1. Introduction

It is estimated that there are 250,000 to 500,000 species of plants on Earth. Relatively small percentages (1 to 10%) of these are used as foods by both humans and other animal species. It is possible that even more are used for medicinal purposes ^[1]. Hippocrates (in the late fifth century B.C.) mentioned 300 to 400 medicinal plants ^[2].

Black pepper (Piper nigrum L.) is a flowering vine of the Piperaceae family that cultivated for its fruit, which is usually dried and used as a spice and seasoning. P. nigrum native of south India and popularly known as “King of Spices”. It is commonly used in curry recipes, as masalas and also included in the prescriptions of Ayurvedic and other traditional medicinal systems. Pepper is also used in folk medicine as aphrodisiac, carminative, stomachic, antiseptic diuretic and for the treatment of cough, rheumatoid arthritis, peripheral, acetic acid, neuropathy, melanoderma and leprosy due to the presence of volatile compounds, tannins, phenols, alkaloids, and other unknown substances ^[3].

According to (Friedman et. al., 2008) ^[4] alkaloids play a significant role in plant physiology, agriculture, host-plant resistance, entomology, the diet and medicine. Among them piperine is the major chemical constituent responsible for the bitter taste of the black pepper. Phenolic compounds comprise an aromatic ring, bearing one or more hydroxyl groups and range from simple phenolic molecules to highly polymerized compounds ^[5]. Polyphenols may be associated with various carbohydrates and organic acids ^[6]. These compounds exhibit a wide range of physiological properties, such as anti-inflammatory, antimicrobial and antioxidant effects ^{[7, 8]}. Although a great amount of research has been performed to determine the antibacterial activity of medicinal plants, optimal extraction of bioactive compounds has not been well established for most plants. Unfortunately, the development of bacterial resistance to these antimicrobials quickly diminished this optimism. Therefore, the objectives of this study were to optimize the concentrations of Pepper nigrum alkaloids and phenols antimicrobial activity against some of human pathogenic bacteria.

2. Materials and Methods

The Piper nigrum fruits were purchased from local market. After the fruit were air derided and powdered, it kept at 4°C until further investigations.

a-Alkaloids

The extraction was prepared according to the method of (Harborne, 1984) ^[9] were considered. Quantities of 100 g dried materials were homogenized in electrical shaker with 350 mL of (4:1) ethanol: Distilled Water, then filtered through muslin. Then through a filter paper in Bouknner funnel, the filtrate was concentrated to quarter of original volume, then acidified by drops of 2% H2SO4 until the pH became between 1 and 2, then it was extracted with chlorophorm 3 times in the separating funnel; alkaloids were precipitated by the addition of drops of concentrated NH₄OH, pH became 9.0 and 10.0. Then extracted with chloroform-methanol (1:3) twice and with chloroform once, 2 layers appeared, lower layer was neglected because it containing weak alkaloids, while the upper layer, aqueous layer was dried by rotary evaporator; dried residue was extracted with methanol. Then kept in refrigerator until use.

b-Phenols

The extraction was made according to ^[10] and ^[9], 200 g of dried materials were divided into 2 equal quantities, one was mixed with 300 mL of Distilled Water. and another one was mixed with 300 mL of 1% HCl. Then samples were homogenized in electrical shaker for 5 min., and warmed by using centrifuge. The supernatants were mixed with equal volume of N-propanol and saturated with amount of NaCl in separating funnel, 2 layers were appeared: The lower one (aqueous layer) was extracted with amount of ethyl acetate and concentrated by using rotary evaporator. The upper layer was dried by rotary evaporator at 40°C. Dry material for both layers was dissolved with 5 mL of 96% ethanol, the 2 layers were dried using electrical oven at 40°C, and then kept in the refrigerator until use.

Stock solutions were prepared by mixing 2 g from the dried extract with 20 mL Ethylene glycol, and then it was sterilized with Millipore membrane filter (0.22 µm). Then different concentrations of (0.002, 0.004, 0.02, 0.04, 0.2, and 0.4%) mg. mL^-1 were prepared by mixing known volume from the stock solution with Ethylene glycol using the following equation:

C1V1 = C2V2.

C1 = Concentration of stock solution.

V1 = Volume that obtained from stock solution.

C2 = Final concentration.

V2 = Final volume.

Ethylene glycol was the solvent which used as diluent solution.

Test organisms were obtained from laboratory of Biotechnology Dept., College of Science, University of Baghdad; (Table 1). Four gram-negative E. coli strain, while four gram-positive bacteria were Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pyogenes. For the purpose of antimicrobial evaluation, the microorganisms were cultured in Tryptone Soya Broth (TSB) (Oxford, England) at room temperature for 24 hr. and were adjusted to 10⁷ cfumL^-1 with sterile saline.

