Assessment of Phytochemical Screening, Antioxidant and Antibacterial Potential of the Methanolic Extract of Ricinus communis l.

Nidhi Rao¹, Sandhya Mittal¹*, Sudhanshu¹, and Ekta Menghani²

¹Suresh Gyan Vihar University, Jaipur

²Mahatma Gandhi Institute of Applied Sciences, JECRC Campus, Jaipur-22. India

*Corresponding Author E-mail: nidhiscorpian03@gmail.com

ABSTRACT:

The medicinal plants represent a massive reservoir of probable microbial compounds that could be useful as a substitute to synthetic microbicides as well as are being used to develop drugs. In the present study, the powder extracts of Ricinus communis L. (Euphorbiaceae) were tested for their antioxidant activity by radical scavenging activity using 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) method and antibacterial acitivity against different bacteria i.e. Shigella flexneri, Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus vulgaris, Enterobacter aerogenes, Aspergillus niger, Candida albicans, Trichophyton rubrum by disc diffusion method. Also, Phytochemical screening of methanolic extracts of Ricinus communis have been screened. Further, the antioxidant activity shows moderate to potent antioxidant activity, with the ED50 value i.e. 0.838 μg/ml. When screening for antimicrobial activity, the results were expressed in terms of the diameter of the inhibition zone: The maximum efficacy of methanolic extract was showed against Enterobacter and Aspergillus niger.

KEY WORDS: Antioxidant activity, Antimicrobial activity, DPPH Scavenging, Ricinus communis.

INTRODUCTION:

Natural history has been a source of remedial agents ever since times immemorial. Though, the harmonizing components give the plant as entire a safety as well as efficiency much finer to that of its isolated in addition to pure active components¹. There are numerous reports on the phytochemical screening, antioxidant and antimicrobial activity of different herbal extracts in different regions of the world^2-5. As of the side effects and the resistance that pathogenic microorganisms build against antibiotics, in recent times much attention has been compensated to extracts and geologically active compounds isolated from plant species which are used in herbal medicine⁶. Roughly 20% of the plants found in the world have been submit to pharmacological or biological test, furthermore a considerable number of new antibiotics introduce on the market are obtained from natural or else semi-synthetic resources⁷.

The fourth largest family of the angiosperms i.e. Euphorbiaceae, is comprising over 300 genera in addition to about 7500 species disseminated widely in tropical Africa⁸. The euphorbiaceae plants are shrubs, trees, herbs or else hardly ever lianas⁹. The family provides food¹⁰ as well as diverse medicinal properties used in ethnobotany^11-14. For example, ricin contained in Ricinus communis is a well known toxic compound that elicits aggressive purgative accomplishment in man¹⁵. The Castor oil plant, Ricinus communis, is a species of flowering plant in the spurge family, Euphorbiaceae. It belongs to a monotypic genus, Ricinus, and subtribe, Ricininae. Its seed is the castor bean which, despite its name, is not a true bean. Castor is indigenous to the southeastern Mediterranean Basin, Eastern Africa, and India, but is widespread throughout tropical regions (and widely grown elsewhere as an ornamental plant). Castor seed is the source of castor oil, which has a wide variety of uses. The seeds contain between 40% and 60% oil that is rich in triglycerides, mainly ricinolein. The seed contains ricin, a toxin, which is also present in lower concentrations throughout the plant.

Thus current study was worn to calculate the antioxidant potential of methanolic extract of Ricinus communis. Plant extracts were tested for different free radical scavenging activities including the 1,1-diphenyl 2-picryl hydrazyl (DPPH) and their total antioxidant capacity. Also in this study, the drugs have been used owing to the various bacterial infections. They have been screened for their antibacterial activity against Shigella flexneri, Staphylococcus aureus, Klebsiella pneumonia, Proteus vulgaris, Enterobacter aerogenes, Chryseobacteriu gleum, Bacillus subtilis and antifungal activity against Candida albicans, Aspergillus niger, Aspergillus fumigates, Aspergillus flavus. The crude drug was the Ricinus communis used as antimicrobial drug. The antimicrobial activity of this plant extract was compared with standard antibacterial drug Tetracycline.

