In Vitro Antimicrobial Activity of Fruit Extracts of Lagenaria Siceraria (Mol.)

 

Dahikar S. B.

Department of Microbiology, Sanjivani Arts, Commerce and Science College, Kopargaon-423603, INDIA

*Corresponding Author E-mail: sbdahikar10@gmail.com

 

ABSTRACT:

In the present study  Petroleum ether, chloroform, ethanol and aqueous  extract of fruit of Lagenaria siceraria were prepared  and antimicrobial  activity were studied by agar well diffusion method against pathogenic bacteria and fungus such as Escherichia coli, Staphylococcus griseus, Bacillus macerans, Proteus mirabilis, Aspergillus niger, Aspergillus flavus, Candida albicans. Result shows antimicrobial activity of chloroform extract and absolute alcohol extract against Bacillus macerans, Escherichia coli and Proteus mirabilis. From the investigation, the results confirm the presence of therapeutically potent compound in absolute alcohol extract of bottle gourd. The present study suggests that the fruit extract of Lageneria siceraria can be used in treating diseases caused by the test organisms.

 

KEYWORDS: Lagenaria siceraria, antimicrobial activity, microbial pathogens.

 

 


INTRODUCTION:

Bottle gourd is one of the excellent fruits gifted by nature to human beings having composition of all the essential constituents that are required for good health of human life [1]. Lagenaria siceraria (Cucurbitaceae) popularly known as bottle gourd, lauki or ghiya, is a climbing plant, which bears hard-shelled and bottle-shaped gourds as fruits. It forms an excellent diet being rich in vitamins, iron and minerals. The fruit is reported to contain the triterepenoide cucurbitacins B, D, G, H, two sterols viz., fucosterol and campesterol, aerpene byonolic acid (an allergic compound), flavones-C glycosides (a ribosome inactivating protein) and lagenin. Extract of the ghiya seeds show antibiotic activity [2].

 

About 80% of individuals from developed countries use traditional medicine, which involves compounds derived from medicinal plants [3].There are different species of this plant found worldwide and both mature and immature fruits are employed in medicinal practices [4]. According to WHO (World health organization) medicinal plant would be the best source to obtain a variety of drug [5]. A Triterpene,   Bryonolic acid an anti allergic compound was reported from callus culture of Lagenaria siceraria roots [6]. Two sterols were isolated from petroleum ether fraction of dried fruit pulp of Lagenaria siceraria namely fucosterol and campesterol [7]. The HPLC analysis of the extract of flowering plant of Lagenaria siceraria shows presence of flavones C–glycosides [8]. The work on different parts of this plant were reported in so many research papers but there is very few work on fruit so that the attempt of this work was evaluation of antimicrobial activity of sun dried fruit powder extract with different solvent against pathogenic bacteria and fungus.

MATERIAL AND METHODS:

Collection and processing of samples:

Fresh and green bottle guard (Lagenaria siceraria) was collected from the local market. The seeds were removed. Remnant was sun dried until dryness, grinded into fine powder and stored in airtight container at 40C for further analysis.

 

Preparation of extract:

A 30gm of bottle guard powder was taken in Soxhlate assembly and extracted successively in petroleum ether, chloroform absolute alcohol and distilled water such that the polarity of solvents would leach out compounds soluble in the particular solvent. Each fraction was concentrated in water bath at its specific temperature. These extracts were stored at 40C and used as sample for further analysis

 

Microbial Pathogens:

The standard pathogenic bacterial and fungal cultures used in the study are listed in Table 1. The typed cultures of bacteria and fungi were sub-cultured on nutrient agar (Qualigens) and potato dextrose agar (HIMEDIA M403) slants respectively and stored at 40C until required for study. The inoculums size of the bacterial culture was standardized according to the National Committee for Clinical Laboratory Standards [9] guideline. The pathogenic bacteria was inoculated into sterile Nutrient broth and incubated at 37°C for 3hr until the culture attained a turbidity of 0.5 McFarland units.

 

Table 1:  Microorganisms used in the study

S. No.

Name of organism

Code Number

1

Staphylococcus griseus

NCIM 2621

2

Bacillus macerans

NCM 5037

3

Proteus mirabilis

NCIM 2300

4

Aspergillus niger

ARIFCC1456

5

Aspergillus flavus

ARIFCC 1371

6

Candida albicans

MTCC 227

7

Escherichia coli

NCIM 2739

 

Antimicrobial activity using agar well diffusion method:

A 0.1ml of microbial activated suspension was inoculated on Nutrient agar petri plate. The inoculum was spread by glass spreader until totally absorbed in agar layer for the development of uniform microbial growth. A 8mm diameter wells were punched into agar with sterilized cork borer and wells so obtained was filled with 50 µl of extract (experimental), 50µl solvent (Negative control) and 10 µl of drug Campicillin 500 (Positive control). The plates were incubated for 24 hours at 370C. The antimicrobial activity was evaluated by measuring the diameter of the inhibition zone formed around the well. The experiments were performed in triplicate and the mean diameter of the zone of inhibition was calculated.

