Characterization of Antifungal Metabolites Produced by Aeromonas Hydrophila and Analysis of its Chemical Compounds Using GC-MS

 

Sabreen A. Kamal¹, Lena Fadhil Hamza²*, Israa Adnan Ibraheam¹

¹Department of Biology, College of Science for women, University of Babylon, Iraq

¹College of Pharmacy, University of Babylon, Iraq

 

ABSTRACT:

Bioactives were analyzed using gas chromatography-mass spectroscopy (GC-MS) techniques, then the in vitro antibacterial and antifungal activity of the methanolic extract was evaluated. Twenty three bioactive compounds were identified in the methanolic extract of Aeromonas hydrophila. GC-MS analysis of Aeromonas hydrophila revealed the existence of the Tricyclo[4.3.1.1(3,8)]undecan-1-amine 1-Methyl-4-[nitromethyl]-4-piperidinol, Bicyclo[2.2.1]heptan-2-one, 4,7,7-trimethyl-, semicarbazone, D-Streptamine, O-6-amino-6-deoxy-α-D-glucopyranosyl-(1-4), 4-(2,5-Dihydro-3-methoxyphenyl)butylamine, 3-Cyclohex-3-enyl-propionic acid, Benzenemethanol, 2-(2-aminopropoxy)-3-methyl-, Benzene, 1-methyl-4(4-morpholyl)ethenylsulfonyl-, Indan-1,2-dione, 4-methyl-,2,3-Dihydroindole-4-ol-2-one, 5,7-dibromo-3,3-dimethyl-, Benzamide, 3-amino-N-[4,5-dihydro-5-oxo-1-(2,4,6-trichlorop, 2-Acetonyl-9-[3-deoxy-β-d-ribouranosyl]hypoxanthine, DL-Leucine ,N-glycyl-, 3-(2,5-Dimethylanilinomethyl)-5-(3-fluorobenzylidene)-2,4-thia, Benzenepropanoic acid, α-(hydroxyimino)-, Butanoic acid, 4,4'-dithiobis [2-amino-,[S-(R*,R*)]- N-Propionyl-D-glucosamine Actinomycin C2, N-(O-Nitrophenylthio)-l-leucine, D-Fructose, diethyl mercaptal , pentaacetate , 1H-Indole , 4-(3-methyl-2-butenyl)-, Spiculesporic acid, 2,5-Piperazinedione, 3,6-bis(2-methylpropyl). Nerium olender (Alkaloids) was very highly active (5.408±0.24) mm. The results of anti-fungal activity produced by Aeromonas hydrophila showed that the volatile compounds were highly effective to suppress the growth of Aspergillus flavus (6.009±0.22). Aeromonas hydrophila produce many important secondary metabolites with high biological activities. Based on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases, the purification of compounds produced by Aeromonas hydrophila can be useful.

 

 

INTRODUCTION:

Aeromonas hydrophila is a heterotrophic, Gram-negative, rod-shaped bacterium mainly found in areas with a warm climate. Aeromonas hydrophila is the most well-known of the six species belonging to the genus Aeromonas. They usually grow from 0.3 to 1.0 μm in diameter and 1.0 to 3.5 μm in length. Aeromonas hydrophila does not form endospores, and can grow in temperatures as low as 4°C.

This bacterium is motile by polar flagella. It can survive in aerobic and anaerobic environments, and can digest materials such as gelatin and hemoglobin A. hydrophila is not as pathogenic to humans as it is to fish and amphibians. One of the diseases it can cause in humans, gastroenteritis, occurs mostly in young children and people who have compromised immune systems or growth problems. Because of its structure, it is very toxic to many organisms. When it enters the body of its victim, it travels through the bloodstream to the first available organ. It produces aerolysin cytotoxic enterotoxin that can cause tissue damage. Aeromonas hydrophila colonies on agar plates are smooth, convex, and rounded, and they are tan/buff-colored on trypticase soy agar 1-4. This bacterium can be found in fresh, brackish, estuarine, marine, chlorinated and unchlorinated water supplies worldwide, with highest numbers obtained in the warmer climates 5-11.