MATERIALS AND METHODS:

Collection:

Authentic samples: Various market samples of Ricinus communis L. were procured from Chunnilal Attar Ayurvedic Store, Ghat Gate, Jaipur in the month of March, 2010.

Identification:

All the samples were authenticated and were given identification number. The identification was as follows:

These samples were authenticated and submitted in Ethnomedicinal Herbarium, Centre of Excellence funded by DST, MGiaS, Jaipur (Rajasthan).

Processing of plant materials:

During the course of the study each sample was screened for its foreign matter and milled, before use.

Experimental details:

Present studies were performed on Ricinus communis L. for the following studies-.

1. Phytochemical test of plant extract

2. Antioxidant Potentials of Methanolic extract of plant

3. Antimicrobial activity

1. PHYTOCHEMICAL SCREENING

Phytochemical screening was performed using standard procedure:

TEST FOR REDUCING SUGARS (FEHLINGS TEST)

The aqueous ethanol extract (0.5gm in 5 ml of water) was added to boiling fehling’s solution (A and B) in a test tube. The solution was observed for a colour reaction.

TEST FOR TERPENOIDES (SALKOWSKI TEST)

To 0.5 gm each of the extract was added to 2ml of chloroform. Concentrated sulphuric acid (3ml) was carefully added to form a layer. Reddish brown coloration of the interface indicates the presence of terpenoides.

TEST FOR FLAVONOIDES

4ml of extract solution was treated with 1.5ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated Hydrochloride acid was added and red colour was observed for flavonoids and orange color for flavons.

TEST FOR TANNINS

About 0.5 g of the extract was boiled in 10ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration.

TEST FOR SAPONINS

To 0.5 g of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigorously. And observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.

TEST FOR ALKALOIDS

Alkaloids solutions produce white yellowish precipitate when a few drops of Mayer’s reagents are added. Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s regent.

The alcoholic extract was heated on a boiling water bath with 2% hydrochloric acid. After cooling, the mixture was filtered and treated with a few drops of mayer’s reagent. The sample was then observed for the turbidity or yellow precipitation.

2. ANTIOXIDANT ACTIVITY

Preparation of test extracts

All the test plant sample and their adulterants were milled and refluxed in ethanol for 36 h, filtered, concentrated to dryness in vacuo. A portion of ethanolic extract was further successively extracted in pet. ether, benzene, chloroform, alcohol and water, concentrated and stored at minimum temperature, until used.

Preparation of DPPH

DPPH (1, 1'-diphenyl-2-picrylhydrazl, C₁₈H₁₂N₅O₆; Hi media) 0.8 mg was dissolved in 10 ml methanol to obtain a concentration of 0.08 mg/ml for antioxidative (qualitative and quantitative) assay.

Qualitative assay

Each successive extract (10 mg) was dissolved in 10 ml of its suitable solvent to get a concentration of 1 mg/ml and from this, 0.25µl was taken with the help of micropipette, applied on silica gel G coated plates. These circular spots were sprayed with DPPH solution, allowed to stand for 30 min. When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced, and the changes in colour (from deep- violet to light- yellow on white) were recorded at 517 nm on a UV spectrophotometer (Varian Cary PCB 150, Water Peltier System).

Quantitative assay

A concentration of 1 mg/ml of ethanolic extract of each test sample was prepared to obtain different concentrations (10²µg to 10^-3 µg/ ml).

Each diluted solution (2.5 ml each) was mixed with DPPH (2.5ml). The samples were kept in the dark for 15 min at room temperature and then the decrease in absorption was measured. Absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured. The UV absorbance was recorded at 517 nm. The experiment was done in triplicate and the average absorption was noted for each concentration. Data were processed using EXCEL and concentration that cause 50% reduction in absorbance (RC₅₀) was calculated. The same procedure was also followed for the standards- quercetin and ascorbic acid.

