The knowledge and use of traditional medicinal plants with potential curative
properties have now been investigated for the development of common drugs and
herbal remedies (Rahalison et al., 1991). Now
a day, it is emerging as an important research area because of their potent
pharmacological activities, low toxicity and economic viability (Auddy
et al., 2003). Prevention of fungal diseases by synthetic antifungal
chemicals causes high mortality despite of treatment due to their unavoidable
harmful side effects (Lorthoraly et al., 1999;
Vincent, 1999). Identification of strong bioactive allelochemicals
are a useful source for the development of biological herbicides and fungicides
and their implication in control of diseases would be environmentally friendly
as natural chemicals are renewable and easily degradable.
Piper chaba, Hunter (Piperaceae) is a relatively less well-known spice.
This was not even mentioned by Pruthi (1998). Some spices
possess phenolic compound and antioxidant properties (Muchuweti
et al., 2007). This plant bears both medicinal and spicy properties.
The edible part of the plant is the swollen stem whose taste is better than
that of Piper nigrum, according to some consumers. The stem of the plant
is very much effective against cold and cough and also enhances immunity against
this disease. We are therefore interested to isolate and identify the compound
responsible for its medicinal properties.
There is currently immense interest in natural anti-oxidant and their role
in health and diseases, considerable data has been generated around the globe
(Aruoma, 1994), however traditionally used medicinal
plants awaits such screening and Piper chaba is one such plant. Vaghasiya
and others (Vaghasiya et al., 2007) studied Anti-Bacterial
and Anti-Inflammatory properties of some Piper species on 15 clinically important
bacterial strains. In recent time, major research emphasis has been on the plants
with anti-oxidant and antimicrobial properties (Arora et
al., 2011; Uddin et al., 2008; Aisha
et al., 2011; Gupta and Sharma, 2010; Duru
and Onyedineke, 2010; Jimenez et al., 2009).
The literature studies have shown very scanty information on this plant and
thus we are prompted to work on anti-oxidant and antimicrobial properties of
MATERIAS AND METHODS
Isolation and characterization of Active hot pungent compound of Piper chaba:
Samples were collected from Habra, Bongoan and Dumdum (North 24-parganas, West
Bengal) in the month of January, 2010 and present study continued for 2-years.
Two hundred grams of chai (Piper chaba), stem dust was dried and powdered
using Sample Miller Machine (Cyclotec 1093. Sample Mill, TECATOR) and then soaked
in 500 mL of methanol for 7 to 10 days. The entire mixture was then vortexed
in high speed (3000 rpm) using Mechanical Stirrer (Model No. DC Stirrer NZ-1000s
AC220V, EYELA) for 1 h and then filtered through sintered disc funnel. The brown
colored extract was collected and concentrated in a rotary vacuum evaporator
(EYELA, Model No. N1-NW) and subsequently extracted with hexane, ethyl acetate,
acetone and methanol, respectively. Finally the compounds were purified by column
chromatography and thin layer chromatography. Here we put emphasis on Acetone
fraction of Piper chaba (henceforth referred to as PCAF or Piper chaba
acetone fraction or Fraction-3) because highly potent bioactive compound was
obtained in this fraction (Fig. 1).
Thin layer chromatography (TLC): TLC plates (20x20 cm) were used for
this work. Silica gel G of TLC grade was used as a coating material and the
plates were coated uniformly with 0.5 mm thick layer of silica gel.
||Schematic representation of extraction of compound from the
concentrated mathanolic extract of Piper chaba Hunter
A solvent mixture in the ratio of 20: 80:: Ethyl Acetate: Hexane was used
as solvent system (Stahl, 1969). Plates were loaded with
20 μL solution (500 ppm of PCAF) and developed up to a height of 18 cm
within the glass chamber, pre-saturated with desired solvent system. TLC plates
were then taken out and dried under a stream of hot air. Finally compounds were
detected by exposing the plates under iodine vapor or under UV light (365 nm).