 

A. hydrophila, A. caviae, and A. sobria are all considered to be opportunistic pathogens, meaning they rarely infect healthy individuals. A. hydrophila is widely considered a major fish and amphibian pathogen, 12 and its pathogenicity in humans has been recognized for decades 13-15. The genomic insights of aeromonads could be a stepping stone into understanding of them 16. Aeromonas hydrophila is very toxic to many organisms because of its structure. When it enters the body of fish, amphibians, or humans, it travels via the bloodstream to the first available organ. It produces aerolysin cytotoxic enterotoxin (Act) which is one of the major virulence factors. Its toxin is produced and secreted by the cell from a type II secretion system. The toxin binds to high- affinity receptors and undergoes oligomerization to form a heptameric pore- forming complex which allows passage of small molecules in the plasma membrane, resulting in permeabilization of the cell, cell death, and eventually tissue destruction. Extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. However, the pathogenic mechanisms are unknown. The recently proposed type-III secretion system (TTSS) has been linked to Aeromonas pathogenesis. It also causes diseases such as myonecrosis and eczema in people with compromised or suppressed (by medication) immune systems 17. In very rare cases, A. hydrophila can cause necrotizing fasciitis 18. A. hydrophila infections occur most often during environmental changes, stressors, changes in temperature, in contaminated environments, and when an organism is already infected with a virus or another bacterium. It can also be ingested through food products contaminated with the bacterium, such as seafood, meats, and even certain vegetables such as sprouts. It can also be transmitted by leeches 19-23. Strains of Aeromonas hydrophila are capable of causing disease in fish and amphibians as well as in humans who may acquire infections through open wounds or by ingestion of an adequate number of the organisms in water or food. Aeromonas hydrophila has the oxygen- sensitive IscSUA- HscBA- Fdx system for the biosynthesis of iron- sulfur clusters rather than the oxygen- resistant SUF system, typically associated with aerobic and facultatively anaerobic organisms 24-29. Though Aeromonas hydrophila is considered a pathogenic bacterium, scientists have not been able to prove that it is the actual cause of some of the diseases it is associated with. It is believed that this bacterium aids in the infection of diseases, but do not cause the diseases themselves 30-35. The most common treatment for Aeromonas hydrophila infection in humans are broad-spectrum antibiotics, like tetracycline 36-39. It is particularly susceptible to fluoroquinolones (a family of antibiotics). The most effective were levofloxacin, gatifloxacin, ciprofloxacin, and moxifloxacin. Resistance to these antibiotics is rare 40-47. Aeromonas hydrophila is also known as an opportunistic pathogenic bacterium, meaning they only infect hosts with weakened immune responses 48-53. Other virulence functions include a surface layer which inhibits complement- mediated killing, type IV pili for attachment, a set of extracellular proteases which can cause tissue damage, the ability to form capsules, and polar and lateral flagella 54-57.

 

MATERIALS AND METHODS:

Growth conditions and determination of metabolites

Aeromonas hydrophila strain was isolated from bronchitis patients and obtained from Maternity and children hospital. Subcultures were obtained on the Nutrient Agar for 48 hrs. at 22°C. The mixture was incubated at 4ºC for 10 min and then shook for 10 min at 130 rpm. Metabolites was separated from the liquid culture and evaporated to dryness with a rotary evaporator at 45ºC. The residue was dissolved in 1 ml methanol, filtered through a 0.2 μm syringe filter, and stored at 4ºC for 24 h before being used for GC-MS 58-60. The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library as well as on comparison of their retention indices either with those of authentic compounds or with literature values. The studied fungi, Microsporum canis, Streptococcus faecalis, Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Saccharomyces cerevisiae, Penicillium expansum, Trichoderma viride, Trichoderma horzianum, Aspergillus niger, and Aspergillus terreus were isolated and maintained in potato dextrose agar slants. Spores were grown in a liquid culture of potato dextrose broth (PDB) and incubated at 25ºC in a shaker for 16 days at 130 rpm. The extraction was performed by adding 25 ml methanol to 100 ml liquid culture in an Erlenmeyer flask after the infiltration of the culture.

 

Materials of Plants Collection and Preparation:

In this study, the leaves were dried at room temperature for ten days and when properly dried the leaves were powdered using clean pestle and mortar, and the powdered plant was size reduced with a sieve 61. The fine powder was then packed in airtight container to avoid the effect of humidity and then stored at room temperature.

