INTRODUCTION:

Drumstick is a plant belonging to the family Moringaceae, a genus of Moringa with 13 different species. M.oleifera, M. arborea, M. borziana, M. concanensis, M. drouhardii, M. hildebrandtii, M. longitula, M. ovalifolia, M. peregrina, M. pygmaea, M. rivae, M. ruspoliana, and M. stenopetal.¹ It has been naturalized in many tropic and subtropics regions (native to Africa, Arabia, South Asia, South America, the Himalayas region, India, Nepal, Pakistan, the pacific, and Caribbean Islands) worldwide.²

The plant is referred to by several names such as horseradish tree, drumstick tree, ben oil tree, miracle tree, and "Mother's best friend."³ Moringa oleifera is a small, fast-growing evergreen or deciduous tree that usually grows up to 10 to 12m in its height, open crown of drooping fragile branches, feathery foliage of trip innate leaves and thick corky, whitish bark.⁴ It is widely cultivated for its young seed pods and leaves. Moringa is one of the world's most valuable plants and most nutritious crops. Moringa produces more leaves during the rainy season, which can be harvested from 6 to 12 months after planting, depending on the tree's growth. Harvest leaves by snapping leaf stems from branches. Harvesting young shoot tips will promote the development of side branches where cuts along the main branches are made. Allow plants to develop new shoots and branches before subsequent harvests.⁵ Moringa leaves should be dried in an area protected from light to prevent the loss of vitamins and protected from dust and pests to prevent contamination. Moringa leaf powder should be stored in airtight containers protected from heat, humidity, and light.⁶ Moringa represents an available source of essential nutrients and proteins all year. It is a potent immune booster and has the best antioxidant potential.^7-9 It is a mean to cure and prevent malnutrition in infants and pregnant and lactating women.¹⁰ The rationale for choosing the plant lies in its more vitamin c content than oranges¹¹ and potassium content than bananas.¹² The main aim of this research is to carry out in vitro studies and the antioxidant properties of the Moringa plant and to project the future aspects of research in vivo.

MATERIALS AND METHODS:

Plant’s leaf collection and authentication:

The leaves of Moringa were locally collected from Laxmipur Kotwali, Bara (District), Nepal, in February 2021 in the morning time and authenticated by the Department of Botany, Post Graduate Campus, Biratnagar, during February, 2021.

Microbial organism availability:

The fresh pathogenic bacteria species (Staphylococcus aureus, Salmonella typhi, Escherichia coli, and Klebsiella pneumonia) were collected from the Department of Microbiology, BPKIHS, Dharan and it was subcultured, preserved in the microbiology laboratory at Sunsari Technical College, Dharan-01.

Sample processing:

The collected plant samples were washed thoroughly under running tap water to remove debris, and leaves were separated and shade dried at room temperature for about two weeks to attain a constant weight. Exposure to direct sunlight was avoided to prevent the loss of active compounds from the sample. The dried samples were mechanically grinded by grinder and sieved through mesh size 80, and powder was collected and stored in an airtight container.

Extraction processes:

In the successive extraction process, the powdered plant material (50g) was extracted with non-polar solvents, n-Hexane and polar solvents, Ethanol, Methanol, and Water (300ml each). Based on the polarity of content in the plant material, extracted out in particular solvents like non-polar in hexane and polar in ethanol, methanol, and water.^13,14 On the laboratory scale, the Soxhlet apparatus was used. It consists of a flask, a Soxhlet extractor, and a reflux condenser. The raw material was usually placed in a thimble made of filter paper and inserted into the vast central tube of the extractor. Alternatively, after imbibition with menstruum, the drug may be packed in the extractor, ensuring the bottom outlet for the extract is not blocked. The solvent was placed in the flask and brought to its boiling point. Its vapors pass through the larger right-hand tube into the upper part of the extractor and then to the condenser, where they condense and drop back onto the drug. During this period, the soluble constituents are extracted. When the extract level reaches the top of the siphon tube, the entire volume of extract siphons over into the flask. The process continued until the drug was completely extracted. The extract in the flask was then processed.

