In Vitro Antimicrobial and Phytochemical Analysis of Dichloromethane Extracts of Piper nigrum (Black Pepper)

Introduction

The genus piper, a member of family piperaceae, is an perennial herb, which has over 700 species distributed in both hemispheres. This family has shown a promising effect in therapeutics (Reshmi S.K et al., 2010).  Various piper species have been used as spice and in folk medicine due to attributed physiological activities and thus bear a great commercial, economic and medicinal potential (Krishnamurthi A., 1969; Parmar V.S et al., 1997; Kiuchi F et al., 1998; Siddiqui B.S et al., 1997; Nadkarni., 1954).

Black pepper (Piper nigrum L.) “The king of spices” contributes its major share in Indian Spice Export scenario (Zachariah T.J et al., 2010). It is known that piper nigrum has biological activity such as CNS depressant, antioxidant, radical scavenging, anti-insecticidals, antibacterial, allelopathy, anticonvulsant, anti-tubercular, antipyretic, anti-inflammatory, antioxidant and exterofective (C.F.Su, Helen, et al., 1981; Dorman, H.J.D., 2008; Siddiqui, Z.S., 2007; Daniel, M., 2006).

Plant derived substances has obtained greater attention in the recent years to prevent and cure

human diseases as they are considered to be more bio-friendly. It is generally estimated that over

6000 plants in India are in use in traditional, folk, and herbal medicine, representing about 75% medicinal needs of the third world Phyto-chemicals are nonnutritive plant chemicals that have protective or disease preventive properties. Plant produces these chemicals to protect itself but research works demonstrates that many phytochemicals can protect humans against diseases. Knowledge of the chemical constituents of plants is desirable because such information will be of value for the synthesis of complex chemical substances. countries (Veerachari, U et al., 2011).

The main objective of entire study is to perform the complete analysis of various extracts of Piper nigrum to isolate some new compounds.

Experimental

General

The oils were analyzed by using Varian 4000 GC-MS. The instrument operates at the following conditions: equipped with fused silica 30m (CP-Sil-8,Varian) capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25µm, the Helium carrier gas had a delivery rate of 1 ml/min, a capillary injector operating at 280°C in the split mode (1:150),flame ionization detector (FID) running at 300°C, the column oven temperature programming was 50°C for 5 min and then increased from 50 to 250°C at the rate of 3°C/min and hold for 7 min.

Plant material

During our research for novel, bioactive natural products, seeds of  Piper nigrum were purchased from an authentic seed shop at Jammu’s district and classified systematically by Dr. Gurdev Singh of the botany department at lovely professional university.

Extraction and Isolation

The dried and crushed seeds (one kg) of Piper nigrum were soxhalated and distilled with different solvents such as light petroleum ether, toluene, dichloromethane, chloroform and methanol according to their polarity gradient. Oily fraction was obtained from dichloromethane extracts after repeated treatment with ethanol. These oily fractions were subjected to GC-MS for identification of components

Analysis of oily fraction

Compounds were identified by their GC retention time relative   to known compounds and by comparison of their mass spectra with those present in IIIM library. The GC-MS spectra of the oily fraction of dichloromethane extracts of Piper nigrum unveiled the presence of following components.

Table 1: List of Secondary Metabolites identified in Dichloromethane extracts of Piper nigrum

Compound number	RT (min)	Compound Name	Area	Amount/Rf
1	25.176	Formylpiperidine	2658	0.446
2	27.230	4-Terpeneol	633	0.106
3	28.362	A-phellandrene epoxide	1213	0.204
4	32.761	Carvacrol	1852	0.311
5	33.629	2-Methyl naphthalene	618	0.104
6	34.732	Piperonal	15416	2.587
7	35.000	Dimethoxyphenol	2359	0.396
8	38.367	Caryophyllene	3855	0.647
9	41.686	2,4-di-t-butylphenol	3216	0.540
10	41.832	β–bisabolene	1590	0.267
11	42.351	δ –Cadinene	1359	0.228
12	43.573	Elemol	3059	0.513
13	43.853	Nerolidol	992	0.166
14	46.763	4,4-dimethyl-3-(3-methylbut-3-enylidene)methylenebicyclo[4.1.0] heptane	2047	0.343
15	47.471	c-murrolene	6958	1.168
16	47.901	α- Eudesmol	1436	0.241
17	56.881	Cyclopropanebutanoic acid	776	0.130
18	57.384	Bicyclo(3.3.1) Nonan-2-one	1044	0.175
19	59.074	Ethyl palmitate	1897	0.318
20	64.226	Ethyl linoleate	1202	0.202

 Table:  GC-MS of the various components extracted from the hot dichloromethane extracts of Piper nigrum

Figure 2:  Structures of various components isolated from dichloromethane extracts of  Piper nigrum. Click here to View figure

 

Antimicrobial activity

Cultures

Gram positive bacteria: Bacillus subtilis, Staphylococcus aureus; Gram negative bacteria: Escherichia Coli, Pseudomonas aeruginosa, Salmonella typhi and Fungi Aspergillus niger were obtained from Biotech Research Laboratory, Lovely Professional University.