 

Spectral analysis of bioactive natural chemical compounds of Aeromonas hydrophila using (GC/MS) Analysis was conducted using GC-MS (Agilent 789 A) equipped with a DB-5MS column (30 m×0.25 mm i.d., 0.25 um film thickness, J&W Scientific, Folsom, CA). The oven temperature was programmed as for the previous analysis. Helium was used as the carrier gas at the rate of 1.0 mL/min. Effluent of the GC column was introduced directly into the source of the MS via a transfer line (250 Cº). Ionization voltage was 70 eV and ion source temperature was 230oC. Scan range was 41- 450 amu. The components were identified by comparing their retention times to those of authentic samples of WILEY MASS SPECTRAL DATA BASE Library 62.

 

Determination of Antibacterial and Antifungal Activity:

Five-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 25 μl of the samples solutions (Zingiber officinale  (Crude), Nerium olender  (Alkaloids), Ricinus communis  (Alkaloids), Datura stramonium(Alkaloids), Linum usitatissimum (Crude), Anastatica hierochuntica (Crude), Cassia angustifolia   (Crude), Euphorbia lathyrus   (Crude), Rosmarinus oficinalis   (Crude), Mentha viridis  (Crude), Quercus infectoria  (Crude), Citrullus colocynthis  (Crude), Althaea rosea  (Crude), Coriandrum sativum  (Crude), Origanum vulgare  (Crude), Urtica dioica  (Crude), Foeniculum vulgare  (Crude), Ocimum basilicum  (Crude) and Punica granatum  (Crude) were delivered into the wells. The plates were incubated for 48 h at room temperature. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test microorganisms.  Methanol was used as solvent control. Amphotericin B and fluconazole were used as reference antifungal agent. The tests were carried out in triplicate. The antifungal activity was evaluated by measuring the inhibition-zone diameter observed after 48 h of incubation.

 

Data Analysis:

All the measurements were replicated three times for each assay and the results are presented as mean ± SD and mean ± SE. IBM SPSS 20 version statistical software package was used for statistical analysis of percentage inhibition and disease incidence and disease severity in each case.

 

RESULTS AND DISCUSSION:

Gas chromatography and mass spectroscopy analysis of compounds was carried out in methanolic extract of Aeromonas hydrophila, shown in Table 1.

 

Table 1. Bioactive chemical compounds identified in methanolic extract of Aeromonas hydrophila.