3. ANTIMICROBIAL ACTIVITY

Sources of test organisms

Bacteria-Pure culture of all test organisms, namely Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Shigella flexneri, Proteus vulgaris, Enterobactor aerogenes and fungi Candida albicans, Aspergillus niger, Trichophyton rubrum were obtained through the courtesy of Mahatma Gandhi Institute of applied Sciences (MGiaS), Jaipur, which were maintained on Nutrient broth media. Culture of test microbes: For the cultivation of bacteria, Nutrient Agar Medium (NAM) was prepared by using 20 g Agar, 5 g Peptone, 3 g beef extract and 3 g NaCl in 1 L distilled water and sterilized at 15 lbs pressure and 121°C temperature for 25-30 min. Agar test plates were prepared pouring approximately 15 ml of NAM into the Petri dishes (10 mm) under aseptic conditions. A saline solution was prepared (by mixing 0.8% NaCl) in distilled water, followed by autoclaving and the bacterial\ cultures were maintained on this medium by regular sub-culturing and incubation at 37°C for 24-48 h. To prepare the test plates, in bacteria, 10-15 ml of the respective medium was poured into the Petri plates and used for screening. For assessing the bactericidal efficacy, a fresh suspension of the test bacteria was prepared in saline solution from a freshly grown Agar slant.

Preparation of test extracts

Crushed powder (50 g) of all the species were successively soxhlet extracted with Methanol. Later, the homogenates was filtered and the residue was re-extracted twice for complete exhaustion, the extract was pooled. The filtrate was concentrated to dryness in vitro and re dissolved in respective solvents, out of which 80mg/10disc i.e. 8mg/disc concentration were stored at 4°C in a refrigerator, until screened for antibacterial activity.

Bactericidal assay

For both, bactericidal in vitro Disc diffusion method was adopted (Gould and Bowie, 1952), because of reproducibility and precision. The different test organisms were proceeded separately using a sterile swab over previously sterilized culture medium plates and the zone of inhibition were measured around sterilized dried discs of Whatman No.1 paper(6 mm in diameter), which were containing 8 mg of the text extracts, its control (of the respective solvent) and tetracycline as reference drugs(standard disk) separately. Such treated discs were air-dried at room temperature to remove any residual solvent, which might interfere with the determination, sterilized and inoculated. These plates were initially placed at low temperature for 1 h so as to allow the maximum diffusion of the compounds from the test disc into the agar plate and later, incubated at 37°C for 24 h in case of bacteria, after which the zones of inhibition could be easily observed. Five replicates of each test extract were examined and the mean values were then referred.

The inhibition zone (IZ) in each case were recorded and the activity index (AI) was calculated as compared with those of their respective standard reference drugs (AI = Inhibition Zone of test sample / Inhibition zone of standard).

RESULTS AND DISCUSSION:

1. Phytochemical screening:

The phytochemical screening of Ricinus communis as given in Table 1 shows the occurrence of saponins whereas it shows the absence of tannin and flavonoides respectively. The screening of the Ricinus communis make only a a small amount of differences in the constituent of the toughened plants. The drug shows the confirmation of strong antioxidant activity in more or in a less important amount. The existence of these compounds in this plant is credible to be scrupulous for the free radical scavenging effects hardnosed.

2. Antioxidant Activity:

Table 2: Showing Optical density of Ricinus communis on different concentrations.

CONCENTRATION (µg/ml)	O.D (nm)
0.001	1.764
0.01	1.729
0.1	1.651
1	1.411
10	1.261
100	1.187
1000	1.117

In the present investigation it was showed in Table 2 that the maximum optical density comes out to be 1.764 nm which is at the concentration 10^-3 µg/ml and the smallest optical density is 1.117 nm which is at the concentration 10³µg/ml where as the other shows comparable O.D at different concentrations i.e.1.729 nm at 10^-2µg/ml, 1.651 nm at 10^-1 µg/ml, 1.411 nm at 1 µg/ml, 1.261 nm at 10¹ µg/ml, 1.187 nm at 10²µg/ml.

Fig 1: Graph showing Antioxidant Activity of Ricinus communis at different concentration.

Table 3: Showing Antibacterial activtiy of Ricinus communis on different concentrations.

