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ORIGINAL ARTICLE
Year : 2013  |  Volume : 4  |  Issue : 1  |  Page : 71-75  

Absence of antibiotic activities of Cenchrus setigerus and cenchrus ciliaris seed extracts in different polar solvents


1 Department of Botany, Laboratory of Tissue Culture and Secondary Metabolites, University of Rajasthan, Jaipur, Rajasthan, India
2 Department of Animal Husbandry, Government of Rajasthan Veterinary Hospital, Pahari (Bharatpur), Rajasthan, India

Date of Web Publication21-Aug-2013

Correspondence Address:
Premlata Singariya
Dr. D. S. Kothari Post Doctoral Fellow, Shri Kalyan Nagar, Kartarpura, Jaipur, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.116756

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   Abstract 

Aim: The aim of present study is to investigate the antibiotic activity of seeds of Cenchrus setigerus and Cenchrus ciliaris extracts, in order to use them as a possible source of new anti-microbial substances against important human pathogens. Materials and Methods: Crude extracts of seeds of both species of Cenchrus grass were evaluated against some important G-ve bacteria. Escherichia coli, Raoultella planticola, Enterobacter aerogenes and one fungus Aspergillus flavus. The dried and powdered seeds were successively extracted with hexane, toluene, isopropyl alcohol, acetone and ethanol using the soxhlet assembly. The antimicrobial activity assay was done by both disc diffusion and serial dilution methods. Results: The results indicate that all the extracts, in different polar solvents did not show any antibacterial activity against R. planticola or any antifungal activity against A. flavus. Conclusion: All extracts in the different polar solvents did not have or had very less antibacterial and antifungal activities.

Keywords: Antibiotic, Aspergillus flavus, Cenchrus, hexane, toluene


How to cite this article:
Singariya P, Kumar P, Mourya KK. Absence of antibiotic activities of Cenchrus setigerus and cenchrus ciliaris seed extracts in different polar solvents. J Pharm Negative Results 2013;4:71-5

How to cite this URL:
Singariya P, Kumar P, Mourya KK. Absence of antibiotic activities of Cenchrus setigerus and cenchrus ciliaris seed extracts in different polar solvents. J Pharm Negative Results [serial online] 2013 [cited 2019 Feb 17];4:71-5. Available from: http://www.pnrjournal.com/text.asp?2013/4/1/71/116756


   Introduction Top


Thar desert occupies diverse habitat and landscape that support distinctive plant species. About 682 plant species have been identified including 107 grasses. [1] Cenchrus setigerus and Cenchrus ciliaris (C 4 grasses) are gaining attention in various field of research, as they are best suited to the present environmental conditions. These grasses are more competitive under the conditions of high temperature, solar radiation and low moisture [2] and are more efficient at gathering CO 2 and utilizing nitrogen from the atmosphere and recycled N in the soil. [3],[4] Cenchrus L. (Poaceae) is highly nutritious grass and considered excellent for pasture in hot, dry areas and is valued for its production of palatable forage and intermittent grazing during droughty periods in the tropics. The grass, fed green, turned into silage, or made into hay is said to increase flow of milk in cattle and impart a sleek and glossy appearance. This grass has excellent soil binding capacity which helps to conserve soil in desert areas. [5] However, Cenchrus is most suitable and highly nutritive grasses for desert environmental conditions, still no antimicrobial work yet have been done on this grass.

E. coli is the culprits for human urinary tract infections. [6] Raoultella planticola has been determined to cause severe pancreatitis in one case. [7] E. aerogenes is a nosocomial and pathogenic bacterium that causes opportunistic infections including most types of infections. Aspergillus species are the most common mold causing severe infections. [8],[9]


   Materials and Methods Top


Experimental design

Crude extracts of seeds of Cenchrus ciliaris (CAZRI-358) and Cenchrus setigerus (CAZRI-76) were prepared with a series of non polar to polar solvents by hot extraction method [10] in soxhlet assembly. Different extracts were then screened for antimicrobial activity by DDA [11] against a few medically important bacteria and fungi. The fraction showing best activity was then used for determining of Minimum inhibitory concentration (MIC) by tube dilution method [12] and minimum bactericidal/fungicidal concentration (MBC/MFC).

