Vasai Taluka, Dist. Thane in the state of Maharashtra,
India. Testa samples were collected from Sawantwadi
region of Goa, India. The plant specimens were
authenticated and a herbarium of the plant specimen
(voucher number no. YOGA1/No.BSI/WC/Tech/2008/69)
was submitted at the Botany Department of Botanical
Survey of India, Pune; (M.S), India. Reference Catechin
was purchased from Sigma–Aldrich (Germany). HPLC
grade and analytical grade solvents were obtained from
Merck (Mumbai, India).
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The identification of Catechin was carried out by chemical
and spectral studies and its structure was elucidated by P-
NMR, MS and IR spectral analyses. Chromatographic
analysis as well as some physical properties were also
determined and compared with literature data24-25.
2.7Chromatographic conditions:
2.7.1 HPLC fingerprinting:
The HPLC analysis was done on a TOSOH-CCPM system
using a quaternary CCPE Tosoh pump. A UV-Visible,
absorbance detector of the model LINEAR UVIS-205 was
used to perform HPLC analysis. The HPLC fingerprinting
was carried out on a C18 column (Phenomenex C18,
4.6mm×250mm, 5µm) equipped with an extended guard
Research J. Pharmacognosy and Phytochemistry 2010; 2(5): 372-376 Pratima A. Tatke et.al.

column at ambient temperature with a sample injection 1H-NMR (400 MHz, acetone-d6): 1H-NMR spectra showed
volume of 10 µL. An isocratic elution was carried out with peaks at TMS 4.56 [H-2, d, J(H- 2, H-3a) 7.8 Hz], 4.00 [H-
methanol: water (70:30 v/v). Flow rate was 1ml/min flow 3, ddd, J(H-3a, H-4e) 5.58 Hz, J(H-3a, H-4a) 8.50 Hz, J(H-
rate. The fingerprint chromatograms were recorded at an 3a, H-2a) 7.80 Hz], 2.54 [H-4a, dd, J(H-4a, H-3a) 8.50 Hz,
optimized wavelength of 254 nm. J(H-4a, H-4e) 16.10 Hz], 2.90 [H-4e,dd, J(H-4e, H-3a) 5.50
Hz, J(H-4e, H-4a) 16.10 Hz], 5.87 [H-6, d, J(H-6, H-8) 2.3
2.7.2 Quantitation of Catechin in various extracts by Hz], 6.01[H-8, d, J(H-8, H-6) 2.3 Hz], 6.89 [H-2 , d, J(H-2 ,
HPLC: H-6 ) 1.95 Hz], 6.79 [H-5 , d, J(H-5 , H-6 ) 8.07 Hz], 6.73
Standard stock solutions were prepared by dissolving the [H-6 , dd, J(H-6 , H-2 ) 1.94 Hz, J(H-6 , H-5 ) 8.19 Hz] and
reference standard in methanol to obtain a concentration of 8.00 (phenolic protons, m).
1mg/mL for Catechin. The concentrations of Catechin
reference standards used for calibration were 0.6, 0.7, 0.8,
0.9, 1.0 µg/µL in methanol, respectively. The peaks in
HPLC fingerprints were identified by comparing the
retention times in the chromatograms of extracts with those
of reference standard Catechin peak.

2.7.3 Statistical analysis:

The statistical analysis was performed using GRAPH PAD
Instat software (version 3.01).

3. RESULTS AND DISCUSSION:

3.1 Structure elucidation and spectral analysis of isolated
Catechin:
Isolation of Catechin was obtained by simple preparative
thin layer chromatography from methanol extract of cashew
leaves. Identity of isolated Catechin was confirmed by
chemical and spectral studies and comparison of reference
Catechin with marker Catechin. In order to establish the
selectivity of the method the bands corresponding to
standard Catechin were separated, purified by repeated
recrystallisation and subjected to P-NMR, IR and MS Figure 2. UV–Vis spectra of Catechin marker and Catechin in
spectral analysis. NMR, IR and MS spectra obtained are various extracts.
presented below. The structure of Catechin is represented as
in (Fig.1).
P-NMR, IR and MS signals obtained for the bands of
Catechin were compared with those of reference
compounds. All the signals obtained in the NMR spectra
were found to belong to Catechin which indicated the
identity and purity of isolated Catechin.