Table 1. The microbial isolates used in the study

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Bacterial cultures were activated in test tubes containing 5 mL of nutrient broth, and sabaraud dextrose broth for the bacteria, and then incubated for 24 hours at 37°C; nutrient agar was used for bacterial strains storage at 4°C.

The anti-bacterial activity of the extracted alkaloids and phenols compounds were tested against bacteria by using agar-well diffusion method as follows:

1. 25 mL of sterilized Mueller Hinton agar was poured into numbers of petri-dishes as required.

2. Bacterial broth of 18 hr. was prepared; a sterile cotton swab dipped into the broth, the entire agar surface of each plate was inoculated with this swab, first in the horizontal direction then in vertical direction to ensure even distribution of the organism over the agar surface.

3. Wells were prepared in plates with help of a sterile cork borer (8 mm in diameter).

4. 0.2 mL of different concentrations of the extracted oil was introduced into those wells; a control well was introduced by the diluent of oils (Ethylene glycol) instead of the oil. Replicates were made for each test.

5. Plates were incubated at 37°C for 24-48 hrs.

6. The anti-bacterial activity was recorded by measuring the diameters of clear zones of inhibition ^{[11, 12]}.

The analysis of the chemical composition was made by fast liquid chromatography (FLC). FLC consists from a mobile phase which is polar and consists of a mixture of solvents such as water and acetonitrile, while the stationary phase comprises of a column which is usually stainless steel and packed with silica particles, a sample of 50 µl was injected into the mobile phase using procedure outlined by Hartley and Buchan and it passes along the stationary phase, the time taken for a sample to pass through the system is recorded as its retention time and is one of the characteristic used to identify the compound, all the compounds were separated and identified using FLC with separation conditions C-18, 3 cm particle size, 50 × 4.6 mm internal diameter of the column, detection U.V. set at 275 nm, flow rate 0.7 mL/min. and 30°C temperature, but the differences were in mobile phase which was 0.1 % acetic acid and acetonitrile using linear gradient from 0-100% B in 10 min. with phenolic compounds, However, it was deionized water: methanol 40:60 V/V in alkaloids case and 0.1% acetic acid in deionized water: acetonitrile (20-80V/V) in terpens. The area under a peak is used for calculating the concentration of a sample as the following formula:

Analysis of the chemical composition was made by injecting 20 µl of the extract of each sample in FLC for identification. The procedure that used outlined by Hartley and Buchan. The conditions of separation were listed in Table 2 and Table 3. The peaks were detected by UV detector. The analysis was carried out in the laboratories of Ministry of Science and Technology ^[13].

Table 2. Conditions of Fast Performance Liquid Chromatographic used for analysis of alkaloid compounds of the plants extracts

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Table 3. Conditions of Fast Liquid Chromatographic used for analysis of phenolic compounds of the plants extracts

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Complete Randomized Design (C.R.D.) was used as an experimental design. Data were analyzed by using statistical analysis system-SAS (2001) to study the effect of different factors on the diameters of inhibition zones. Least significant difference (LSD) was used to compare the significant difference between means at P ≤ 0.05.

3. Results and Discussion

It is suggested that plant extracts exhibiting diameters of zones of inhibition ≥ 10 mm were considered active ^{[14, 15]}. There for the alkaloid extracts showed significant zone of inhibition against Gram-positive and Gram-negative bacteria (Table 3). The maximum average for inhibition zone was 26.4 mm for E. coli (PBR 322 standard strain), while the minimum average was 11 mm for S. aureus which isolated from dermal infection (Table 4). Also for alkaloid extract concentrations the maximum average for inhibition zone 43.7 mm was for 0.4%, but the minimum was 0.6 mm for 0.002%. However interaction observation result from the Table 3 indicates that the maximum inhibition zone was 60 mm for Escherichia coli (PBR 322 standard strain) which treatment with 0.4% concentration, Further the minimum inhibition zone was 1 mm for the Escherichia coli (AMP resistance TET resistance Contain plasmid standard strain) treated with alkaloids 0.002% concentration.

