INTRODUCTION:

Moringa oleifera (MO) has an important position in Ayurveda- an Indian indigenous system of medicine. It belongs to the family of Moringaceae, commonly known “Sahajan” in Hindi. It is also known as “Horse-radish tree”, “Drumstick tree”. It is a small, fast-growing, evergreen and deciduous tree that usually grows upto 10 or 12 m in height⁶. It has an impressive range of medicinal uses with high nutritional values. Different parts of this plant contain a profile of important minerals, and are a good source of protein, vitamin, beta-carotene, amino acids and various phenolics⁷.

Taxonomic Classification:

Kingdom - Plantae

Sub kingdom -Tracheobionta

Super Division - Spermatophyta

Division -Magnoliophyta

Class -Magnoliopsida

Subclass – Dilleniidae

Order - Capparales

Family - Moringaceae

Genus - Moringa

Species – Oleifera

Moringa oleifera is an ornamental plant native in tropical and subtropical areas, and commonly cultivated in all regions of Indonesia, North-eastern Pakistan, North-eastern Bangladesh, Srilanka, East and West Africa and West Asia. The specie is native to India. It is distributed among Sub-Himalayan tracts from Chenab to Oudh. It grows at elevation from sea level to 1400 hundred metres¹.

The plant and its parts are used as antispasmodic, stimulant, expectorant and diuretic. It is mainly used as antioxidants and as antimicrobial agent for various infections. It shows ulcer protective effects. It shows hepatoprotective activity².

The antioxidant properties of Moringa may be due to the presence of phenolic compounds that can be confirmed by phytochemical screening of hydroethanolic extract. In this respect Moringa pods contain important bio-active compounds including glucosinolates, isothiocyanates, thiocarbamates, and flavonoids. These compounds quench ROS, chelate metal ions and regenerate membrane-bound antioxidants³.

The biochemical basis of the chemopreventive potency of M. Oleifera extract may be attributed to the synergistic action of the constituent of the extract and the induction of Phase- II enzymes (GSTs) and antioxidant enzymes, which might be implicated in the anticarcinogenic activity. The aqueous extract of M. Oleifera exhibited strong scavenging effect on 2,2-diphenyl-2-picryl hydrazyl (DPPH) free radical, superoxide, nitric oxide radical and inhibition of lipid per oxidation. The extract of M. Oleifera both mature and tender leaves have potent antioxidant activity against free radicals, prevent oxidative damage to major biomolecules and afford significant protection against oxidative damage. The M. Oleifera hydro-alcoholic leaf extract (1000mg/kg) and pod (fruit) extract (750 mg/kg) contain high amount of tannin, phenolic compounds and flavonoids⁴.

The antimicrobial activities of Moringa oleifera leaves, roots, bark and seeds in vitro against bacteria, yeast, dermatophytes and helminths by a disk-diffusion method. The fresh leaf juice and aqueous extract from the seeds inhibit the growth of Pseudomonas aeruginosa and Staphylococcus aureus.

Antimicrobial activities of various oleifera seeds were investigated against Gram positive – Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Sarcinalutea: Gram negative – Escherichia Coli, Acid fast mycobacterium phlei.

Free radical and reactive species are well known as inducers of cellular and tissue pathogenesis which is causing some diseases like diabetes, cancer, inflammatory and also cardiovascular. Free radical reactions take place in human body and food system causing injury and death¹. Free radicals are one of the main factors which are necessary to cause DNA mutation, which is involved in the initial stage of carcinogenesis². An over production of Reactive Oxygen body can occur the imbalance of defense system. Hence, the study of antioxidants is needed which focuses on natural compounds from natural sources.

Discovery of novel antimicrobial agent is very important for the control of pathogenic microbes, especially for the treatment of infections caused by resistant microbes.

MATERIALS AND METHODS:

Hexane -99%, Laboratory Bargoyneurbidges and Co. India., Methanol:-Laboratory Burgoyne reagent, Peptone Water:-Himedia. (M028-500G), Agar: Agar, Type 1- Himedia, Beef extract powder- clualigens. All other reagents were of analytical grade. Stock solutions were prepared fresh when required.

Plant and extraction:

Moringa oleifera leaves and seeds were collected in the Month of January 2018 from the town of Fulchur Peth, Gondia, Maharashtra. The plant was identified by its vernacular name and later it was compared with the herbarium of the Department of studies in Botany and also was authenticated. The authentication no of plant Moringo oleifera is 1823A. The test organisms were selected based on their availability. Hence, two isolated bacteria (Staphylococcus aureus, Bacillus substilis and E.coli) were used for antimicrobial test. The bacteria were maintained on nutrient agar slant and stored in the refrigerator at 4° C. The bacteria were sub cultured onto fresh media at regular interval until it was used for the test.