Molecular ion peak, obtained from MS spectra of these

bands further confirmed the purity and identity of the
isolated Catechin.
Figure 1. Structure of Catechin.
The purity of isolated Catechin was confirmed by carrying
out HPTLC and HPLC analysis of Catechin in different
Catechin: mobile phases to obtain a single, well isolated peak. The
m.p.:1790C; peak areas of marker and isolated Catechin were compared
UV spectra: UV spectra ( max [methanol]) showed and the purity of Catechin was found to be 99.30% ± 0.05.
maxima at 278 nm (Fig. 2). The HPLC and HPTLC chromatograms of isolated
IR spectra: IR spectra [ max (KBr)] showed band at 2600- Catechin are shown in Fig. 3 and 4.
3400 (broad), 1620, 1520, 1470, 1380, 1280, 1240, 1150,
1120, 1080, 1020, 820 cm-1. 3.2 Calibration parameters:
Mass spectra: The mass spectra showed maximum at 290 Different concentrations of the reference compound
and minimum at 55. The other fragments were seen at 139, (Catechin) were analysed by HPLC method under the
138, 110, 152, 151 and 123. The molecular mass optimized conditions. The analysis was performed in
corresponding to 290 was observed. triplicates and mean peak area responses to the
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Research J. Pharmacognosy and Phytochemistry 2010; 2(5): 372-376
concentrations were recorded at 254 nm to establish linear
regression correlation. The regression equation for Catechin
was y = 219763x + 459879 when |x| is between 0.6 and 1.0
µg/ µL. For the given range of concentration of Catechin
the correlation coefficient was 0.99 showing good
correlation with calibration equations.
Figure 3. HPTLC chromatogram of isolated Catechin
Figure 4. HPLC chromatogram of isolated Catechin
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Pratima A. Tatke et.al.
3.3 LOD and LOQ:
The limits of detection and quantification were determined
as signal to noise ratio using the equations LOD= 3.3 /S
and LOQ=10 /S where, is standard deviation of
response and S is the slope of calibration curve. The LOD
and LOQ were respectively 0.2 and 0.6 µg for Catechin.
3.4 Optimization of HPLC conditions for fingerprinting:
The choice of detection wavelength is crucial for
developing a reliable fingerprint and for accurate
quantitative analysis of marker compounds in the herb. The
optimal detection wavelength in the HPLC analysis was
determined to be 254 nm. At this wavelength, more
characteristic peaks in the chromatogram were observed,
with Rt 2.5 min for Catechin, which was sensitively
detected in the HPLC quantitation. The HPLC separation
conditions, such as choice of mobile phase and isocratic
program, were further optimized. A number of mobile
phases with different gradients were screened in order to
obtain a reliable chromatogram with most peaks at
acceptable resolution and balance for the HPLC
fingerprinting and to obtain baseline separation of Catechin
in a relatively short analytical time for the HPLC
quantitation. Finally, an isocratic elution was carried out
with methanol:water (70:30 v/v) as the mobile phase, and
1ml/min flow rate for the HPLC fingerprinting and
quantitative analyses of the herb.
3.5 Quantitative analysis of extracts of testa and leaves of
Cashew by HPLC:
Extracts of testa and leaves were quantitatively determined
using the developed reverse phase HPLC method. Each
sample was analysed in triplicate to determine the mean
content of Catechin. It was observed that Catechin was
more abundantly present in cashew testa extracts as
compared to leaves extracts. Significant amount was
present in aqueous extracts as compared to other extracts.
The results of quantitative analysis of extracts for Catechin
content by HPLC are indicated in Table 1.
Table 1: Determination of catechin content in extracts by HPLC
method
Extract Catechin content (mg/100mg)
Cashew leaf extracts
Ethanol extract 4.95 ± 1.0
Aqueous extract 5.83 ± 0.9
Cashew testa extracts
Methanol extract 12.95 ± 0.7
Ethanol extract 13.20 ± 1.1
Aqueous extract 13.95 ± 0.5
4. CONCLUSIONS:
A method for isolation of a bioactive polyphenol-Catechin
by preparative TLC was developed. The method proves to
be simple, efficient and cost effective as compared to other
alternative advanced chromatographic techniques.
Research J. Pharmacognosy and Phytochemistry 2010; 2(5): 372-376 Pratima A. Tatke et.al.

