Scientific Validation of Piper nigrum by HPLC and Anti-oxidative Assay Markers
The aim of the present investigations is to investigate the HPLC profile and antioxidative assay of P. nigrum L. (A-authentic; M-market samples) and its adulterant (Carica papaya L.). This study will be able to establish libraries as HPLC chromatograms, where such sample tracing can be compared with reference as fingerprints in the evaluation of authentication and standardization methods. The Genus Piper is in great demand as it is known for medicinal properties and newer compound formulations are continually appearing in the market, thus, strict quality inspection of these formulation is necessary. 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. 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. The qualitative anti-oxidative 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) and 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). During present study, HPLC analysis and anti-oxidative assay have proved to be effective markers in the quality inspection of the authenticated and its adulterants. The present studies revealed that Piper nigrum as pepper powder can be checked for its adulteration with papaya seeds by these two methods which are helpful in generation of quick and easy methods for quality control to check the purity at quarantine levels.
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
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).
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
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.
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).
The quantitative evaluation of adjoining elution curves was done by calculating the resolution (R = 2Δt/wa+wb), where Δt is the difference between peak of interest and preceding peak and wa and wb 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.
||Comparison of HPLC profile (rt v/s area %) of P. nigrum
(A and M) and C. papaya
||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.
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
The authors are thankful to the University Grants Commission, New Delhi, India, for partial financial support.
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