 

Bioactive compounds isolation:

After screening the antimicrobial activity of each solvent extract, the maximum activity showing fraction (absolute alcohol fraction of bottle gourd) was selected for further process. Bioactive components were isolated by Thin Layer Chromatography [10].

 

Standardization of solvent system for TLC:

Various combinations of solvents like methanol, toluene, distilled water, ethyl acetate, and diethyl ether were checked for the 2 proper separation of sample. Ten fold diluted sample (absolute alcohol fraction of bottle gourd) in absolute alcohol was used. Three distinct spots of 20µl, 30µl, 50µl were loaded on the silica gel plate and TLC performed in various combinations of solvent system. Bands were developed in iodine chamber.

 

Quantification of distinct spot:  

After the proper separation of sample the spots were quantified by using Glass TLC plates coated with silica gel G60 (20cm × 20cm) were used as stationery phase. The maximum activity showing sample of the active components (absolute alcohol fraction of bottle gourd) mixed in equal volume of same solvent was applied on stationary phase. The quantity of the sample used was 750μl for each plate. A 30ml of mobile phase used was Methanol: Ethyl acetate in ratio of 1:1 v/v. All reagents and solvents used for TLC were of analytical grade. After running TLC in TLC chamber saturated with mobile phase the bands were visualized in UV transilluminator and marked with help of cutter [11].

 

Calculation of Retardation factor:

Retardation factor is calculated by using the formula:

 

RF =

Distance moved by sample from origin    

Distance moved by solvent front from origin

 

Purity determination of spots by Spectrophotometer analysis:  

Each band so obtained after TLC was checked for purity. Each band was analyzed by scanning in 200nm-600nm range using double beam spectrophotometer 2202 to check the purity of sample.

 

Sample preparation for Bioautography:

Each marked bands were scratched from the silica gel plate and collected in the vial separately and mixed properly in known amount of absolute alcohol for dissolution of the separated band. The mixture was centrifuged at 5000 rpm for 5 minutes. Two washings of absolute alcohol were given to silica gel pellet for complete extraction of band. The supernatant’s so obtained were concentrated at room temperature or in hot air oven at 900C and used as sample for bioautography.

 

Bioautography:

The antibacterial activity of each band extract was rechecked on Bacillus macerans NCIM 5037 and Proteus mirabilis NCIM 2300 by disc diffusion method. A 15μl band extract so obtained was applied on Whatman filter paper (No.40) disc along with control disc of solvent (absolute alcohol) and positive control disc of antibiotic (Campicillin).

 

RESULTS AND DISCUSSION:  

The data pertaining to the antibacterial potential of the plant extracts are presented in Tables 2 and 3. Table 2 showed antimicrobial activity of different solvent extracts of Bottle gourd at 20μl. The absolute alcohol extract showed dominant antimicrobial activity against E. coli, B. macerans, P. mirabilis while chloroform extract showed antimicrobial activity against B. macerans, P. mirabilis and aqueous extract showed antimicrobial activity against E. coli.  Similarly, Egwaikhide et al., (2010) reported the antimicrobial assay of Lagenaria siceraria seeds extract was antibacterial against both gram positive and gram negative bacteria and against fungal organisms [12]. This antibacterial potency may be due to the presence of many potent compounds such as flavonoids, terpenes, phenolics and alkaloids etc.

 


 

 

 

Table 2: Antimicrobial activity of soxhlate fractionated extract in different solvent

Test organism/ Extracts

E. coli McMB301

S. griseus NCIM2621

B. macerans NCIM 5037

P. mirabilis NCIM 2300

A. flavus ARIFCC 1248

A. niger ARIFCC 1456

C. albicans MTCC227

Petroleum ether

-

-

-

-

-

-

-

Chloroform

-

-

+

+

-

-

-

Ethanol

+

-

+

+

-

-

-

Aqueous

+

-

-

-

-

-

-

 ‘+’ sign indicates presence of antibacterial activity.   ‘-’   sign indicates absence of antibacterial activity.

 

 

 

 


From the result, it was observed that absolute alcohol fraction was more active hence it was further tested with comparison of known antibiotic Campicillin 500 (Table 3).