Tricyclo[4.3.1.1(3,8)]undecan-1-amine RT=3.161 Mw=165.15175 Pharmacological activity: anti-inflammatory and anti-allergy agents	1-Methyl-4-[nitromethyl]-4-piperidinol 3.3330 174.100442 Pharmacological activity: anti-histaminic agent	Bicyclo[2.2.1]heptan-2-one , 4,7,7-trimethyl-, semicarbazone 3.590 209.152812 Pharmacological activity: anti-Candida, anti-inflammatory
D-Streptamine , O-6-amino-6-deoxy-α-D-glucopyranosyl-(1-4) 3.619 482.258793 Pharmacological activity: Anti-Bacterial Agent	4-(2,5-Dihydro-3-methoxyphenyl)butylamine 3.859 181.146665 Pharmacological activity: anti-inflammatory	3-Cyclohex-3-enyl-propionic acid 4.368 154.09938 Pharmacological activity: anti-thrombotic
Benzenemethanol, 2-(2-aminopropoxy)-3-methyl- 4.952 195.125929 Pharmacological activity: anti-inflammatory	Benzene,1-methyl-4(4-morpholyl) ethenylsulfonyl- 5.353 267.092915 Pharmacological activity: anti-inflammatory agent	Indan-1,2-dione , 4-methyl- 5.747 160.052429 Pharmacological activity: antitumor, anti-inflammatory, antiviral
2,3-Dihydroindole-4-ol-2-one , 5,7-dibromo-3,3-dimethyl- 5.850 332.900003 Pharmacological activity: anti-pain compound	Benzamide, 3-amino-N-[4,5-dihydro-5-oxo-1-(2,4,6-trichlorop 6.142 395.994759 Pharmacological activity: Anti Inflammatory	2-Acetonyl-9-[3-deoxy-β-d-ribouranosyl]hypoxanthine 6.537 308.11207 Pharmacological activity: Unknown
DL-Leucine ,N-glycyl- 6.543 188.116093 Pharmacological activity: anti-cancer agent	3-(2,5-Dimethylanilinomethyl)-5-(3-fluorobenzylidene)-2,4-thia 6.686 356.099478 Pharmacological activity: anti-bacterial, antifungal	Benzenepropanoic acid , α-(hydroxyimino)- 7.332 179.058243 Pharmacological activity: anti-bacterial, antifungal
Butanoic acid , 4,4'-dithiobis [ 2-amino-,[S-(R*,R*)]- 7.910 268.05515 Pharmacological activity: anti-inflammatory agent, an anti-cancer agent	N-Propionyl-D-glucosamine 11.343 235.105587 Pharmacological activity: Anti-Bacterial Agent	Actinomycin C2 15.154 1268.64413 Pharmacological activity: Anti-Infective Agent
N-(O-Nitrophenylthio)-l-leucine 15.623 284.083078 Pharmacological activity: Unknown	D-Fructose , diethyl mercaptal , pentaacetate 16.058 496.14369 Pharmacological activity: anti-inflammatory	1H-Indole, 4-(3-methyl-2-butenyl)- 16.253 185.120449 Pharmacological activity: Anti-inflammatory
Spiculesporic acid 16.447 328.18859 Pharmacological activity: Anti-Infective Agent	2,5-Piperazinedione, 3,6-bis(2-methylpropyl)- 17.186 226.168128 Pharmacological activity: Unknown

 

The GC-MS chromatogram of the twenty nine one peaks of the compounds detected was shown in Figure 1. Peaks were determined to be Tricyclo[4.3.1.1(3,8)]undecan-1-amine 1-Methyl-4-[nitromethyl]-4-piperidinol, Bicyclo [2.2.1] heptan-2-one , 4,7,7-trimethyl-, semicarbazone, D-Streptamine , O-6-amino-6-deoxy-α-D-glucopyranosyl-(1-4), 4-(2,5-Dihydro-3-methoxyphenyl)butyl-amine, 3-Cyclohex-3-enyl-propionic acid, Benzenemethanol , 2-(2-aminopropoxy)-3-methyl-, Benzene , 1-methyl-4(4-morpholyl)ethenylsulfonyl-, Indan-1,2-dione , 4-methyl-,2,3-Dihydroindole-4-ol-2-one,5,7-dibromo-3,3-dimethyl-, Benzamide , 3-amino-N-[4,5-dihydro-5-oxo-1-(2,4,6-trichlorop, 2-Acetonyl-9- [3-deoxy-β-d-ribouranosyl] hypoxanthine, DL-Leucine ,N-glycyl-, 3-(2,5-Dimethylanilinomethyl)-5-(3-fluorobenzylidene)-2,4-thia, Benzenepropanoic acid, α-(hydroxyimino)-, Butanoic acid , 4,4'-dithiobis [ 2-amino-,[S-(R*,R*)]- N-Propionyl-D-glucosamine Actinomycin C2, N-(O-Nitrophenylthio)-l-leucine, D-Fructose, diethyl mercaptal, pentaacetate, 1H-Indole, 4-(3-methyl-2-butenyl)-, Spiculesporic acid, 2,5-Piperazinedione, 3,6-bis(2-methylpropyl). The results of anti-fungal activity produced by Aeromonas hydrophila showed that the volatile compounds were highly effective to suppress the growth of Aspergillus flavus. Aeromonas hydrophila produce many important secondary metabolites with high biological activities. Based on the significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases, the purification of compounds produced by Aeromonas hydrophila can be useful. Maximum zone formation against Aspergillus flavus (6.009±0.22). mm, Table 2.

 

Table 2. Antifungal activity of Aeromonas hydrophila metabolite products.

 

The values ( average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude extract and 30 μg/mL of (Amphotericin B; Fluconazol and Miconazole nitrate). In agar well diffusion method the selected medicinal plants were effective against Aeromonas hydrophila, Table 3.

