INTRODUCTION

The fruit of Piper nigrum L. (Piperaceae), the king of spices, is one of the oldest and most popular spice in the world. It is a perennial, climbing vine Indigenous to Malabar Coast of India and used as an aromatic stimulant in cholera, dyspepsia, flatulence, antiperiodic in malarial fever and arthritic diseases (Nayar et al., 1956; Nadkarni, 1954; Warrier, 1989).

The phytochemistry of the genus Piper is rich, where the studies have revealed volatile oil, terpenes, alkaloids and the same has been adequately documented and reviewed (Sengupta and Ray, 1987; Parmar et al., 1993; Siddiqui et al., 2004). A rapid method for isolation of piperine from fruits of Piper nigrum was performed by Kanaki et al. (2008) and detailed review on disease physiological effect was studied (Srinivasan, 2007). Antispermatogenic and antifertility effect of fruits (Mishra and Singh, 2009) in vitro antioxidant activity of volatile oil (Kapoor et al., 2009) and oleoresin and blood pressure lowering and vasomodulator effects of piperine (Taqvi et al., 2008) were studied. Various bioactives were also isolated from Piper nigrum (Ee et al., 2009; Rai et al., 2009; Siddiqui et al., 2008). Since, black pepper (P. nigrum ), is in great demand and new products viz. bioprine are continually appearing in the market but for want of chemical standardization method, a quality inspection of these products is urgently needed to identify the adulterants and its substitutes. Although, the determination of piperine in P. nigrum by HPLC has been carried out (Rathnawathie and Buckle, 1983) but any quick and reliable method to detect the difference between black pepper and papaya is lacking. There are several challenges as standardization of herbal product is considered like controversial identity of various plants, deliberated adulteration of plant material, problems in storage and transport, which should be considered (Bhutani, 2003; Thatte, 2003). Chemical analysis is so far best method for standardization and to detect contamination and for plant identification and authentication of medicinal plants. Molecular biology techniques can also applied to authentication of medicinal plants as complimentary techniques (Shinde and Dhalwal, 2007). Consistency of biologic activity are essential requirements for the safe and effective use of therapeutic agents. However, botanical preparations rarely meet this standard, as a result of problem in identifying plants and above all, the lack of information about active pharmacologic principles (Donald and Arthur, 2002). Hence, there is an urgent need of quality control and standardization of herbal products by various parameters which can check the quality, efficacy and purity by simple, quick and easy methods that even lay man can identify the difference between pure and adulterated (Shinde et al., 2008; Anonymous, 2008).

Therefore, the aim of this study is to investigate the HPLC profile and anti-oxidative assay of P. nigrum L. (A-authentic; M-market samples) and its adulterant (Carica papaya L.). This study will be able to establish libraries where such sample tracing can be compared with reference as fingerprints in the evaluation of authentication and standardization methods.

MATERIALS AND METHODS

Plant Material
Authenticated samples were procured: Piper nigrum Linn. and Carica papaya Linn. from M/s. Suttind Seeds Pvt. Ltd., Delhi; P. longum Linn., P. chaba Hunter P. belle Linn. and Embelia ribes Burm. from NIA pharmacy, Jaipur, during the course of studies. Market samples: Various market samples of Piper nigrum were procured from M/s. Ajeet Singh Bhag Singh, Jawahar Nagar, Jaipur in the month of April, 2004 and Lantana camara Linn. fruits were collected from the Campus of University of Rajasthan, Jaipur, in the month of March-April, 2004 and market samples of other Piper species were procured from M/s. Chunilal, Johri Bazar, Jaipur, in the month of May, 2004. All the samples were authenticated and voucher specimen have been deposited and all the practical work has been performed in medicinal Plants and Biotechnology Laboratory, Department of Botany, University of Rajasthan, Jaipur, India. The whole experiments were part of thesis work during 2004-2007.

Extraction and Isolation
The fruits of P. nigrum (A and M sample) and C. papaya (the adulterant) were individually extracted with ethanol for 36 h, filtered and concentrated to dryness. Later from each, 10 mg extract of P. nigrum and C. papaya was dissolved in 5 mL MeOH separately and used for HPLC analysis (Rai et al., 2009).

Biological Activity
2, 2- Diphenyl-1- picrylhydrazyl (DPPH; C₁₈H₁₂N₅O₆ ; HiMedia) 0.08 mg mL^-1 in MeOH was used as followed by Tomohiro et al. (1994) for antioxidative assay.s

Qualitative Assay
Various sequential extracts of P. nigrum (A and M) and its adulterant (C. papaya ) were prepared and applied on TLC plates (silica gel G). Each plate was sprayed with DPPH solution, allowed to develop for 30 min and the changes in colour (purple on white) were recorded.

Quantitative Assay
The ethanolic extracts of P. nigrum (A and M) and its adulterant (C. papaya ) were developed in MeOH to obtain a concentration of 1 mg mL^-1. Dilutions were made to obtain concentration of 10² to 10^-3 μg. To 2.5 mL of each solution, DPPH (2.5 mL) was added and allowed ½ h for any reaction to occur. The UV absorbance was recorded at 517 nm.