Spectral analysis: Mass Spectrometric analysis was done at Indian Institute
of Chemical Biology (IICB), Jadavpur, Kolkata, 700032. Fraction-3 of Piper
chaba, Hunter was subjected to MS analysis for detecting the exact molecular
weight of the compound. Mass Spectra (ESI) was recorded on a Micro mass Q-TOF
Micro TM Spectrometer using positive ion mode.
1HNMR (300 MHz, CDCl3) available at Indian Institute
of Chemical Biology, Jadavpur, Kolkata 700032, was used for analysis of extra-purified
PCAF of Piper chaba. For IH, Pulse Programme is Zg and number
of scan is 64. 1HNMR spectra were observed on δ ppm (0-10) scale
with end sweep at 0 ppm. Samples were analyzed at ambient temperature. CDCl3
was used as solvent for PCAF compound.
CNMR (150 MHz, CDCl3) available at Indian Institute of Chemical
Biology, Jadavpur, Kolkata 700032, was used for analysis of extra-purified PCAF
of Piper chaba. For 13CNMR, Pulse Programme is Zgdc and number
of scan is IK (1024). 13CNMR spectra was observed on δ ppm (0-10)
scale with end sweep at 0 ppm. Samples were analyzed at ambient temperature.
CDCl3 was used as solvent for Fraction-3.
FTIR analysis was conducted to confirm the important functional group in the
extracted and purified PCAF of Piper chaba with the help of FTIR Spectrometer
(Model No. QC/FTIR/006 available at Chembiotek, Kolkata 700054).
Antioxidant activity of PCAF of Piper chaba: The antioxidant
activity (free radicle scavenging activity) of the extracts [as shown in Flow-diagram
i.e., Hexane fraction (PCHF), ethyl acetate fraction (PCEtoAcF), Acetone Fraction
(PCAF) and methanol fraction (PCMF)] (Ravishankara et
al., 2002) on the stable radicle 1,1-diphenyl-2-picrylhydrazyl (DPPH)
was determined by the method of Brand-Williams (Brand-Williams
et al., 1995). In the experiment, 2.0 mg of pure PCAF compound was
dissolved in methanol. Solution of varying concentrations such as 500, 250,
125, 62.50, 31.25, 15.62, 7.8125, 3.91, 1.95 and 0.98 μg mL-1
were obtained from serial dilution technique. Two milliliter of the methanol
solution of PCAF of each concentration was mixed with 3 mL of a DPPH-methanol
solution (20 μg mL-1) and was allowed to stand for 20 min for
the reaction to occur. Then the absorbance was determined at 517 nm and from
these values the corresponding percentage of inhibition were calculated by using
the following formula:
Then percentage inhibitions were plotted against respective concentrations
used and from the graph IC50 was calculated by using tert-butyl-1-hydroxytoluene
(BHT), ascorbic acid, potential antioxidant, were used as positive control.
Effects of PCAF of Piper chaba at different concentrations on fungi:
Inhibition zone test technique was performed for testing the impact of extracted
PCAF against four different fungal species viz., Aspergillus niger, Aspergillus
fumigatus, Aspergillus tamarii and Penicillium chrysogenum. Few fungal spores
of test fungi were transferred to PDA (Potato Dextrose Agar Media) slants and
incubated for one week for colony growth. After one week, one loop full of fungal
spore of each species was added separately to the sterile saline water and mixed
well. Fungal spore suspension (1 mL) in water was then poured in a sterile Petri
dish containing molten PDA and allowed to solidify the plates. Four cups (25
mm2 size) were cut at equidistant positions and in these cups 0.5
mL solution of PCAF at 500, 1000, 1500 and 2000 ppm was added. Treated plates
were incubated at 28±1°C for 24-48 h. After 48 h plates were taken
out and observations were recorded for colony growth inhibition. Area of inhibition
zone was calculated as: Area of inhibition at x ppm = 3.14 (TRx2-r2)
where x = concentration used; r = radius, TR = Total radius of the inhibition
zone at specific concentration (Biswas et al., 2009).