 

 

Figure 1: GC-MS chromatogram of methanolic extract of Aeromonas hydrophila.

 

Table 3. Zone of inhibition (mm) of test different bioactive compounds and standard antibiotics of medicinal plants to Aeromonas hydrophila.

S. No.	Plant	Zone of inhibition (mm)
1.	Zingiber officinale  (Crude)	4. 507±0.20
2.	Nerium olender  (Alkaloids)	5.408±0.24
3.	Ricinus communis  (Alkaloids)	3.081±0.19
4.	Datura stramonium(Alkaloids)	4.073±0.21
5.	Linum usitatissimum (Crude)	4.930±0.23
6.	Anastatica hierochuntica (Crude)	5.004±0.21
7.	Cassia angustifolia   (Crude)	4.731±0.20
8.	Euphorbia lathyrus   (Crude)	5.000±0.20
9.	Rosmarinus oficinalis   (Crude)	4.911±0.21
10.	Mentha viridis  (Crude)	5.391±0.23
11.	Quercus infectoria  (Crude)	5.063±0.22
12.	Citrullus colocynthis  (Crude)	3.930±0.18
13.	Althaea rosea  (Crude)	4.591±0.20
14.	Coriandrum sativum  (Crude)	5.371±0.22
15.	Origanum vulgare  (Crude)	4.969±0.20
16.	Urtica dioica  (Crude)	4.078±0.20
17.	Foeniculum vulgare  (Crude)	2.592±0.16
18.	Ocimum basilicum  (Crude)	4.082±0.20
19.	Punica granatum  (Crude)	5.081±0.22
22.	Control	0.000

Nerium olender (Alkaloids) was very highly active (5.408±0.24) mm against Aeromonas hydrophila. Aeromonas hydrophila was found to be sensitive to all test medicinal plants and mostly comparable to the standard reference antifungal drug Amphotericin B and fluconazole to some extent. Recently, it was demonstrated that volatile organic compounds (VOCs) of bacteria such as terpenoids, phenylpropanoids and fatty acid derivatives can influence the growth of some fungi and, in general, the inter- and intra-organismic communication signals. Because of its structure, it is very toxic to many organisms. When it enters the body of its victim, it travels through the bloodstream to the first available organ. It produces aerolysin cytotoxic enterotoxin that can cause tissue damage.

 

CONCLUSION:

Twenty three bioactive chemical constituents have been identified from methanolic extract of the Aeromonas hydrophila by gas chromatogram mass spectrometry (GC-MS). In vitro antifungal and antibacterial evaluation of secondary metabolite products of Aeromonas hydrophila forms a primary platform for further phytochemical and pharmacological investigation for the development of new potential antimicrobial compounds.

 

ACKNOWLGEMENTS:

I am thankful to Dr.  Omer for helping me through the various analysis stages, and for providing helpful criticism and feedback throughout the writing process.

 

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42.   Kadhim MJ, Mohammed GJ, Hameed IH. In vitro antibacterial, antifungal and phytochemical analysis of methanolic fruit extract of Cassia fistula. Oriental Journal of Chemistry. 2016; 32(2): 10-30.

43.   Altameme HJ, Hameed IH, Idan SA, Hadi MY. Biochemical analysis of Origanum vulgare seeds by fourier-transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Journal of Pharmacognosy and Phytotherapy. 2015; 7(9): 221-237.

44.   Hussein HM. Determination of phytochemical composition and ten elements content (CD, CA, CR, CO, FE, PB, MG, MN, NI AND ZN) of cardaria draba by GC-MS, FT-IR and AAS technique. Int. J Pharm Bio Sci. 2016; 7(3): (B) 1009 –1017.

45.   Hussein HM. Analysis of trace heavy metals and volatile chemical compounds of Lepidium sativum using atomic absorption spectroscopy, gas chromatography-mass spectrometric and fourier-transform infrared spectroscopy. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2016; 7(4): 2529 – 2555.

46.   Hameed IH. A new polymorphic positions discovered in mitochondrial DNA hypervariable region HVIII from central and north-central of Iraq. Mitochondrial DNA. 2016; 27(5): 3250-4.