Microorganisms	Disc Concentration
	A(1mg/disc)		B(5mg/disc)		C(10mg/disc)		Standard
	Inhibition zone (in mm)	Activity index (inhibition zone/ standard	Inhibition zone (in mm)	Activity index (inhibition zone/ standard	Inhibition zone (in mm)	Activity index (inhibition zone/ standard	Standard
Proteus vulgeris	Nil	Nil	Nil	Nil	Nil	Nil	19
Cryseobacterium galacum	Nil	Nil	Nil	Nil	Nil	Nil	18
Staphylococcus aureus	Nil	Nil	Nil	Nil	Nil	Nil	22
Enterobacter	6	0.37	6	0.37	12	0.75	16
Klebsiella pneumoniae	Nil	Nil	Nil	Nil	Nil	Nil	14
Bacillus subtilis	5	0.23	6	0.28	6	0.28	21
Shigella flexneri	Nil	Nil	Nil	Nil	Nil	Nil	13

Fig 2: Graph showing Antibacterial Activity of Ricinus communis at different concentration.

Table 4: Showing Antifungal activity of Ricinus communis on different concentrations.

Microorganisms	Disc concentrations
	A(1mg/disc)		B(5mg/disc)		C (10mg/disc)		Standard
	Inhibition zone (in mm)	Activity index (inhibition zone/ standard	Inhibition zone (in mm)	Activity index (inhibition zone/ standard	Inhibition zone (in mm)	Activity index
Aspergillus niger	17	1.06	16	1	9	0.56	16
A.fumigatus	Nil	Nil	Nil	Nil	Nil	Nil	19
A. flavus	Nil	Nil	Nil	Nil	Nil	Nil	9
Candida	Nil	Nil	Nil	Nil	Nil	Nil	12

Fig 3: Graph showing Antifungal Activity of Ricinus communis at different concentration.

In the present investigations, Fig 1 shows the antioxidant activity of Ricinus communis which showed appreciable activity against the DPPH assay method where the regression line clear cut showed the effectiveness of it as it’s have potentials which are comparable to ascorbic acid. The antioxidant activity of Ricinus communis in methanolic extract using DPPH assay method shows appreciable activity comparable to standard ascorbic acid. The straight line showed y=-0.182x+1.776 and regression=0.838 whereas, in above drug the straight line is y =-0.122x+1.933 and regression = 0.959.

3. Antimicrobial Activity:

Ø Antibacterial activity of Ricinus communis:

Table 3 showing the the alcoholic extract showing antibacterial activity against Enterobacter sps. and Bacillus subtilis microorganisms, expect Klebsiella pneumonia, P. vulgaris, Cryseobacterium galacum, and Shigella flexneri has no activity.

Ø Antifungal activity of Ricinus communis:

When antifungal activity of Ricinus communis was performed against above four fungus it was found that activity was appreciable but showed positive activity against Aspergillus niger as shown in Table 4.

CONCLUSION:

From the above results and discussion it can be accomplished that the methanolic extract of Ricinus communis possesses the effectual antioxidant and antimicrobial substances and a which may be rationalize on the basis of using this plant’s extract as folkloric remedies. The noteworthy test systems i.e. wholly free radical scavenging next to with reducing power, were used for the chemical analysis. In the current study it indicates that the saponins are present in Ricinus communis. The occurrence of saponins in huge quantity is rationally proportional to the antioxidant activity so it is evidently show that it will prove the antioxidant activity and promote a drug for treatment of various infectious disease. Further, the results of antibacterial activity were quite good, Enterobacter and Bacillus subtilis showed very good results Whereas antifungal activity of Ricinus communis was found that activity was appreciable but showed positive activity against Aspergillus niger.

ACKNOWLEDGEMENT:

Author acknowledge with thanks the financial support from Department of Science and Technology, Government of Rajasthan, in the form of Centre with Potentials for Excellence in Biotechnology, sanction no F 7(17) (9) Wipro/Gaprio/2006/7358-46(31/10/2008).

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Received on 03.12.2012 Accepted on 25.12.2012

Asian J. Pharm. Tech. 3(1): Jan.-Mar. 2013; Page 20-25

*Ricinus* *communis* L.
TEST	Reducing Sugar	Saponin	Tannin	Terpenoides	Flavonoides	Alkaloides
	-	+	-ve	-	-ve	-