Similar results on antibacterial activity were reported on Table 4 which indicates that there are significant variations of the inhibition zone for all tested bacteria with Phenols compounds. The average for maximum and minimum inhibition zone were 22.4 and 13.4 mm for S. aureus isolated from Dermal infection and Escherichia coli PBR 322 standard strain, receptively. Also the highest and lowest average inhibitory zone for concentrations were (48.1 and 1 mm) exhibited by 0.4 and 0.002%, respectively. Although the interaction result showed the maximum inhibition zone was 53 mm for both Staphylococcus aureus isolated from Urinary tract infection and Escherichia coli isolated from the same Source (Urinary tract infection) with 0.4% concentrations, while the minimum was 1 mm for three strain bacteria treated with phenols 0.002% concentration (Table 4).

The antibacterial activity results often pointed that 0.4% was the most concentration active against all tested bacteria, Especially S. pyogenes (gram positive) and three E. coli strain (gram negative) (Figure 1, Figure 2, Figure 3 and Figure 4). Beside the minimum inhibition concentration was 0.002%.

Table 4. Antibacterial activity of the Alkaloids plant extracts against some of pathogenic bacteria

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Table 5. Antibacterial activity of the Phenolic plant extracts against some of pathogenic bacteria

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Figure 1. Antimicrobial activity of the alkaloids plant extracts, tested against different clinical microorganisms

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Figure 2. Antimicrobial activity of the alkaloids plant extracts, tested against different clinical isolated microorganisms

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Figure 3. Antimicrobial activity of the phenolic plant extracts, tested against different clinical isolated microorganisms

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Figure 4. Antimicrobial activity of the phenolic plant extracts, tested against different clinical isolated microorganisms

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Results have been shown important and significant differences between the concentrations of each secondary metabolic compound among the extracted cruds.

Alkaloids present in the extracted plants were also identified by FLC, as elaborated in Table 6 and the peaks in Figure 5 and Figure 6. The total concentration of alkaloids in the extracted P. nigrum was 156.8 μgmL^-1. Capsaicin (137.2 μgmL^-1) was the major alkaloid, while 2-dihdrocapsaicin (19.6 μgmL^-1) was the minors in the P. nigrum.

Table 6. Types and concentration of alkaloids in plant extracts

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Figure 5. FLC profile of alkaloids standards of Piper nigrum (1) Capsaicin, (2) 2-dihdrocapsaicin

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Figure 6. FLC profile of P. nigrum alkaloids (1) Capsaicin, (2) 2-dihdrocapsaicin

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Table 7. Types and concentration of phenols in plant extracts

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Figure 7. FLC profile of phenols standards of Peper Nigrum (1) Gallic acid, (2) Trans-p-feruloyl-â-D-glucopyranoside, (3) Trens–p-sinapyl-â-D-glucopyranoside, (4) Quercetin3-O-R-L-rhamnopyranoside-7-O-â-D-glucopyranosyl, (5) Quercetin3-O-R-L-rhamnopyranoside, (6) Luteolin 6-C-â-D-glucopyranoside-8-C-R-L-arabinopyranoside, (7) Luteolin 7-O-[2-(â-D-apiofuranosyl)-4-(â-D-glucopyranosyl), (8) Luteolin 7-O-[2-(â-D-apiofuranosyl)-â-D-glucopyranoside-8-C-R-L-arabinopyranoside, (9) Kaempferol, (10) Coumarins

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Figure 8. FLC profile of phenols of P. nigrum (1) Gallic acid, (2) Trans-p-feruloyl-â-D-glucopyranoside, (3) Trens–p-sinapyl-â-D-glucopyranoside, (4) Quercetin 3-O-R-L-rhamnopyranoside-7-O-â-D-glucopyranosyl, (5) Quercetin 3-O-R-L-rhamnopyranoside, (6) Luteolin 6-C-â-D-glucopyranoside-8-C-R-L-arabinopyranoside, (7) Luteolin 7-O-[2-(â-D-apiofuranosyl)-4-(â-D-glucopyranosyl), (8) Luteolin 7-O-[2-(â-D-apiofuranosyl)-â-D-glucopyranoside-8-C-R-L-arabinopyranoside, (9) Kaempferol, (10) Coumarins

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Results of FLC (fast liquid chromatography) analysis indicated the presence of ten phenolic compounds in P. nigrum (Table 7) and Figure 7 and Figure 8. All the isolated compounds appeared to have different retention time. Chrysophanol-1-O-B-glucopyranoside (55.08 µg mL^-1) and Trens–p-sinapyl-â-D-glucopyranoside (147.4 µgmL^-1) were the highest phenolic compounds in P. nigrum, while Anthraquionone (4.76 µgmL^-1) and Trans-p-feruloyl-â-D-glucopyranoside (3.68 µg/mL) were the lowest concentration in P. nigrum.