The application of HPLC method for the quantification of chromatography on fingerprinting of Chinese traditional medicine
Catechin in cashew leaves and testa was established. HPLC J. Chromatogr. A 2004, 1022;139-144.
18. Zhao, L. et.al.Fingerprint analysis of Psoralea corylifolia L. by
fingerprinting method bears the advantages of specificity, HPLC and LC–MS. J.Chromatogr. B. 2005, 821;67-74.
powerful separation ability and ability to derive detailed 19. Ji, Y.B., et.al. Development, optimization and validation of a
chemical information. These methods may be fingerprint of Ginkgo biloba extracts by high-performance liquid
recommended for quality assurance and establishment of chromatography.J. Chromatogr. A. 2005, 1066; 97-104.
the authenticity of cashew leaf and testa samples, extracts of 20. Nederkassel, van A.M. et.al. Development of a Ginkgo biloba
fingerprint chromatogram with UV and evaporative light
the herb and its formulations using Catechin as marker.
scattering detection and optimization of the evaporative light
These methods provide more chemical information for scattering detector operating conditions. J. Chromatogr. A 2005,
analysis of pharmacologically active marker – Catechin. 1085; 230-239.
The methods can be applied for analysis of plant extracts, 21. Nederkassel A.M., et.al. Prediction of total green tea antioxidant
herbal formulations and Pharmaceuticals. capacity from chromatograms by multivariate modeling. J.
Chromatogr. A. 2005,1096;177-186.
22. Ji, Y.B. et.al. Sequential uniform designs for fingerprints
5. ACKNOWLEDGEMENTS: development of Ginkgo biloba extracts by capillary
The authors thank ICMR, New Delhi, India for funding the electrophoresis. J. Chromatogr. A. 2006, 1128; 273-281.
research project. 23. Capasso, R. et.al. Phytotherapy and quality of herbal medicines.
Fitoterapia. 2000,71; S58-S65.
6. REFERENCES: 24. Ulubelen, A. and Topc¸ G. Studies in Natural Product Chemistry,
1. Halliwell H. Free radicals, antioxidants and human disease: Structure and Chemistry, Elsevier Science, 1998.
curiosity, cause or consequence?. Lancet 1994; 344: 721. 25. Mogib, M.A. Albar, H.A. and Batterjee, S.M. Chemistry of the
2. Akinpelu D. Antimicrobial activity of Anacardium occidentale genus Plectranthus. Molecules. 2002, 7; 271–301.
bark. Fitoterapia. 2001; 72(3): 286–287.
3. Goncalves J, Lopes S, Oliveira D, Costa S, Miranda M, Romanos
M. In vitro anti-rotavirus activity of some medicinal plants used
in Brazil against diarrhea. J Ethnopharmacol. 2005; 99(3): 403–
407.
4. Mota M, Thomas G, and Barbosa Filho J. Anti-inflammatory
actions of tannins isolated from the bark of Anacardium
occidentale L. J Ethnopharmacol. 1985; 13(3): 289–300.
5. Schmourlo G, Mendonc_a-Filho and Alviano R. Screening of
anti-fungal agents using ethanol precipitation and bioautography
of medicinal and food plants. J Ethnopharmacol. 2005; 96(3):
563–568.
6. Kamtchouing P, et.al. Protective role of Anacardium occidentale
extract against streptozotocin induced diabetes in rats. J.
Ethnopharmacol. 1998; 62(2): 95–99.
7. Runnie I, et.al. Inhibition of low density lipoprotein oxidation
and upregulation of the low density lipoprotein receptor of
human liver HEPG2 cells by tropical plant extracts, Clin Exp
Pharmacol and Physiol. 2003; 30(A8): 5–6.
8. Kornsteiner M, Wagner K, and Elmadfa I. Tocopherols and total
phenolics in 10 different nut types. Food Chemistry. 2006;
98(2):381–387.
9. Trevisan M, et.al.Characterization of alkyl phenols in cashew
(Anacardium occidentale) products and assay of their antioxidant
capacity. Food and Chemical Toxicology. 2006; 44(2): 188–197.
10. Kubo I, Masuoka N and Tsujimoto K. Antioxidant activity of
anacardic acids. Food Chemistry.2006; 99(3): 555–562.
11. Abas F. et.al. Antioxidant and nitric oxide inhibition activities of
selected Malay traditional vegetables. Food Chemistry. 2006, 95;
566–573.
12. Runnie I. et.al. Inhibition of low density lipoprotein oxidation
and upregulation of the low density lipoprotein receptor of
human liver HEPG2 cells by tropical plant extracts, Clin Exp
Pharmacol and Physiol 2003, 30; (A8): 5–6.
13. Mathew, A. and. Parpia, H Polyphenols of cashew skin. J. Food
Science. 1970, 35: 140–143.
14. Pillai M. Kedlaya K. and Selvarangan, R. Cashew seed skin as a
tanning material, Leather Science. 1963, 10; 317.
15. Mahady, G.B. Fong, H.H.S. and Farnsworth, N.R. Botanical
Dietary Supplements: Quality, Safety and Efficacy, Swets and
Zeitlinger Publishers, Lisse, The Netherlands, 2001.
16. Liang, Y.Z. Xie, P. and Chan, K. Quality control of herbal
medicines J. Chromatogr. B. 2004, 812;53-70.
17. Gu, M. Ouyang, F. and Su, Z. Comparison of high-speed counter-
current chromatography and high-performance liquid

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