 

Table 3: Antimicrobial activity of soxhlate fractionated extract in Absolute alcohol

Test organism

Zone of inhibition (cm) of Absolute alcohol  Soxhlate extract

Zone of inhibition (cm) of antibiotic (Campicillin 500)

B. macerans NCIM 5037

1.6

1.7

E. coli

1.2

1.5

P. mirabilis NCIM 2300

1.4

 

1.7

 

 

From the Table No. 3, the maximum activity of absolute alcohol fraction of bottle gourd was observed against B. macerans NCIM 5037. The traditional healers use primarily water as the solvent.

 

Table 4:  RF value obtained from TLC

Band Number

Retardation factor

Compound expected

1

0.93

Tannins

2

0.16

Glutamic acid

3

0.38

Cucur B

4

0.56

Saponin

5

0.50

Saponin

6

0.42

Flavonoids

 

 

 

From TLC six different spots were obtained having RF value shown in table 4. Rf value of band 1 is 0.93 which is similar to Tannins as reported by [13], band 2 have RF value of 0.16 which is similar to glutamic acid present in bottle gourd Rf value of band 3 is 0.38 which is very close to cucur B (stander value), band 4 and 5 RF value is very close to different types of saponin reported by Yamunadevi et al., (2012) and band 6 value is 0.42 which is resembles with flavonoids reported by [14]. These compounds are reported in bottle gourd so from TLC we can conclude that the antimicrobial activity found may be due to above mentioned components.

 

The results obtained indicate the existence of antimicrobial compounds in the absolute alcohol extracts and showed a good correlation between the reported uses of the plant in Medicine against different diseases and the experimental data of such extracts toward the most common pathogens.

 

CONCLUSION:

The result of antimicrobial experiment shows the presence of therapeutically potent compound in ethanol extract of fruit Lagenaria siceraria. The study supports the folkloric uses of plant and clearly proves that plant soxhlate extracts have great potential as antimicrobial compounds against pathogenic and nonpathogenic organism and it can be used as new drug for therapy of infectious disease caused by microorganism.

 

REFERENCES:

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2.       Parle M, Kaur S.(2011). Is bottle is a natural guard?? International Research Journal of Pharmacy. 2(6):13-17.

3.       Kiritikar KR and Basu BD. (1987). Indian medicinal plants. 2nd edn. International Book Distribution Vol. III. Dehradun.1664–1666.

4.       JeffreyC, (1978). Cucurbitaceae. Flora of Zambesiacca, 4:437-440.

5.       Santos PRV,Oliveira ACX and Tomassini TCB. Controle microbiogico de produtos fitoterapicos, Rev.Farm.Bioquim, 1995; 31:35-38.

6.       Tabata M, Taluka S, Shimakura J. Ito M.(1993). Production of an antiallergic triterpene bryonolic acid by plant tissue cultures. J. Nat. Products. 56 (2):165-175.

7.       Shirwaikar A, Sreenivasan KK.(1996). Chemical investigation and antihepatotoxic activity of fruit of Lagenaria siceraria. Indian Journal of Pharma. Science, 58:197-202.

8.       Baranoswka KM,Cisowski W.(1994). HPLC determination of Flavone C glycocides in some species of cucurbitaceae family. J. Chromatogram A, 675:240-243.

9.       Stahl E. (1969).Thin Layer Chromatography, Springer-Verlag, New York.

10.     Egwaikhide PA, Bulus T, and Emua SA. (2010). Antimicrobial activities and phytochemical screening of extracts of the fever tree, Eucalyptus globulus. Electronic Journal of Environmental, Agricultural and Food Chemistry, 9(5): 940-945.

11.     Chougale AD, Padul MV, Arfeen MS, Kakad SL. (2009). Antibacterial activity directed fractionation of Woodfordia fruticosa kurz. Leaves. Journal of Medicinal Plants,8(31):75-81

12.     NCCLS, (2002). (National Committee for Clinical Laboratory Standards), Performance Standards for antimicrobial susceptibility testing. 8th Informational Supplement. M100 S12. National Committee for Clinical Laboratory Standards, Villanova, Pa.

13.     Yamunadevi M, Weselye G, Johnson M. (2012). Chromatographic finger print studies on saponins of  Aerva  lanata  (L.) Juss. Exschultes by using  HPTLC,  International Journal of Current Pharmaceutical Research.

14.     Gordana S, Cetkovic, Sonja M. Dilas, Jasna M. (2003). Canadanovic-Brunet and Vesna T. Tumbas T.Thin layer chromatography analysis and scavenging activity of marigold (Calendula officinalis L.) extracts. Original scientific paper, APTEFF; 34:1–148.

 

 

 

 

 

Received on 22.03.2018       Modified on 08.04.2018

Accepted on 13.04.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(2): 183-186.

DOI: 10.5958/0975-4385.2018.00029.8