Collection of plant material

Seeds of C. ciliaris and C. setigerus were collected in the month of August from the CAZRI, Jodhpur, Rajasthan. Selected plant seeds were separately shade dried and powdered with the help of grinder. [13] Fine powder of each sample was stored in clean container to be used for Soxhlet extraction following the method of Subramanian and Nagarjan [14] in different polar solvents selected.

Extraction procedure

Seeds were sequentially extracted with different solvents according to their increasing polarity (hexane <toluene <isopropyl alcohol <acetone <ethanol) by using the Soxhlet apparatus for 18 hours at a temperature not exceeding the boiling point of the respective solvent. The obtained extracts were filtered by using Whatman No. 1 filter paper and then concentrated at 40°C by using an evaporator and stored in amber color bottle for subsequent use in the further antimicrobial and phytochemical analysis. [15]



Drugs and chemicals used

Drugs

Gentamycin (for bacteria) and ketoconazole (for fungi)

Chemicals

Hexane, toluene, isopropyl alcohol, acetone and ethanol, Nutrient Agar (NA medium for bacteria), Sabouraud Dextrose Agar (SDA medium for fungi).

Micro-organisms

The organisms used in this study were namely [Table 1]. Test pathogenic microorganisms were procured from Microbial Type Culture Collection, IMTECH, Chandigarh, India. The reference strains of bacteria were maintained on nutrient agar slants, sub cultured regularly (every 30 days) and stored at 4°C as well as at -80°C by preparing suspensions in 10% glycerol.
Table 1: Name of the tested pathogens (bacteria and fungi)

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Screening for antimicrobial activity

Bacterial colonies were maintained on NA medium and fungi were maintained on SDA medium. DDA [16] was performed for screening. NA and SDA base plates were seeded with the bacterial and fungal inoculum, respectively (inoculum size 1 × 10 8 CFU/ml for bacteria and 1 × 10 7 cell/ml for fungi). DDA was performed for screening by standard method. [12] Bacterial growths were seen after a minimum of 18 h and occasionally until 24 h. [17] Activity index for each extract was calculated.



Serial dilution method

MICs are considered as the "gold standard" for determining the susceptibility of the organisms to antimicrobials. [18] MIC of antibiotics was evaluated (thrice) using standard micro broth dilution method against inoculum  Escherichia More Details coli organisms. [19] Bacterial and fungal suspensions were used as negative control, while broth containing standard drug was used as positive control.

Determination of minimum bactericidal/fungicidal concentration (MBC/MFC)

Equal volume of the various concentration of each extract and nutrient broth mixed in micro-tubes to make up 0.5 ml of solution. 0.5ml of McFarland standard of the organism suspension was added to each tube. [20],[21] The tubes were incubated aerobically at 37°C for 24 h for bacteria and 28°C for 48 h for fungi. Two control tubes were maintained for each test batch. These include tube-containing extract without inoculum and the tube containing the growth medium and inoculum. The MBC was determined by sub culturing the test dilution on MHA and further incubated for 24 h. The highest dilution that yielded no single bacterial colony was taken as the MBC. [22] MBC was calculated for some of the extracts showed high antimicrobial activity against highly sensitive organisms.