Determination of Percentage Yield:

The percentage yield of solvent extracts was calculated using the formula:

W1 –W2/ W0 × 100%

Where, W1 is the final weight of the extract in the Petri plate after the complete evaporation of the solvent, W2 is the weight of the empty Petri plate, and W0 is the initial weight of the sample.

Pharmacognostic Analysis:

Microscopy study:

Fresh leaves were collected and preserved in a glycerine and alcohol mixture (1:1) for softening. Section of fresh leaves was cut in transverse planes and mounted on a microscope for microscopical characters.

Macroscopic study:

The macroscopical study was carried out with the naked eye, which gave details concerning the plant aspect, like general appearance, color, and odor.

Loss on drying:

About 5-10gm of the powdered crude plant was accurately weighted in a tarred dish and dried in Hot Air Oven at 100-105℃ for 15 min. It was then cooled in a desiccator and again weighed. The loss on drying was calculated with reference to the amount of dried powder taken. This process was repeated three times for more accurate results

Calculated by the following formula: LOD (%) = (Initial sample weight – sample weight after drying) / Initial sample weight x 100%

Phytochemical screening of drumstick leaves extract:

The leaves extracts were screened for some secondary metabolites like saponins, tannins, alkaloids, anthraquinones, phlobatannins, flavonoids, terpenoids, reducing sugar and poly phenols, etc.^15-19

Antimicrobial Activities:

Preparation and sterilization of Culture Media (Mueller Hinton Agar):

Suspend 19gm of the medium in 500ml of distilled water. Heat with frequent agitation and boil for one minute to completely dissolve the medium. Autoclave at 121℃ for 15 minutes. Cool to room temperature. Pour cooler Mueller Hinton Agar into sterile Petri dishes on a level, horizontal surface to give uniform depth (4mm thickness). Allow cooling to room temperature. Store the plates at 2-8℃

Procedure (Agar well diffusion method):

Each petri dish was filled to a depth of 4-5mm with MHA (Mueller Hinton Agar) medium and allowed to solidify. The petri dish was specially selected with a flat bottom and placed on a level surface to ensure that the medium layer was uniformly thick. The Petri dishes were sterilized at 160-170℃ in a hot air oven for 30 minutes before use. The media was inoculated with suitable inoculums of the suitable test organism. The tiny sterile borer of uniform size was placed approximately at 10cm height, having an internal diameter of approximately 4-6mm, and made of aluminum (or) stainless steel. To each portion, one cylindrical cavity was made in medium with the help of a sterile borer. Four cavities for test compounds (for different concentrations of sample, i.e.,200mg/ml, 400mg/ml, 600mg/ml, 800mg/ml) and one cavity for the negative control sample and centrally antibiotic disc (Ciprofloxacin 0.005mg/ml kit) was used as the reference standard. The Petri-dishes were incubated at 37℃ for 18-24 hours. The diameter of the zone of inhibition was measured, and the average diameter of each sample was calculated. The diameter obtained by the test sample was compared with that produced by reference standard Ciprofloxacin 5mcg/ml kits.^20-22

Determination of Antioxidant Activity:

The antioxidant activity of Moringa leaf extract was carried out by using DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging activity. Ascorbic acid was used as the standard, and DPPH with methanol was used as a solvent.^23-26

Preparation of stock solution of drumstick leaf extract:

Preparation of primary and secondary stock solution:

Primary stock solution was prepared by weighing 100 mg Moringa leaves extract and dissolving it in 100ml methanol having concentration of 1mg/ml(1000mg/ml). Secondary stock solution was prepared by taking 4 ml primary stock solution and dissolving in 1 ml methanol to prepare 800mcg/ml. From the secondary stock solution, different concentrations of 400µg/ml, 200µg/ml, 100µg/ml, and 50µg/ml were prepared by serial dilution. Above each concentration, the sample compound was added to 1ml DPPH solution and was incubated at 37℃ for 30 minutes