4. Discussion

Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. Structurally, the plant extracts contains at least two classes of secondary metabolites namely alkaloids, phenols. However, it should be noted that the activity does not depend on the number of classes of detected bioactive compounds, but mostly on their concentration. The inhibitory activity of P. nigrum was previously reported against some bacteria such as Staphylococcus aureus, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Salmonella typhi and Escherichia coli ^[16]. Some studies showed that the pharmacological and biology properties were mostly due to alkaloids ^[17]. The inhibitory activity of the system originated from the synergistic actions of both capsaicin and 2-dihdrocapsaicin. While the capsaicin microcapsules displayed potential antimicrobial applications in the food storage ^[18]. The phytoconstitutes of P. nigrum fruits include volatile oil, other minor alkaloids such as piplartin, piperlogumine, piperidine, starch, resin and pungent alkaloid piperine. The piperine present in the skin and seed of the black pepper which is responsible for the antimicrobial activity ^{[19, 20]}.

Generally, the reason could be attributed to the presence of extra outer membrane in their cell wall acting as barrier for the compound(s) to diffuse into the bacterial cells. Or may be genetic content of their plasmids ^[21] or to the differences in the composition and the mechanism of action of the bioactive compounds ^[22].

The alkaloids sanguinarine, berberine, jatrorrhizine and palmatine are known to inhibit the multiplication of bacteria, fungi and viruses ^[23]. Therefore the antibacterial activity observed in the present investigation is attributed to the alkaloids berberine, palmatine and jatrorrhizine which have been widely known to occur in different species of this genus. Otherwise the biological phenolic compounds activities are related to the molecules structures; by their hydroxyl groups or by phenolic ring, phenolic compounds have capacity to link with proteins and bacterial membrane to form complexes ^[7].

The variation in the inhibition among the gram positive and gram negative bacteria is due to the cell wall and cell membrane compositions. However the differences in sensitivity between Gram-positive and Gram-negative bacteria to the extract can probably be attributed to the structural and compositional differences in membranes between the two groups ^[24]. The Gram-negative bacteria have an outer membrane that serves as an impermeable barrier for many small molecules.

The resistance between bacteria from same spices may due to antibiotic resistance genes and the later may be on chromosome or on plasmids. It is well known that plasmids are major vectors for the dissemination of both antibiotic resistance and virulence determinants among bacterial populations ^[25]. The exchanging of genetic material between microorganisms through transformation, conjugation or transduction processes or may by mobile genes (transposons) have been proposed as a major contributor in the rapid evolution of microorganisms resistant to antibiotics ^[26]. On the other hand, using inaccurate concentration of antibiotics or drug or unnecessary of medicine appointment leads to the resistance of sensitive bacteria. In addition to weakening the immunity system in some human due to poor nutrition or heredity factors make bacteria to be more resistant ^[26].

The above results open the possibility of finding new clinically effective drug and could be useful in understanding the relationship between traditional cures and current medicines. An understanding of these properties would be invaluable in the development of alternative, natural, and safe methods of controlling bacterial infections. The crude extract of P. nigrum demonstrated a significant antibacterial activity against the microorganisms investigated and could therefore be added to the potential list of antibacterial agents.

This result suggests the need for further studies on this substance to identify, isolate, characterize and elucidate the structure of the active ingredient(s) using some spectroscopic techniques such as nuclear magnetic resonance (NMR), infrared spectrophotometry (IR) and mass spectrometry (MS).