The experiment was done in triplicate and the average absorption was noted for each concentration. Data were processed using EXCEL and the concentration that caused a 50% reduction in absorbance (RD₅₀) was calculated. The same procedure was followed for the standards (quercetin and ascorbic acid).

Chromatographic Conditions and Detection of HPLC
The HPLC analysis was performed using a Shimadzu Model-VP 135P2 equipped with a UV spectrophotometric detector set at 254 nm, column: Luna 5μ C₁₈(2) 100Å (250x4.6 mm; 5 particle diameter), flow rate: 1 mL min^-1, injection volume 20 μL in methanol (HPLC grade).

RESULTS

The quantitative evaluation of adjoining elution curves was done by calculating the resolution (R = 2Δt/w_a+w_b), where Δt is the difference between peak of interest and preceding peak and w_a and w_b are the width of peaks, respectively using HPLC analysis of MeOH extracts of fruits of P. nigrum (A and M) and its adulterant (C. papaya).

An easier interpretation of the HPLC tracing was achieved when the peak area was divided by the area of a reference peak and the retention time (rt) was plotted against normalized peak area (Fig. 1). The retention times were affected by a mean percentage error of ±0.01 that has taken in P.nigrum in C. papaya, a closer peak in rt to piperine is also present, which otherwise is of piperine (33%) in P. nigrum. Resolution value of P. nigrum genuine (0.09), market sample 1 (0.06), market sample 2 (0.11) and C. papaya (0.06) was calculated. These results with high resolution value of market samples are closure to C. papaya value thus, indicative of adulteration.

Fig. 1:	Comparison of HPLC profile (rt v/s area %) of P. nigrum (A and M) and C. papaya

Fig. 2:	Effect of the concentration of P. nigrum (A and M) and C. papaya extracts on DPPH (0.08 mg mL-1)

Similarly theoretical plates value further make a clear cut demarcation between P. nigrum genuine (0.80), market 1 (0.16), market 2 (0.08) and its adulterant i.e., C. papaya (0.64). The market sample 2 with same theoretical plate value indicates more purity as compared to market sample 1 Resolution (0. 685, 0. 678, 1.33 ) and theoretical plates value ( 0.901, 1.75, 0.663) further make a clear cut demarcation between P. nigrum (A and M) and its adulterant (C. papaya), respectively.

The qualitative antioxidative assay using DPPH spray on TLC plates bearing the spot of various test extracts of P. nigrum (A and M) and its adulterant (C. papaya) indicates the presence of antioxidant compounds in P. nigrum (A)<P. nigrum (M)<C. papaya. Different samples of P. nigrum A ( RD₅₀ 0.15 mg mL^-1), M ( RD₅₀ <1 μg mL^-1) and C. papaya ( RD₅₀< 1 μg mL^-1) demonstrated antioxidant properties and compared with the standards ( quercetin RD₅₀ 0.003 mg mL^-1, ascorbic acid RD₅₀ 0.006 mg mL^-1) (Fig. 2).

From these studies, it is evident that both the HPLC and antioxidative assay can be used as markers in evaluating the authentic and their adulterants. Even though the tracings in HPLC were quite similar in three samples, there were sufficient characteristics that allow these to be distinguished. The normalized tracings that are the ratio between the areas of the peaks also show a characteristic fingerprint for each kind of plant material.

In antioxidative studies of P. nigrum, market sample showed better efficacy than authentic where former was quite close to that of C. papaya, thus, indicative of greater degree of adulteration.

DISCUSSION

Accordingly, many analytical technologies based on IR, HPLC and NMR have been enlisted to profile the herbal drugs (metabolome), but there are difficulties in quality control of herbal due to different kinds, types, various geographical origins, names, cultural usage, dubious adulterated and sub-standard products. Now-a-days identification and authentication of the herbals is necessary as the herbals are loosing their relevance in the world trade. The HPLC is a new emerging technology used for fingerprintings of herbals (Rai et al., 2009) which is effective in giving computerized fingerprints of each herbal characteristic of its chemical appearance, in the form of which the absorbance of a compound at a particular point in UV detector. These chemical profiles are effective markers in the identification of purity of the drug. The HPLC fingerprinting is a step forward in the enhancement of standardization ladder which is easy, reliable, quick and reproducible to perform, even results obtained in few minutes. Therefore, using HPLC chemical markers for various fruit extracts of P. nigrum and its adulterants could be established. An overlay view of these fingerprints showed that the market samples are not exactly resembling to its genuine drug but closer to its adulterants, indicative of adulteration of C. papaya. Such systems may be effectively used in quarantines to check the quality and ensure the safety of the product. Earlier, the use of HPLC as a tool for standardization of herbals was performed by few workers (Philipp and Isengard, 1995) but no such HPLC standardization in Piper species was carried out so far and thus, it is the first report of this nature to generate HPLC chromatograms of genuine v/s adulterants.

ACKNOWLEDGMENT

The authors are thankful to the University Grants Commission, New Delhi, India, for partial financial support.