Preparation of 0.1mM DPPH (Control sample):

The Molecular weight of DPPH is 394g/mol. To make a 0.1mM solution, 3.94mg of DPPH was dissolved in 100ml of 99% methanol. From this, 10ml of solution was taken, and the volume was made up to 100ml with 99% methanol. Then the concentration of 0.1mM DPPH was prepared and stored protected from light. Then, 1 ml of DPPH was added to all the test samples and incubated at 37^oC for 30 minutes.

Preparation of (Ascorbic acid) standard sample in methanol:

Preparation of primary and secondary stock solution:

Primary stock was prepared by weighing 100mg ascorbic acid and dissolving in 100ml methanol having a concentration of 1mg/ml(1000µg/ml). Secondary stock was prepared by taking 4 ml primary stock solution and dissolving in 1ml methanol to prepare 800mcg/ml. From the secondary stock solution, different concentrations of 400mcg/ml, 200mcg/ml, 100mcg/ml, and 50mcg/ml were prepared by serial dilution. Above made each concentration was added 1ml of DPPH and was incubated at 37c for 30 minutes

Measurement of DPPH free radical scavenging activity:

The different concentration of moringa leaf extract was prepared in methanol, and DPPH in methanol was also prepared. Similarly, Ascorbic acid was taken as standard, and DPPH solution without any sample served was taken as blank. The absorbance of the sample was measured at 517nm in UV-Spectrometer. UV- Spectrometer was Calibrated using Potassium dichromate. Each concentration of sample and standard measure five times absorbance.^27-31 Free radical scavenging activity was expressed as the percentage of inhibition (I%) of DPPH radical was calculated using the following formula: % Inhibition = Abs (control) −Abs (Sample)/Abs (control) x 100%. Where, Abs control is the absorbance of DPPH radical solution, Abs sample is the absorbance of sample solution

RESULTS:

Pharmacognostical Study:

Macroscopic and microscopic identification:

On observation, leaves are bipinnate or commonly tripinnate up to 9 - 26cm long, and the leaflets are hairy, green, and almost hairless on the upper surface. The twigs are hairy and green with compound leaves with leaflets of 1–2cm in height and 0.9 – 4cm wide. The macroscopic identification was made by the organoleptic test of fresh leaves on M. oleifera, which showed green color, Characteristic Odour but not aromatic, and Characteristic taste. The microscopic identification was made by T.S. section (Transverse Section) of fresh drumstick leaves observed under the microscope. Under observation, T. S of the leaf through mid-rib was slightly curved at the upper surface while at lower surface curve was deep. The lower epidermis was single-layered, the central region occupied by 2-4 layers of collenchyma cells. The leaflet showed dorsiventral structure; the epidermis and unicellular hairs were present on both surfaces; the palisade single layered; both upper and lower epidermis was single layered. Phloem cells were on the exterior side enclosing the xylem cells and were visualized by a pink stain. Xylem cells were scattered in the pith, consisting of large hexagonal pith parenchyma. Palisade Ratio ranges from 6-10 cells beneath each epidermal cell.

Fig. 1: T.S. of M. oleifera leaf

Loss on drying:

Initial weight of sample = 5gm; Weight of empty Petri plate = 29.76gm; Final weight (wt. of Petri plate + plant powder after drying in oven) before drying = 34.76gm; Weight of sample after drying = 34.32gm – 29.76gm = 4.56gm

Therefore, LOD (%) = (initial sample weight – sample weight after drying)/Initial sample weight x 100%

= (5 - 4.56)/5 x 100% = 8.8%

Extractive value:

The yield percentage (%) for four extracts were calculated, and % yield was found to be highest in the case of the Methanol extract