The leaves and seeds of M. oliefera plant were washed with distilled water, dried in shade and then grinded to powder. About 20gms of leaf powder was extracted with 250 ml of solvent (methanol) and about 10gms of seed powder was extracted with 200 ml of solvent (n-hexane). This can be done by inserting the sample in soxhlet apparatus and poured the solvent in round bottom flask. This can be kept for 50 cycles. After which crude extracts obtained was decanted and filtered with filter paper. Filtrates are evaporated to remove the solvent and get four crude extract.

Determination of antioxidant activity:

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is chemical reaction that can produced free radicals, leading to chain reaction that may damage cells. Free radicals and other reactive oxygen species generated in living organisms leads to many diseases including cancer, cardiovascular diseases, cataracts, asthma, hepatitis, liver injury and immunodeficiency diseases. The use of antioxidant activity is an old practice and their safety could be questioned by the consumers. The antioxidants activity was evaluated by DPPH radicals scavenging methods, and by FRAP method.

DPPH Radical Scavenging Assay:

The radical scavenging activity of Moringa oliefera extracts against the DPPH radical was determined by the method. Determination procedures were as follow:

1ml of 6×10^-5 M DPPH radical solution (prepared daily) was mixed with 33.33µL of methanolic solution of M. oliefera extract (maximum dissolved concentration). After 20 min. Incubation for at 37 ⁰C, absorbance decrease of mixture was monitored at 515nm (As). During reduction by the antioxidant, the solution colour changed from violet to pale yellow. DPPH radicals have an absorbance maximum at 515nm. Blank samples with 33.33µL of methanol in the above DPPH radical solution were prepared and measured daily at same wavelength (Ab). Trolox was used as positive control. The experiment was carried out in triplicate. Radical scavenging activity was calculated using the following formula¹².

Inhibition rate (%) = [Ab-As/Ab] ×100

The 50% inhibitory concentration (IC₅₀) was expressed as the quantity of the extracts to react with a half of DPPH radical.

FRAP Assay:

FRAP assay is based on the ability of antioxidant to reduce fe³⁺ to fe²⁺in the presence of 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), forming an intense blue fe²⁺TPTZ complex with an absorption maximum at 593 nm. This reagent was freshly prepared by mixing 25 ml acetate buffer (300mM, pH3.6), 2.5 ml 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ) solution (10mM TPTZ in 40mM /L HCL) and 2.5 ml feCl₃ (20mM) water solution. Each sample (150 µl) (0.5 mg/ml) dissolved in methanol was added in 4.5 ml of freshly prepared FRAP reagent and stirred and after 5minute, absorbance was measured at 593 nm, using FRAP working solution as blank. This reaction is pH dependent (optimum pH 3.6). The absorbance decrease is proportional to antioxidant content. FeSO₄ is used for calibration. The antioxidant capacity based on the ability to reduce ferric ion of sample is calculated from the linear calibration curve and expressed as mmol. FeSO₄equivalents per gram of sample.

Inhibition rate (%) = [Ab-As/Ab]×100

Determination of antimicrobial activity:

Natural products (secondary metabolites) are the measure sources of drugs. This products greater structural diversity than drugs is compound from standards combinatorial chemistry. The use of medicinal plants for treatment of microbial diseases is well known and well documented since ancient times. Medicinal plants are synthesis, many defensive compounds of protect themselves and predators. This compound have antimicrobial activity. The ability of plant extract to reduce or inhibit the growth of microorganisms are kill the pathogenic microorganisms is known as antimicrobial activity or efficacy. Several plants species have been tested for antimicrobial properties but the vast majority have not yet been adequately evaluated. Antimicrobial activity of plant extracts may be tested by agar diffusion method or Cup plate method.

Sterility test:

The aqueous and the ethanolic extract were tested for growth or contamination. This was carried out by inoculating 1ml each of them on nutrient agar and incubated at 37°C for 24 hours. The plates were observed for growth. No growth in the extract after incubation indicates that the extract were sterile. The extracts were then accessed for antimicrobial activity.

