Isolation and Antimicrobial Screening of Some Phytochemicals from Moringa oleifera Lam

 

Namitha R*, Anjitha A1

Department of Chemistry, RSM SNDP Yogam Arts and Science College, Koyilandy, Kozhikode

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

 

ABSTRACT:

Inhibition of growth of pathogenic gram positive and gram negative micro organisms exposed to two different isolated components of ethanol extract of Moringa oleifera was used as an index for assessment of its antimicrobial activity. Well diffusion method was used against the Staphylococcus aureus (S.a) Escherichia coli (E.c), Candida albicans and Aspergillus flavus at various concentrations. The two components were active towards both bacterial and fungal stains. Response of the bacterial isolates varied with concentration. For both the components the gram negative Escherichia coli responded more markedly than gram positive Staphylococcus aureus. Aspergillus flavus was more active than Candida albicans.

 

KEYWORDS: Moringa oleifera, Escherichia coli, Well diffusion method, TLC, Candida albicans.

 

 


 

INTRODUCTION:

Moringa oleifera” is one of the 14 species of family Moringaceae, native to India, Africa, Arabia, South Asia, South America and the pacific and Caribbean Islands. Because Moringa oleifera has been naturalized in many tropic and subtropics regions worldwide, the plant is referred to number of names such as horseradish tree, drumstick tree, ben oil tree, miracle tree, and “Mothers best friend” 1-4.

 

Moringa oleifera (lam) is considered a complete food as it has an impressive range of medicinal uses with high nutritional value. Its multiple pharmaceutical effects are capitalized as therapeutic remedy for various diseases in traditional medicinal system, the extracts of the leaves are known to have biological properties and these are usually found to vary with the type of solvent used to extract the active components5.

 

Moringa leaves contains phytochemical having potent anticancer and hypotensive activity and are considered full of medicinal properties and used in siddha medicine3. The whole Moringa oleifera plant is used in the treatment of psychosis, eye diseases, fever and as an aphrodisiac, the aqueous extracts of roots and barks were found to be effective in preventing implantation, aqueous extracts of fruits have shown significant anti-inflammatory activity, methanolic extracts of leaves have shown antiulcer activity and ethanol extracts of seeds exhibited antitumour activity6 .In our study we have Identified different components present in ethanol extract of Moringa oleifera using Thin layer Chromatography and separated two phytochemicals by Column chromatography. The components were tested against antimicrobials.

 

EXPERIMENTAL:

Collection of plant materials

Leaves were collected from Moringa oleifera plant and it was ensured that the plant was healthy and uninfected. The leaves were washed under running tap water to eliminate dust and other foreign particles and to cleanse the leaves and dried in the shade for several days.

 

Preparation of leaf extract

Drying process

Fresh leaves of Moringa oleifera were shade dried in chemistry lab until constant weight was obtained.They were kept away from high temperature and direct sunlight to avoid destroying active compounds.

 

Extraction process

The dried leaves were soaked with ethanol for 9 days. The extract was then filtered.     

 

Chromatographic identification and separation

Thin layer chromatography

The appropriate solvent system for isolation of compounds was made after carrying out the TLC analysis of the crude extract in various combination of solvent with increasing polarity.  The solvents were the mixture of petroleum ether and ethyl acetate. The different components were identified by examining the difference in Rf values. The crude extract was dissolved in alcohol and spotted on a TLC plate precoated with silica by using capillary tube.

 

A developing chamber was prepared by placing an amount of the solvent system namely petroleum ether and ethyl acetate. The chamber was then covered with a watch glass to equilibrate the chamber. The TLC plate was carefully placed in the beaker to develop. The spots were visualized in iodine chamber.

 

Column chromatography

Here silica gel is used as stationary phase. Mobile phase or eluent is a mixture of petroleum ether and ethyl acetate. Eluent of 10% and 15% are prepared. The different components can be desorbed and collected separately by adding more solvent at the top and this process is known as elution. The weakly adsorbed component will be eluted more rapidly than the other. The two different components were collected separately. Distillation of the solvent from the different fractions gave the pure component.  

 

Antimicrobial activity

Antibacterial and antifungal activities were studied by Well diffusion method. Different concentrations (25, 50, 75 and 100%) of samples were carefully added into the wells and allowed to diffuse at room temperature for 2 h. The plates were then incubated at 37°C for18–24 h. Gentamycin (10 μg) and Clotrimazole (10 μg) were used as positive controls and the respective solvents as negative control. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone.