Effects of PCAF of Piper chaba at different concentrations on bacteria:
Effects of PCAF against four different bacterial species viz., Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa and Sarcina lutea
was measured by using inhibition zone test. Firstly the strains of test bacteria
were transferred to NA (Nutrient Agar Media) slants and incubated for 24 h.
After 24 h, 1 loop full of bacterial spore of each test species was added separately
to sterile nutrient broth and mixed well and incubated at 37°C for 2.5 h.
Bacterial spore suspension (1 mL) in sterile nutrient broth was then added to
sterile Petri dish containing molten NA medium and allowed to solidify. After
complete solidification four cups (25 mm2 size) were cut at equidistant
positions and in these cups 0.5 mL solution of PCAF at 500, 1000, 1500 and 2000
ppm was added. Treated plates were incubated at 37±1°C for 24 h.
After this period, plates were taken out and observations of inhibition zone
were recorded, using the formula described for fungal effects.
Isolation and characterization of active allelochemical: Fraction-3
of Piper Chaba was purified by column chromatography and thin layer chromatography.
|| Crystals of the Fraction-3 (PCAF) of Piper chaba
By repeatedly following the proposed scheme of fractionation we could extract
the compound of interest (Acetone fraction of Piper chaba, henceforth referred
to as Fraction-3 or PCAF) as a single pure compound (purity of >95%). At
least eight repetitions of the procedure were necessary to achieve this goal.
TLC analysis: After purification PCAF (Piper chaba acetone fraction)
was run on TLC in the solvent system (20: 80 :: Ethyl Acetate: Hexane) showed
single bright yellow spot in iodine vapor and violet color quenching spot under
UV light with Rf value 0.81. Purified PCAF was off-white in color with hot pungent
flavor and physical state of compound was crystalline at room temperature (Fig.
Spectral analysis: Mass spectra (Fig. 3a, b)
of purified PCAF revealed the molecular weight, i.e., M+ 285. The
conclusion is drawn based on the appearance of the (M+H) and (M+Na) peaks at
the spectrum. Though the molecular ion i.e., M+ peak is not visible
in the spectrum but 100% abundance molecular ion peak at 286 (M+H)+
and low abundance peak at 308 corresponding to (M+Na)+ molecular
ion is answering for the difference of 23 (molecular weight of sodium), strongly
suggesting our conclusion. Fragmentation of the original compound plus hydrogen
is 286. Hence molecular weight of the given compound is (286-1) = 285.
The 1H NMR spectrum (Fig. 4a, b)
of this compound exhibited signals at δ 5.97 (s, 2 h), δ 3.62 (s,
4 h), δ 1.58 (brs, 2 h), δ 1.64 (brs, 2 h), δ 1.79 (brs, 2 h),
δ 7.39 (q,J = 9.8 Hz, 1 h), w 7.26 (s, 1 h), δ 6.97 (d, J = 8.2 Hz,
1 h), 6.75-6.90 (m, 3 h), 6.43 (d, 1 h).
13CNMR spectrum of PCAF (Fig. 5a) showed signals
for 17 carbons. In this compound there are six CH2 at δ 101.19,
46.82, 43.16, 26.65, 25.57, 24.58 ppm as evident clearly from the Dept-135 Spectra
(5b). Dept-90 spectra indicated the presence of seven CH at δ 105.58, 108.39,
120.00, 122.41, 125.28, 138.11, 142.38 ppm (5c). Peak at δ 165.34 ppm supported
the presence of >C = O. The triplet signal at δ (77.42, 77.00, 76.58
ppm) occurred for the CHCl3 (solvent used for dissolving the compound).
Benzene carbon atom peak observed at δ 130.94 ppm. Two more peaks at δ
148.11 and 148.30 ppm were due to the other carbons of the aromatic ring.