Chemicals

Nutrient agar and nutrient broth for bacterial cultivation; Potato dextrose agar and potato dextrose broth for fungal cultivation and standard antibiotic like gentamicin were purchased from Hi Media Laboratories Pvt. Ltd., Mumbai.

One gram of the extracts was dissolved in same solvent in such a way that the final concentration of each extract would be 1gm/ml of respective solvent.

Disc diffusion method

The in vitro antimicrobial activity of DCM extracts of pepper were checked by disc diffusion method (Elgavyar, M. et al., 2001). Bacterial culture in log phase was inoculated in nutrient agar and plated. The 5 µl of various extracts were poured on to different discs prepared from whatman No: 1 filter paper. The 2 or 3 discs were then placed on the petriplates containing cultures and incubated bacterias for 24 hours at 37⁰C and fungi in B.O.D incubator for 7 days.  The diameter of zone of inhibition was measured.

Table 2: In vitro antibacterial activity of DCM extracts

Bacterias	Zone of inhibition
Gram (+) Bacillus subtilis	(+) 3mm dia
Gram (-) Escherichia coli	(-)
Gram (-)  Pseudomonas aeruginosa	(-)
Gram (+) Staphylococcus aureus	(-)
Gram (-)     Salmonella typhi	(-)

 

Table 3: In vitro antifungal activity of DCM extracts

Fungus	Zone of inhibition
Aspergillus niger	(+) 5 mm dia

  

Figure 3: Antibacterial activity of DCM extracts against the fungi Bacillus subtilis     Click here to View figur

 

Figure 4: Antifungal activity of DCM extracts against the fungi Aspergillus niger. Click here to View figure

 

Conclusions

Phytochemical screening of DCM extract has shown the presence of large numbers of alkaloids, terpenoids and other organic compounds that are of great importance as a source of new useful drugs. From these studies, it can be concluded that all extracts of Piper nigrum have many beneficial effects with respect to the presence of the above secondary metabolites which are likely to combat many diseases and also boost the immune system. The phytochemical characterization of the extracts, the identification of responsible bioactive compounds and quality standards are necessary for future study.

Acknowledgements

We are very grateful to Dr. Ravikant Khajuria of IIIM, Jammu for GC-MS analysis and Lovely Professional University for lab facility.

References

1. Reshmi, S.K., Sathya, E., Devi, S. Isolation of piperidine from Piper nigrum and its antiproliferative activity. J. of Medicinal Plants Research., 2010; 4(15): 1535-1546.
2. Krishnamurthi, A. The Wealth of India. Counsil of Scientific and Industrial Research, New Delh,i 1969.
3.  Parmar, V.S., Jain, S.C., Bisht K.S., Jain, R., Taneja, P., Jha, A., Tyagi, O.D., Prasad,  A.K., Wengel, J., Olsen, C.E., Boll, P.M. Phytochemistry of the genus piper.  Phytochemistry., 1997;  46: 597-673.
4. Kiuchi, F., Nakamura, N., Tsuda, Y., Kondo, K., Yoshimura, H. Amides from Piper nigrum L. with dissimilar effects on melanocyte proliferation in-vitro.  Chem.Pharm. Bull., 1998; 36: 2452—2465.
5. Siddiqui B. S., Begum S., Gulzar T., Farhat, Noor F. An Amide from fruits of Piper nigrum. Phytochemistry., 1997; 45: 1617—1619.
6. Nadkarni, A.K. Indian Materia Medica. Popular Book Depot, Bombay, 1954.
7. Zachariah,  T.J., Safeer,  A.L., Jayarajan, K.,  Leela,  N.K., Vipin,  T.M., Saji,  K.V., Shiva,  K.N., Parthasarathy, V.A., Mammootty, K.P. Correlations of metabolites in the leaf and berries of selected black pepper varieties. Scientia Horticulturae., 2010; 123: 418-422.
8. C. F. Su, Helen., Horvat,  R. Isolation, Identification and Insecticidals Properties of Piper nigrum Amides. J.Agric.Food.chem., 1981; 29(1): 115-118.
9. Dorman, H.J.D., Deans, S.C. Antimicrobial agents from plants: antibacterial    activity of plant volatile oils. J.of Applied Microbiology., 2008; 2(88): 308-316.
10. Siddiqu, Z.S. Allelopathic effects of black pepper leachings on Vigna mungo(L) Hepper. Acta Physiologiae  Plantarum., 2007; 29(4): 303-308.
11. Daniel, M. Medicinal Plants: Chemistry and Properties. Science Publisher.USA, 2006.
12. Veerachari, U., Bopaiah, A.K. Preliminary phyto-chemical evaluation of the leaf extract of five Cassia Species. J. Chem. Pharm. Res., 2011; 3(5): 574-583.
13. Elgavyar, M., Draughon, F.A., Golden, D.A., Mount, J.R. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Prot., 2001; 64(7): 1019-1024.

---

This work is licensed under a Creative Commons Attribution 4.0 International License.