 

Antibacterial activities of the components Viz., 1 and 2 were against the Staphylococcus aureus (S.a) and Escherichia coli (E.c). Gentamycin was used as the standard for antibacterial studies. The media for anti bacterial study is Nutrient Agar (NA).

 

Antifungal activities of the components were against the Candida albicans and Aspergillus flavus.  Clotrimazole was used as the standard for antifungal studies. .The media for antifungal study is potato dextrose agar (PDA).

 

RESULTS AND DISCUSSION:

We have discussed the efforts which were put forward for identification of different components present in ethanol extract of Moringa oleifera by using thin layer chromatography and separation of two components by column chromatography. Further the isolated components were screened for antimicrobial activity using Well diffusion method against the Staphylococcus aureus (S.a) Escherichia coli (E.c), Candida albicans and Aspergillus flavus at various concentrations.

 

From the IR spectra of the two components the major difference are recorded in the Table1.

 

TABLE 1.  Results from the IR spectra of the two separated components.

Sl.no.

Component 1

Component 2

1.

Showed a strong and intense band at 1727 cm-1 which is indicative of the carbonyl stretch (C=O) it may be in aldehydes.

Showed a strong  but broad band at 1715cm-1 which is indicative of the carbonyl stretch (C=O) in ketone.

2.

The C-H stretch for the carbon-hydrogen  bond on the aldehyde group is showed by the peak at 2862cm-1

and another at 2927cm-1

The C-H stretch for the carbon- hydrogen bond in alkanes is showed by the peak at 2918cm-1.

3.  

Showed a strong and intense band at 1274 cm-1 which is indicative of the  stretching in (C=S)

No such band near 1274cm-1 (absence of C=S)

4.

Showed a intense band at 739 cm-1 which is indicative of the  stretching in (C-Cl)

No such band near 739cm-1 (absence of C-Cl)

5.

Showed a intense band at 1071cm-1 which is indicative of the  stretching in (C-O-C)

No such band near 1071cm-1 (absence of C-O-C)

 

 

From the above data, it reveals that the two components are different. Component 1 contains an aldehyde group (-CHO) whereas component 2 contains a keto group(C=O).

 

Components separated from alcohol extract of Moringa Oleifera, were screened for antimicrobial activity.




TABLE-2 :Antibacterial activity of the components

 

Inhibition zone  in mm

Concentrations

(100µg/)

(75µg/L)

(50µg/L)

(25µg/L)

Standard

Components

1

2

1

2

1

2

1

2

Gentamcin (10µg)

Staphylococcus aureus

17

15

16

14

14

12

13

10

     12

Escherichia coli

18

18

14

15

10

14

10

11

      13

 

TABLE-3:  Antifungal activity of components

 

Inhibition zone  in mm

Concentrations

(100µg/)

(75µg/L)

(50µg/L)

(25µg/L)

Standard

Components

1

2

1

2

1

2

1

2

Clotrimazole (10 µg)

Candida albicans

18

11

16

11

14

11

12

10

15

Aspergillus flavus

22

18

20

14

18

13

16

15

21

 


From the table 2 and 3 it is clear that both the components will be used as antimicrobial drug in mere future.

 

CONCLUSION:

We have isolated two components of Moringa oleifera by column chromatography. Further the isolated components were screened for antimicrobial activity using Well diffusion method against the Staphylococcus aureus (S.a)  Escherichia coli (E.c), Candida albicans and Aspergillus flavus at various concentration. The two components were active towards both bacterial and fungal stains.

 

REFERENCES:

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2.          Farooq Anwar, Sajid Latif1, Muhammad Ashraf and Anwarul Hassan Gilani . Phytother. Res. 2007; 17-25.

3.           Pinal Patel, Nivedita Patel, Dhara Patel, Sharav Desai, Dhananijay Meshram. International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 .2014 ; Vol 6 ; 144-147.

4.          Mehta J., Shukla A., Bukhariya V., Charly. International Journal of Biomedical and Advance Research 2011

5.          Bhumika Dodiya, Bijal Amin. Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 2015;3(3);.421-425.

6.          Patel Rameshwar, K., Manish, M. P., Nilesh, R. K., Kirit, R.V., R.K. Patel. Int. J. Ph. Sci. Res. 2010; 2(1): 457-463.

 

 

 

 

Received on 26.04.2019         Modified on 02.05.2019

Accepted on 05.05.2019       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2019; 11(2):70-72.

DOI: 10.5958/0975-4385.2019.00013.X