Total activity (TA) determination

Total activity is the volume at which the test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in ml/g. [23]




   Results Top


Antimicrobial activity

Antimicrobial activity [assessed in terms of zone of inhibition (ZOI) in mm* and activity index] of the seeds of C. ciliaris and C. setigerus extracts in different polar solvents, tested against selected microorganisms were recorded [Table 2]. In the present study total ten extracts of selected plant were tested for their bioactivity, among which all these extracts showed insignificant antimicrobial potential against test microbes. However, all these extracts showed no activity or very less activity against E. coli, R. planticola, E. aerogens and antifungal activity against A. flavus at tested concentration. Highest antimicrobial activities were recorded for C. ciliaris in hexane (ZOI-9.17 ± 0.24 and AI-0.459) and for C. setigerus in acetone (ZOI-8.50 ± 0.64 and AI-0.425) against E. aerogens and E. coli, respectively.
Table 2: Inhibition zone (mm)* and activity index of seeds of Cenchrus grass in different polar solvents against tested pathogens

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MIC and MBC/MFC

MIC and MBC/MFC values [Table 3] were evaluated for those plant extracts, which were showing activity in disc diffusion assay. The range of MIC and MBC/MFC of extracts recorded was 3.75-15 mg/ml. In the present investigation lowest MIC value 3.75 mg/ml was recorded for C. ciliaris (against E. aerogens) in hexane extract.
Table 3: Minimum inhibitory concentration and (MBC/MFC) of seeds of Cenchrus in different solvents against tested pathogens

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Total activity

Total activity indicates the volume at which extract can be diluted with still having ability to kill microorganism. Seed extracts of C. ciliaris in hexane showed high values of TA 7.73 ml against E. aerogenes [Table 4].
Table 4: Total activity of seeds of Cenchrus in different solvents

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Preliminary phyto-profiling

The preliminary phyto-profiling for the seeds of Cenchrus extracts were carried out according to Farnsworth [24] wherein the consistency was found to be sticky in the hexane and toluene extracts whereas all other extracts were found to be non-sticky. The yield mg/g (w/w) of the extracts was also analyzed wherein the highest yield was found in acetone extract of C. ciliaris (685 ± 11.78 mg/10 g ± S.D.) followed by ethanolic and iso propyl alcohol extract of C. setigerus (346 ± 13.46 mg/10 g ± S.D.) and (346 ± 10.13 mg/10 g ± S.D.) respectively [Table 5].
Table 5: Preliminary phyto-profile for seeds of Cenchrus grass in different polar solvent

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   Conclusion Top


Cenchrus ciliaris and C. setigerus extracts in different polar solvents at a tested concentration were not showing ZOI against the selected pathogens [Table 1] although, water, chloroform and acetic acid extract of seeds of this grass show good activity against these pathogens supported by different researchers. [25],[26],[27] But, in this study these extracts lack of antimicrobial activity well be useful to avoid any study repeated in this direction in the future.

Disclaimer

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   References Top

1.Khan TI, Frost S. Floral biodiversity: A question of survival in the Indian Thar Desert, The Environmentlist 2001;21:231-6.  Back to cited text no. 1
    
2.Agrawal P. Ecophysiological and Biochemical studies Related to drought adaptation in grasses of Indian Desert. Ph.D. Thesis, J. N. Vyas University. 2007.  Back to cited text no. 2
    
3.Bessman SP. Ammonia Metabolism in Animals: Symposium on Inorganic Nitrogen Metabolism. In: W.D. Mcelory and B. Glass (eds.) The Johns Hopkins Press. 1956  Back to cited text no. 3
    
4.Singariya P. Effect of Sub-Optimal Environment and PGR's on Metabolic Pattern of Certain Species of Cenchrus. Ph.D Thesis, J. N. Vyas University. 2009.  Back to cited text no. 4
    
5.Sinha RK, Bhatia S, Vishnoi R. Desertification control and rangeland management in the Thar desert of India. In: Rala Report no. 1996;200:115-23.  Back to cited text no. 5
    
6.Venier AG, Talon D, Party I, Mercier-Girard D, Bertrandx. Patient and bacterial determinants involved in symptomatic urinary tract infections caused by E. coli with and without bacteraemia. Clin Microbial Infect 2007;13:205-8.  Back to cited text no. 6
    
7.Alves MS, Riley LW, Moreira BM. A case of severe pancreatitis complicated by Raoultella planticola infection. J Med Microbiol 2007;56:696-8.  Back to cited text no. 7
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10.Harborne JB. Phytochemical Methods, London, New York: Chapman and Hall; 1984. p. 5-6.  Back to cited text no. 10
    