References

[1]	Moerman, D. E. An analysis of the food plants and drug plants of native North America. J. Ethnopharmacol. 52: 1-22. 1996.
	In article		CrossRef
[2]	Schultes, R. E. The kingdom of plants, p. 208. In W. A. R. Thomson (ed.), Medicines from the Earth. McGraw-Hill Book Co., New York, N.Y. 1978.
	In article
[3]	Algohary, M.E.M., B.M. Mahmoud, H.M. Ali and M.M. Homeida, Medicinal plants of North Africa, pp: 142-144. 1994.
	In article
[4]	Friedman, M., C.E. Levin. Seung-UN Lee, JIN-shik Lee, Mayumi Ohnisi-Kameyama and N. Kozukue. Analysis by HPLC and LC/MC of pungent piperamides in commercial Black, white, green and red whole and ground peppercorns. J. Agric. Chem., 56: 3028-3036. 2008.
	In article		CrossRef
[5]	Sakihama, Y., M.F. Cohen, S.C. Grace and H. Yamasaki. Plant phenolic antioxidant andprooxidant activities: phenolics-induced oxidative mediated by metals in plants. Toxicology, 177: 67-80. 2002.
	In article		CrossRef
[6]	Manach, C., A. Scalbert, C. Morand, C. Rémésy and L. Jiménez. Polyphenols, food sources andbioavailability. Am. J. Clin. Nut, 79: 727-747. 2004.
	In article
[7]	Balasundram, N., K. Sundram and S. Sammar. Phenolic compounds in plants and agri-industrial by-products. Antioxidant activity, occurrence and was active potential uses, Food Chem., 1: 191-203. 2006.
	In article		CrossRef
[8]	Abah, S.E. and G. Abah. Antimicrobial and Antioxidant Potentials of Agaricusbisporus. Advances in Biological Research. 4 (5): 277-282. 2010.
	In article
[9]	Harborne, J.B. 1984. Phytochemical methods. Chapman and Hall. New York 2nd ed. Pp: 288.
	In article		CrossRef
[10]	Ribereau-Gayon, P. 1972. Plant phenoles. Oliver and Boyd. USA. Pp: 254.
	In article
[11]	Vignolo, G. M., Suriani, F., Holgado, A. P. and Oliver, G., Antibacterial activity of Lactobacillus strains Isolated from drfermented sausages. J. APP. Bac., 75: 344-349. 1993.
	In article
[12]	Perez, L., M. Pauli and P.Bazequre. “Antibiotic assay by the agar well diffusion method.” Journal of Actabiology, 15, 113-115. 1990.
	In article
[13]	Perucka, M. M. and Agric, J. Antioxidant activity of main phynolic compounds isolated from hot peppers, Capsicum annum L. 9, 53 (5) 1750-6. 2005.
	In article
[14]	Zwadyk, P., Enteriobacteriaceae in Zinsser Microbiology. 20th Ed. George ThieneVerlag, Stuttgart. pp. 20-32. 1972.
	In article
[15]	Usman, H. and Osuji, J.C. Phytochemical and in vitro anti-microbial assay of the leaf extract of New bouldialeavis. Afr. J. Trad. CAM. 4 (4): 476-480. 2007.
	In article
[16]	Pavithra VK, O Bhagya L: Antibacterial activity of black pepper (Piper nigrum Linn.) with special reference to its mode of action bacteria. Indian J Comp MicrobiolImmunol Infect Dis, 30 (1): 65-66. 2009.
	In article
[17]	Traore-keita, F.,M. Gasquet, C.Di-Giorgio, E. Ollivier and F. Delmas. Antimalarial activity of four plants used in traditional medicine in Mali. Phytother. Res., 14: 45-47. 2005.
	In article		CrossRef
[18]	Xing, F.; Cheng, G. and Yi, K. Study on the antimicrobial activities of the capsaicin microcapsules. Journal of Applied Polymer Science. Vol (102), Issue 2, pages 1318-1321. 2006.
	In article
[19]	Kokate, C. K.; Purohit, A. K. and Ghokhle, S. B. Pharmacognosy. Nirali Prakashan, 315-317. 1994.
	In article
[20]	Khare, C. P. Encyclopedia of Indian medicinal plants. Springer publication, 367-370. 2006.
	In article
[21]	Karamanİ, Şahin F, Güllüce M, Öǧütçü H, Şengül M, Adıgüzel A: Antimicrobial activity of aqueous and methanol extracts of Juniperusoxycedrus L. J Ethnopharmacol 85 (2-3): 231-235. 2003.
	In article		CrossRef
[22]	Ono T, Kashimura M, Suzuki K, Oyauchi R, Miyachi J, Ikuta H, Kawauchi H, Akashi T, Asaka T, Morimoto S: In vitro and in vivo antibacterial activities of the tricyclic ketolide te-802 and its analogues. J Antibiot (Tokyo) 57 (8): 518-527. 2004.
	In article		CrossRef
[23]	Schmeller T, LatzBrunin B, Wink M. Biochemical activities of berberine, palmatine and sanguinarine mediating chemical defence against microorganisms and herbivores. Phytochem, 44: 257-266. 1997.
	In article		CrossRef
[24]	Lambert PA. 2002. Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. J ApplMicrobiol. ; 92 Suppl: 46S-54S. 2002.
	In article		CrossRef
[25]	Hamada TA Mahmood AR and Ahmed IB: Antibiotic resistance in pathogenic bacteria isolated from utis in tikrit province. Tikrit Med. J.; 14 (1): 203-210. 2008.
	In article
[26]	Salah HF: Effect of some medicinal plant extract on antibiotic resistance by plasmids of Escherichia coli isolated from different sources. M.Sc. thesis submitted to College scientific education, University of Salahaddin-Erbil, Iraq; 2007.
	In article