A. occidentale has been used in the treatment of various diseases including malaria, yellow fever and diarrhea^2,3. Cashew is a tropical tree indigenous to Brazil, and is a member of the family Anacardiaceae. The biological activities of this plant are widely reported and it has been reported to possess antidiabetic, anti-bacterial, anti-fungal, anti-oxidant and anti-inflammatory activities^4-7. The antioxidant activities and phenolic content of this plant have been reported mainly in the nuts, leaves and stem barks^8-12. The kernel of cashew nut is covered with a thin reddish-brown skin or testa that has been reported as a good source of hydrolysable tannins with Catechin and epiCatechin as the major polyphenols^13,14.

Plants contain a large amount of structurally and functionally diverse components. Medicinal plants serve as an important source to invent potential drugs and safe antioxidant compounds. Numerous novel bioactive compounds have been isolated and identified from plants.

However, isolation and purification of pure compounds from plants is usually difficult, tedious and expensive process. Reports on the identification of novel compounds from plants are available in significant numbers; however research publications on the quantitative analysis of novel bioactive compounds are relatively few, due to the lack of standard compounds. Recently, chromatographic fingerprint technique has been accepted by WHO as a strategy for the quality assessment of herbal medicines^15-22. Chromatographic techniques like HPLC and HPTLC have recently gained increasing importance due to their emphases on the characterization of the complete sample composition. The methods developed by use of these techniques can also be applied to determination of standard compounds as markers, bioactive components and enhancement of herbal medicinal product quality²³.

The aim of the present research work was to isolate and characterize the bioactive polyphenol – Catechin, from extracts of Anacardium occidentale Linn. and thereby quantitate the amount of Catechin present in the selected extracts of testa and leaves of Cashew by optimised HPLC method.

2. EXPERIMENTAL:

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).

2.2 Preparation of standard solutions:

Standard stock solutions (1 mg/ml) of reference Catechin were prepared in methanol. Working solutions of Catechin were prepared by appropriate dilutions of the stock solutions with methanol. All solutions were prepared freshly prior to analysis.

2.3 Preparation of plant extracts:

Fully matured shade dried leaves, and dried testa of A.occidentale were collected and ground to coarse powder form. The samples were extracted by using Soxhlet extractor, with ethanol and methanol, for 18 h with a mass to volume ratio of 1:6 (g/mL). The methanol and ethanol extracts were evaporated to dryness on the rotary evaporator. Aqueous extract was prepared by refluxing for 18h and mass to volume ratio of 1:6 (g/mL). The aqueous extract was freeze dried and used for analysis.

2.4 Apparatus and operating conditions:

The HPLC analysis was done on a TOSOH-CCPM system using UV-Visible detector. Melting point was determined using Remik melting point apparatus, India. The UV spectrum was recorded using JASCO UV Visible spectrophotometer. The IR spectrum was recorded using JASCO FTIR 5300 using the KBr pellet method. The NMR spectrum was recorded using Bruker 400 Ultra shield TM spectrophotometer in Dimethyl Sulphoxide (DMSO). The MASS spectrum was recorded using micro TOF-Q instrument carried out in the negative mode (M+ -1).

2.5 Isolation and purification of Catechin:

Isolation of Catechin was carried out by Preparative TLC technique, from methanol extract of cashew leaves. The methanol extract was defatted with n-hexane (AR grade). Hexane layer was discarded and methanol layer was concentrated and applied on Silica gel GF₂₅₄ precoated plates for preparative TLC. The mobile phase used for chromatography was Toluene: Ethyl acetate: Methanol: Formic acid (6:6:1:0.1v/v/v/v) with saturation time of 20 min. The Catechin spot was identified by co-chromatography with reference Catechin, extracted and purified by recrystallisation. The recrystallisation process was carried out with hot water.

2.6 Identification of Catechin:

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 data^24-25.

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 column at ambient temperature with a sample injection volume of 10 µL. An isocratic elution was carried out with methanol: water (70:30 v/v). Flow rate was 1ml/min flow rate. The fingerprint chromatograms were recorded at an optimized wavelength of 254 nm.

2.7.2 Quantitation of Catechin in various extracts by HPLC:

Standard stock solutions were prepared by dissolving the reference standard in methanol to obtain a concentration of 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 spectral analysis. NMR, IR and MS spectra obtained are presented below. The structure of Catechin is represented as in (Fig.1).

Catechin:

m.p.:179⁰C;

UV spectra: UV spectra (λmax [methanol]) showed maxima at 278 nm (Fig. 2).

IR spectra: IR spectra [υmax (KBr)] showed band at 2600-3400 (broad), 1620, 1520, 1470, 1380, 1280, 1240, 1150, 1120, 1080, 1020, 820 cm-1.

Mass spectra: The mass spectra showed maximum at 290 and minimum at 55. The other fragments were seen at 139, 138, 110, 152, 151 and 123. The molecular mass corresponding to 290 was observed.

1H-NMR (400 MHz, acetone-d6): 1H-NMR spectra showed peaks at δTMS 4.56 [H-2, d, J(H- 2, H-3a) 7.8 Hz], 4.00 [H-3, ddd, J(H-3a, H-4e) 5.58 Hz, J(H-3a, H-4a) 8.50 Hz, J(H-3a, H-2a) 7.80 Hz], 2.54 [H-4a, dd, J(H-4a, H-3a) 8.50 Hz, 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 Hz], 6.01[H-8, d, J(H-8, H-6) 2.3 Hz], 6.89 [H-2′, d, J(H-2′, H-6′) 1.95 Hz], 6.79 [H-5′, d, J(H-5′, H-6′) 8.07 Hz], 6.73 [H-6′, dd, J(H-6′, H-2′) 1.94 Hz, J(H-6′, H-5′) 8.19 Hz] and 8.00 (phenolic protons, m).

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.

The purity of isolated Catechin was confirmed by carrying out HPTLC and HPLC analysis of Catechin in different mobile phases to obtain a single, well isolated peak. The peak areas of marker and isolated Catechin were compared and the purity of Catechin was found to be 99.30% ± 0.05. The HPLC and HPTLC chromatograms of isolated Catechin are shown in Fig. 3 and 4.

3.2 Calibration parameters:

Different concentrations of the reference compound (Catechin) were analysed by HPLC method under the optimized conditions. The analysis was performed in triplicates and mean peak area responses to the 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.

3.3 LOD and LOQ:

The limits of detection and quantification were determined as signal to noise ratio using the equations LOD= 3.3σ /S and LOQ=10 σ /S where, σ is standard deviation of response and S is the slope of calibration curve. The LOD and LOQ were 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.

The application of HPLC method for the quantification of Catechin in cashew leaves and testa was established. HPLC fingerprinting method bears the advantages of specificity, powerful separation ability and ability to derive detailed chemical information. These methods may be recommended for quality assurance and establishment of the authenticity of cashew leaf and testa samples, extracts of the herb and its formulations using Catechin as marker. These methods provide more chemical information for analysis of pharmacologically active marker – Catechin. The methods can be applied for analysis of plant extracts, herbal formulations and Pharmaceuticals.

5. ACKNOWLEDGEMENTS:

The authors thank ICMR, New Delhi, India for funding the research project.

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Received on 29.05.2010

Accepted on 07.07.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(5): Sept.-Oct. 2010, 372-376