Procedure:

Semisolid extract are dried by placing over water bath. Dimethyl sulphoxide (DMSO), A colourless hygroscopic liquid is used for dissolving various extract by testing antimicrobial efficacy. Prepare nutrient agar or Sabouraudʹs agar petri plates for the growth of bacterial and fungal cultures. Spread the test cultures on the plates by spread plate methods. The test culture used such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli. Prepare well in seeded plates by using cork borer that is sterilized by burning with absolute ethanol. Plant extracts 0.1ml of pure solvents (DMSO). Bacterial test culture is incubated at 32-37°Cfor 48 hrs. And fungal test culture plates at 25-30°C for 96 hrs. (Yeast culture, 30-35°C for 48 hrs.). The sensitivity of test organism to each of extract is indicated by clear zones of inhibitions. Around the well and measure the diameter of clear zones of inhibitions.

Determination of minimum Inhibitory concentration (MIC):

The minimum inhibitory concentration was determined against bacteria after the antimicrobial test have been performed. This shows the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. The isolates were cultured on Mueller Hinton agar and agar diffusion method was used for this purpose. Sterile cork borer of diameter 6mm was used to bore holes on the plates after seeding the plates with the bacterial strength being tested. It was left for above 1 hour at room temperature and subsequently incubates at 37°C. Result was read after 24 hours of incubation.

Qualitative Method of Analysis:

Preliminary test / Preparation test:

The Moringa oleifera leaves filtrates used for this study were prepared by boiling 20 gm of the fresh plant in distilled water. The solution was filtered through a vaccum pump. The sample source obtained were used for phytochemical screening for alkaloids, tannins, saponins, flavonoids, reducing sugar, anthraquinones.

(1) Test for Alkaloids:

Alkaloid was determined by about 0.2 gm of Moringa oleifera sample was warmed with 2% of H₂SO₄ for two minutes. It was filtered and few drops of Dragendorff’s reagent were added. Orange red precipitate indicates the present of Alkaloids.

(2) Test for Tannins:

The test was performed by following a standard procedure Maxson and Rooney. In doing this one millilitre of the filtrate was mixed with 2 ml of FeCl. A dark green colour indicated a positive test for the tannins.

(3) Test for Saponin:

One millilitre of Moringa oleifera leave filtrate were diluted with 2ml of distilled water; the mixture were vigorously shaken and left to stand for 10min during which time, the development of foam on the surface of the mixture lasting for more than 10min, indicates the presence of saponins.

(4) Test for Flavonoids:

One millilitre of Moringa oliefera plant filtrate was mixed with 2ml of 10% lead acetate; a brownish precipitate indicated a positive test for the phenolic flavonoids. While for flavonoids, 1ml of the plant filtrate were mixed with 2ml of dilute NaOH ; a golden yellow colour indicated the presence of flavonoids.

(5) Test for Reducing Sugars:

The reducing sugar in the sample source was determined by measuring one millilitre of the plant filtrate into which Fehling A and Fehling B and a green colour with Fehling A indicate the presence of reducing sugars.

(6) Test for Anthraquinone:

A Borntranger test, was performed whereby one millilitre of the plant filtrate was shaken with 10ml of benzene; the mixture was filtered and 5ml of 10% (v/v) ammonia were added, the shaken and observed. A pinkish solution indicates a positive test.

(7) Determination of Total Phenolic content:

Total phenolic content (TPC) in extracts was determined by Folin-ciocalteuʹs colorimetric method as described by Adedapo et al. (2009b). Extracted solution(0.3 ml in triplicate) was mixed with 1.5 ml of 10% Folin-Ciocalteuʹs reagent and 1.2 ml of 7.55 (w/v) sodium carbonate. The mixture was kept in the dark for 30 min and absorbance was measured a 765 nm. Quantification was done on the basis of a standard curve of gallic acid. The results were expressed as gallic acid equivalent (GAE), that is, mg gallic acid/100ml. All tests were performed in triplicate.

RESULTS AND DISCUSSION:

Qualitative method of analysis:

Quantitative analysis of the phytochemical screening of Moringa oleifera was determined. This confirms the presence of alkaloids, cynogenicglucoside, steroid, anthroquinone, phenol, tannins, saponins and flavonide (Table 1).

Antioxidant activity:

The antioxidant activity of the different parts of plant extract measured by the ability to scavenging DPPH free radical, was compared with standard / Trolox (Tochopherol). It was observed that extract of leaves had higher activity than seeds extract. At a concentration 1mg/ml, scavenging activity of extract of leaves and seeds reached 51.62 % and 30.77 % respectively (table 4). Though the DPPH radicals scavenging abilities of extract less than Trolox (65.96%) at 1mg/ml. the study showed that the extract proton donating ability and could act as free radical inhibitor or scavenging, acting possibly primary antioxidant (Fig 3).