11.Singariya P, Kumar P, Mourya KK. Antimicrobial Activity of Fruit coat (Calyx) of Withania somnifera against some Multi Drug Resistant Microbes. Int J Biolog Pharm Res 2012;3:252-8.  Back to cited text no. 11
    
12.Singariya P, Kumar P, Mourya KK. Evaluation of Antibacterial Activity and Preliminary Phytochemical Studies on the Stem of Cenchrus ciliaris and Cenchrus setigerus. Asian J Pharm Clin Res 2012;5:163-7.  Back to cited text no. 12
    
13.Hussain A, Zaman MK, Ramteke AM. Antibacterial Activity of Trunk Bark of Alstonia scholaris, Asian J of Pharm and Clin Res 2010;3:46-7.  Back to cited text no. 13
    
14.Subramanian SS, Nagarjan S. Flavonoids of the seeds of Crotolaria retusa and Crotolaria striata. Curr Sci 1969;38:65.  Back to cited text no. 14
    
15.Dey AC. Indian Medicinal Plants Used in Ayurvedic Preparations. In: Singh B, Singh MP, Editors. Dehradhun: 1980. p. 202.  Back to cited text no. 15
    
16.Andrews JM. BSAC standardized disc susceptibility testing method. J Antimicrob Chemother 2001;4:43-57.  Back to cited text no. 16
    
17.Brindha D, Arthi D. Antimicrobial activity of white and pink Nelumbo nucifera gaertn flowers. JPHRC 2010;2:147-55.  Back to cited text no. 17
    
18.Moideen MMJ, Raffick MM. Antibacterial, Antifungal Activity and Phytochemical Analysis of Sanseviveria roxburghiana Root. Int J Phytopharmacol 2012;3:21-6.  Back to cited text no. 18
    
19.Liu CP, Tsai WJ, Lin YL, Chen CF, Kuo YC. The extracts from Nelumbo nucifera suppress cell cycle progression, cytokine genes expression, and cell proliferation in human peripheral blood mononuclear cells. Life Sci 2004;75:699-716.  Back to cited text no. 19
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20.Singariya P, Mourya KK, Kumar P. Comparative Microcidal Activity of Withania somnifera and Cenchrus setigerus against the Pathogenic Micro-organisms. Int J Pharmacy Pharm Sci 2011;3:511-5.  Back to cited text no. 20
    
21.Joshi B, Sah GP, Basnet BB, Bhatt MR, Sharma D, Subedi K, et al. Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica (Neem). J Microbiol Antimicrobials 2011;3:1-7.  Back to cited text no. 21
    
22.Cheesbrough M. Medicinal laboratory manual for tropical countries, ELBS reprinted Edition. 1993.  Back to cited text no. 22
    
23.Singariya P, Mourya KK, Kumar P. Bio Activity of crude extracts of Leaves of Cenchrus Grass in different polar solvents against some pathogenic microbes. Int J Pharm Sci Rev Res 2011;11:124-9.  Back to cited text no. 23
    
24.Farnsworth NR. Biology and Phytochemical screening of plants. Pharm Sci 1966;55:225-76.  Back to cited text no. 24
    
25.Singariya P, Mourya KK, Kumar P. Preliminary Phyto-profile and Pharmacological Evaluation of some Extracts of Cenchrus grass against Selected Pathogens. J Pharm Sci Res 2011;3:1387-93.  Back to cited text no. 25
    
26.Singariya P, Mourya KK, Kumar P. Antimicrobial Activity of the Crude Extracts of Withania somnifera and Cenchrus setigerus In-vitro. Pharmacog J 2011;3:60-5.  Back to cited text no. 26
    
27.Singariya P, Kumar P, Mourya KK. Phyto-chemical Screening and Antimicrobial Activities of Dhaman grass and Indian Ginseng. J Pharmacy Res 2012;5:135-9.  Back to cited text no. 27
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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