47.   Jaddoa HH, Hameed IH, Mohammed GJ. Analysis of volatile metabolites released by Staphylococcus aureus using gas chromatography-Mass spectrometry and determination of its antifungal activity. Orient J Chem. 2016; 32(4).

48.   Hameed IH, Salman HD, Mohammed GJ. Evaluation of antifungal and antibacterial activity and analysis of bioactive phytochemical compounds of Cinnamomum zeylanicum (Cinnamon bark) using gas chromatography-mass spectrometry. Orient J Chem. 2016; 32(4): 2107-2116.

49.   Hameed IH, Jebor MA, Ommer AJ, Abdulzahra AI. Haplotype data of mitochondrial DNA coding region encompassing nucleotide positions 11,719–12,184 and evaluate the importance of these positions for forensic genetic purposes in Iraq. Mitochondrial DNA. 2016; 27(2): 1324-1327.

50.   Kadhim MJ, Mohammed GJ, Hussein HM. Analysis of bioactive metabolites from Candida albicans using (GC-MS) and evaluation of antibacterial activity. International Journal of Pharmaceutical and Clinical Research. 2016; 8(7): 655-670.

51.   Mohammad A, Imad H. Autosomal STR: From locus information to next generation sequencing technology. Research Journal of Biotechnology. 2013; 8(10):92-105.

52.   Hameed, I.H., Abdulzahra, A.I., Jebor, M.A., Kqueen, C.Y., Ommer, A.J. Haplotypes and variable position detection in the mitochondrial DNA coding region encompassing nucleotide positions 10,716-11,184. Mitochondrial DNA. 2015; 26(4): 544-549.

53.   Ubaid JM, Hussein HM, Hameed IH. Analysis of bioactive compounds of Tribolium castaneum and evaluation of anti-bacterial activity. International Journal of Pharmaceutical and Clinical Research. 2016; 8(7): 655-670. 

54.   Altaee N, Kadhim MJ, Hameed IH. Detection of volatile compounds produced by Pseudomonas aeruginosa isolated from UTI patients by gas chromatography-mass spectrometry. International Journal of Toxicological and Pharmacological Research. 2017; 8(6): 462-470.

55.   Altaee N, Kadhim MJ, Hameed IH. Characterization of metabolites produced by E. coli and analysis of its chemical compounds using GC-MS. International Journal of Current Pharmaceutical Review and Research. 2017; 7(6); 107-113.

56.   Hussein JH, Ubaid JM, Hameed IH. Gas chromatography – mass spectrum analysis of volatile components of methanolic leaves extract of Cordia myxa. International Journal of Current Pharmaceutical Review and Research. 2017; 7(6): 93-99.

57.   Kadhim MJ, Kaizal AF, Hameed IH. Medicinal plants used for treatment of rheumatoid arthritis: A review. International Journal of Pharmaceutical and Clinical Research. 2017; 8(12): 1685-1694.

58.   Hameed, I.H., Al-Rubaye A.F., Kadhim, M.J. Antimicrobial Activity of Medicinal Plants and Urinary Tract Infections. International Journal of Pharmaceutical and Clinical Research. 2017; 9(1): 44-50.

59.   Kadhim WA, Kadhim, MJ, Hameed, I.H. Antibacterial Activity of Several Plant Extracts Against Proteus Species. International Journal of Pharmaceutical and Clinical Research. 2017; 8(12): 1673-1684

60.   Kadhim MJ. In Vitro antifungal potential of Acinetobacter baumannii and determination of its chemical composition by gas chromatography-mass spectrometry. Der Pharma Chemica, 2016; 8(19): 657-665.

61.   Al-Yaseri A, Kadhim WA, Hameed IH. Detection of volatile compounds emitted by Proteus mirabilis isolated from UTI patients and its anti-fungal potential. Der Pharma Chemica, 2016; 8(19): 671-678.

62.   Ubaid JM, Kadhim MJ, Hameed IH. Study of bioactive methanolic extract of Camponotus fellah using Gas chromatography – mass spectrum. International Journal of Current Pharmaceutical Review and Research. 2017; 8(6); 434-439.

 

 

 

Accepted on 19.09.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(11): 3845-3851.