Antioxidant activity also measured by FRAP method. The results are given in table 2. In this methods leaves extract given higher antioxidant activity than seeds extract.

Table 1: Quantitative analysis of the phytochemical screening of medicinal plants.

Test	Observation
Alkaloid	+ve
C. Glucoside	+ve
Steroid	+ve
Anthraquinone	+ve
Phenol	+ve
Tannins	+ve
Saponins	+ve
Flavanoids	+ve

Keys - +ve= presence of constituents

Table 2: Antioxidant activity of Moringa oliefera by DPPH Radical Scavenging Assay

Samples	Absorbance (nm)	Inhibition rate (%)
Control (Methanol)	0.4449	---
Leaves extract (1mg/ml)	0.2152	51.62
Seeds extracts (1mg/ml)	0.3080	30.77
Standard (Trolox) (1mg/ml)	0.1514	65.96

Fig: 1. Comparative study on Antioxidant activity of Moringa oliefera by DPPH+

Table 3. Antioxidant activity of Moringa oliefera by FRAP Assay

Samples	Absorbance (nm)	Inhibition rate (%)
Control (Methanol)	0.4089	--
Leaves extract (1mg/ml)	0.2012	50.79
Seeds extracts (1mg/ml)	0.3096	24.28
Standard (Ferrous Sulfate) (1mg/ml)	0.1099	73.00

Antimicrobial activity of medicinal plant:

The extract of Moringa oliefera show highest zone of inhibition compared to seed extract of Moringa oliefera. The leaves extract of Moringa oliefera show highest measurement zone of inhibition in E.coli, S. aureus, Bacillus subtilis. Seed extract of Moringa oliefera has least antimicrobial effect on selected species (Table 4).

Table 4. Antimicrobial activity of Moringa oliefera on different species

Culture	Zone of inhibition (mm)			Control (DMSO)
Culture	Standard (Streptomycin)	Leaves	Seeds	Control (DMSO)
E.coli	22	18	15	00
S. aureus	20	17	15	00
Bacillus subtilis	18	17	13	00

Minimum inhibitory concentration:

Different concentration of standard, leaves extract and seeds extract were prepared (Table 5). Concentration was 50, 100,150, 200, 250, 300, 350,400,450, 500 ug/ml. Herbal extract prepared from the seeds and leaves of the plant were screened against three bacterial strains for the purpose of (antimicrobial activity) minimum inhibitory concentration range between 50µg to 500µg/ml.

In antimicrobial activity activity both gram +ve and gram –ve organisms were used. In E. coli, the methanolic extract showed minimum inhibitory concentration (MIC) at 100µg/ml, when compared with standard. For Staphylococcus aureus and Bacillus subtilis showed minimum inhibitory concentration (MIC) at 150µg/ml. For seeds extract, E. coli showed minimum inhibitory concentration (MIC) at 150µg/ml while, S. aureus and Bacillus subtilis showed antimicrobial activity at 200µg/ml.

On comparison between leaves and seeds extract, leaves extract showed highest antimicrobial activity as compared with seed extract.

Table 5. Minimum inhibitory concentration of different parts of Moringa oliefera on different species

Concentration (ug/ml)	Zone of inhibition (mm)
	DMSO			Standard			Leaves Extract			Seeds Extract
	*E.coli*	*S. aureus*	*B. Subtilis*	*E. coli*	*S. aureus*	*B. Subtilis*	*E. coli*	*S. aureus*	*B. Subtilis*	*E. coli*	*S. aureus*	*B. Subtilis*
50	00	00	00	00	00	00	--	--	--	--	--	--
100	00	00	00	10	09	08	08	--	--	--	--	--
150	00	00	00	11	10	10	09	09	08	07	--	--
200	00	00	00	14	13	11	10	09	08	09	09	08
250	00	00	00	15	13	12	11	10	09	10	10	08
300	00	00	00	16	15	12	11	11	10	10	11	09
350	00	00	00	18	16	13	12	11	10	13	12	09
400	00	00	00	20	17	15	14	12	16	14	13	11
450	00	00	00	20	19	16	17	16	17	14	13	11
500	00	00	00	22	20	18	18	17	17	15	15	13