Figure 6 showed the IR Spectra of purified PCAF of Piper
chaba. Peak at 2930 and 2857 cm-1 are due to the asymmetrical
stretching (vasCH2) and symmetrical stretching (vsCH2),
|| (a) MS and (b) MS/MS spectra of purified PCAF of Piper
Peak at 1443 cm-1 suggested CH2 bending. Peak at 803
cm-1 strongly supported the benzene ring within the compound. Peak
at 1252 cm-1 indicated the C-O stretching vibration (Aromatic methylene
dioxy compounds). Stretching of -CO-N observed at 1632 cm-1. Peak
at 3435 cm-1 indicated the Intermolecular hydrogen bonding.
Chromatographic and Spectral analysis of PCAF revealed the compound to be 5-Benzo
(1,3) dioxol -5-y1-1-piperidin-1-yl-penta-2,4-dien-1-one or designated as chabbarin
(Fig. 7). About 20-22% (of dry weight with 99.9% purity) of
chabbarin have been isolated and purified from the stem of Piper chaba.
Antioxidant activity of PCAF of Piper chaba: In case of antioxidant
screening (Table 1), the IC50 value of BHT and
ascorbic acid was obtained 26.0 and 5.0 μg mL-1, respectively.
In this investigation, the acetone extract (PCAF) of the plant showed the highest
antioxidant activity with IC50 value of 3.00 μg m-1.
|| (a) 1HNMR and (b) Expended 1HNMR spectra
of purified PCAF of Piper chaba
Methanol soluble fraction (PCMF) of the methanol extract also revealed potent
antioxidant activity (IC50 = 8 μg mL-1). On the other
hand the ethyl acetate (PCEtoAcF) and n-hexane soluble fraction (PCHF) showed
moderate and low antioxidant activity with the IC50 of 126 and 132
μg mL-1, respectively. So we find the Acetone Fraction of Piper
chaba with the maximum antioxidant activity than the other three fractions.
|| (a) 13CNMR, (b) DEPT-135 and (c) DEPT-90 spectra
of purified PCAF of Piper chaba
||IR spectra of purified PEAF compound of Piper chaba,
Hunter spectra name: 78.sp, Accumulations: 16, Resolution: 4.00 cm-1
|| Molecular structure of Fraction-3 (PCAF) of Piper chaba
Effects of PCAF of Piper chaba at different concentrations on fungi:
Chabbarin showed strong antifungal activity against Aspergillus niger,
Aspergillus fumigatus, Aspergillus tamarii, Penicillium chrysogenum
(Fig. 8). Aspergillus niger, Aspergillus fumigatus,
Aspergillus tamarii showed maximum inhibitory activity at 2000 ppm, whereas
in Penicillium chrysogenum maximum inhibitory activity was noticed at
500 ppm. In case of Aspergillus tamarii, MIC is 0.01562 mg mL-1
whereas in other three fungal species, it is 0.03125 mg mL-1.
||Effects of Fraction-3 of Piper chaba (PCAF) at different
concentration on Aspergillus niger, Aspergillus fumigatus,
Aspergillus tamarii, and Penicillium chrysogenum. Correlation
is significant at 0.01 level (Pearson 2-tailed)
Effects of PCAF of Piper chaba at different concentrations on bacteria:
Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa)
are much more effective to Chabbarin than gram positive bacteria (Sarcina
lutea and Staphylococcus aureus). Gram negative bacteria exhibited
maximum inhibitory activity at 1500 ppm. For Escherichia coli, MIC was
found at 0.01562 mg mL-1 and that of Pseudomonas aeruginosa at
0.00391 mg mL-1. In case of Staphylococcus aureus, maximum
inhibitory activity noticed at 500 ppm and Sarcina lutea inhibitory activity
increased with concentration. In both gram positive bacteria, MIC was detected
at 0.00781 mg mL-1 (Fig. 9).
Natural products and their derivatives have historically been invaluable as
a source of therapeutic agents. Piper chaba has been traditionally used as a
prime medicament for cold and cough (Rukachaisirikul et
al., 2002) but the compound responsible for this medicinal property
has never been studied before (Bhandari et al., 1998).
We have isolated and purified the hot pungent compound from the stem of Piper
chaba. About 20-22% (dry weight) of the compound has been recovered with
up to 99.9% purity and was identified as 5-Benzo [1, 3] dioxol 5-y1-1-piperidin-1-
yl-penta-2, 4-dien-1-one or designated as Chabbarin. This compound possesses
strong antimicrobial and antioxidant activity. It is very much effective both
on gram positive and gram negative bacteria with minimum MIC ranging from 0.00391
mg mL-1 to 0.01562 mg mL-1. It also exerted strong antifungal
activity with minimum MIC ranging from 0.01562 to 0.03125 mg mL-1.
Significant antioxidant activity has also been detected in this compound. The
acetone extract (PCAF) of the plant showed the maximum antioxidant activity
with IC50 value of 3.00 μg mL-1 as compared to the
other fractions, with the minimum being in n-hexane (132 μg mL-1).
Hence it is a potent bioactive compound responsible for the medicinal properties
of the plant (Jayashree et al., 2003).
Numerous drugs have entered the international pharmacopoeia via the study of
ethno-pharmacology and traditional medicine (Sunilson
et al., 2009; Joseph and Sujatha, 2011; Zongo
et al., 2009; Kolawole, 2007). For traditional
medicines, newer guidelines of standardization, manufacture and quality control
and scientifically rigorous research on the scientific basis for traditional
treatments will be required (Chopra et al., 1992).
Traditional knowledge can serve as powerful search engine which will greatly
facilitate intentional, focused and safe natural product drug discovery and
help to rediscover the drug discovery process.
Ayurvedic, Indian and traditional Chinese systems are living great traditions.
These traditions have relatively organized database and more exhaustive description
of botanical material that is available and can be tested using modern scientific
methods. Both systems of medicine thus have an important role in bio prospecting
of new medicines. Good botanical practices which can improve the quality control
procedures of monitoring impurities and other toxins in the raw material can
make the ethno pharmacology research more meaning full (Aqil
and Ahmad, 2007).
Natural pharmaceuticals, nutraceuticals and cosmeceuticals (Sanjib,
2009) are of great importance as a reservoir of chemical diversity aimed
at new drug discovery and can be explored as potential antimicrobial, cardiovascular,
immunosuppressive and anticancer drugs.
A golden triangle consisting of Ayurveda-Modern medicine- Modern Science will
converge to form a real discovery engine that can result in newer, safer, cheaper
and effective therapies.
The hot pungent compound, chabbarin, extracted from the acetone fraction of
the stem of Piper chaba, is the key reason for the medicinal property
of the plant. The compound exhibits strong antifungal (MIC range 0.01562 to
0.03125 mg mL-1) and antibacterial (both on gram positive and gram
negative; MIC range 0.00391 to 0.01562 mg mL-1) activity. It also
has a high range of antioxidant activity of IC50 value 3.00 μg
mL-1. Thus we conclude that our study on the isolation and identification
of the active compound supports the fact that Piper chaba, Hunter is
one of the potential sources of natural pharmaceuticals.
We are privileged to convey our sincere gratitude to our respected Director,
Prof. Bimal Roy, Indian Statistical Institute, for providing Laboratory facilities
and financial support. We are indebted to Prof. Anjana Dewanjii, Prof-in-Charge
and Head, Biological Sciences Division, Indian Statistical Institute, for her
affectionate encouragement, valuable advice and enormous laboratory facilities.
We also thank Miss Barnali Das, Mrs. Jaba Mukherjee, Mr. Bharat Bhanderi, Mr.
Nityananda Banik and Mr. Laxman Magranti of Agricultural and Ecological Research
Unit, Indian Statistical Institute, for their valuable assistance in laboratory