Table 1 Extractive yield % of different solvents

Plant Powder Quantity (g)	Solvent quantity (ml)	% Yield
50 grams	N-hexane (200)	6.46% ((3.23gm)
	Ethanol (200)	7.46% (3.73gm)
	Methanol (200)	14.68% (7.34gm)
	Distilled water (200)	9.2% (4.6gm)

Fig. 2: Percent yield value of extraction

Phytochemical Test:

The phytochemical characteristics of M. oleifera leaf extract tested were summarized in Table – 2

Table 2: Phytochemical characteristics of M. oleifera leaf extract

Name of the test	Solvents used for extraction
Name of the test	N-hexane	Ethanol	Methanol	Water
Alkaloid	-	+	+	-
Carbohydrate	-	+	+	+
Cellulose	+	-	-	-
Flavonoid	-	+	+	+
Glycoside	-	-	-	+
Phenols	-	+	+	+
Phlobatannin	-	-	-	-
Protein	+	+	+	+
Quinone	-	+	-	+
Reducing sugar	-	-	-	+
Saponin	-	-	+	-
Starch	-	-	-	+
Tannin	-	+	+	+

+ Positive, - Negative

Fig 3: Phytochemical test (Alkaloid, saponin, phlobatannin, tannin, and reducing sugar) left to right

Fig 4: Phytochemical test (Cellulose, carbohydrate, protein, anthocyanins, starch) left to right

Fig 5: Phytochemical test (phenol, quinone, flavonoid) left to right

Antibacterial activity:

Table 4 Antioxidant activity of the sample (Moringa oleifera leaf extract)

Treatment	Concentration (in µg/ml)	Average absorbance ± SEM	Average % inhibition ± SEM
Moringa extract + methanol + DPPH	50	0.418±0.013	34.684±0.814
	100	0.332±0.010	48.122±1.677
	200	0.264±0.006	59.372±1.082
	400	0.212±0.005	66.871±0.926
	800	0.152±0.006	76.246±1.026
	DPPH +Methanol (control)	0.64	--

Table 5 Antioxidant activity of standard (Ascorbic acid)

Treatment	Concentration (in µg/ml)	Average absorbance ± SEM	Average % inhibition ± SEM
Ascorbic acid + methanol + DPPH	50	0.132±0.008	79.684±1.59
	100	0.120±0.004	81.246±0.626
	200	0.072±0.006	88.746±1.026
	400	0.048±0.005	92.497±0.815
	800	0.018±0.008	98.404±0.702
	DPPH +Methanol (control)	0.64	--

Antioxidant activity (DPPH radical scavenging activity):

Drumstick leaves exhibited a concentration-dependent free radical scavenging activity by inhibiting the DPPH radical. The DPPH method is based on reducing methanolic DPPH solution in the presence of hydrogen-donating antioxidants through the formation of a non-radical form (DPPH-H). As the Drumstick particle reduces the DPPH radical, the reduction capability of the DPPH radical was determined by the decrease in its absorbance at 517 nm.

DISCUSSION:

Moringa oleifera Lam. in the current scenario, is being viewed as a very potent plant in the fields of nutraceuticals and pharmaceuticals. Several well-established companies are producing and processing different parts of the plant for healthcare. The current trend shows enormous future possibilities for the vigorous use of the plant, especially by third-world nations. As it is quite a task for these nations to put in significant finances for healthcare, simple methods, and techniques used here can be an effective tool to identify and standardize this plant.²⁷ This study was used for the extraction of four different solvents (n-hexane, ethanol, methanol, and distilled water) used in an equal quantity of volume and equal weight of powder in the Soxhlet apparatus to find out the maximum extractive yield percentage in methanol (14.68%) whereas minimum extractive yield percentage in n-Hexane (6.46%). Phytochemical screening of Moringa oleifera leaf extract shows the presence of various phytochemicals, such as alkaloids, flavonoids, glycosides, terpenoids, tannins, saponins, reducing sugar and steroids, etc., in different solvents (n-Hexane, Ethanol, Methanol, and distilled water),³² Cellulose presence in n-hexane extract and absent in others extract solvents, Proteins are present in all solvent extract, Steroids are present in all solvents extract except water, Carbohydrates, alkaloids, phenols, flavonoids, saponins, tannins, volatiles oils, and quinone are presence in ethanol and methanol extract, Reducing sugar and starch presence in water extract as shown in Table 2. The phytochemical screening indicated the presence of tannins, flavonoids, glycosides, terpenoids, phenols, etc., in leaf extracts of Moringa oleifera that are responsible for its antibacterial activity. The antibacterial activity of the ethanolic extract was investigated using the agar well diffusion method against gram-positive species (S. aureus) and gram-negative strain (E. coli).^33-35 The results showed that the ethanolic extract of Moringa oleifera has activity against both test bacteria. The maximum zone of inhibition against S. aureus was 25mm, and E. coli was 22mm at 200mg/ml.³⁶ In this study, it was found that the maximum zone of inhibition against Staphylococcus aureus (ATCC25923), Salmonella typhi, Escherichia coli (25922), klebsiella pneumoniae, was 21±0.34mm, 24±0.15mm, 22±0.36mm and 9±0.01mm at 800mg/ml extract concentration, and with standard drug (Ciprofloxacin 5mcg/ml) zone of inhibition was found to be 42±012mm, 45±0.25mm, 35±0.21mm and 6±0.00mm (R) respectively. Hence, Ciprofloxacin is resistant to Klebsiella pneumoniae. The medicinal plant is essential in controlling oxidation caused by free radicals. Thus, it served as a remedy for oxidative damage. DPPH radical scavenging assay was performed to evaluate antioxidant activity. The methanolic leaf extract possesses the highest percentage inhibition of DPPH, i.e., 76.246±1.026% and highest percentage inhibition of standard (Ascorbic acid), i.e., 98.404±0.702% at a concentration of 800µg/ml. The methanolic leaf extract possesses the lowest percentage inhibition of DPPH with 34.68±0.814% and the lowest percentage inhibition of standard with 79.684 ± 1.59% at 50µg/ml. ³⁷

Staphylococcus aureus activity test, Salmonella typhi activity test,

Escherichia coli activity test, Klebsiella pneumoniae activity test

Fig 8.

LIMITATIONS OF THE STUDY:

a. Further study can be done on the antifungal and anti-helminthic activity of the leaf extract of Moringa oleifera.

b. In vivo studies regarding anti-cancer, anti-inflammatory, and anti-diabetic activity can also be carried out in the future.

c. Antibacterial activity can also be studied on other species of bacteria, such as Bacillus subtilis, Pseudomonas aeruginosa.

d. Purification of compounds and elucidation of the molecular mechanism of their antibacterial and antioxidant properties.

CONCLUSION:

The study of phytochemical analysis demonstrates the presence of various phytochemicals like alkaloids, glycosides, tannins, flavonoids compounds, etc., in ethanolic leaf extract of M. oleifera Lam and are responsible for showing considerable antibacterial and antioxidant activity. The ethanolic leaf extract of M. oleifera used in this experiment showed significant antibacterial activity against test pathogens. This supports the fact that M. oleifera contains active phytochemicals with wide-spectrum antibacterial activity, capable of inhibiting the growth of gram-positive (S. aureus) and gram-negative bacteria (E. coli, S. typhi, K. pneumoniae). M. oleifera leaf extract possesses antioxidant activity, which might help prevent the progress of various oxidative stress-related diseases. Thus, it can be conducted that the methanolic extract of M. oleifera leaves can be used as an accessible source of natural antioxidants with consequent health benefits.

ACKNOWLEDGMENT:

We are grateful to all of those with whom we have had the pleasure to work during this and other related projects.

CONFLICT OF INTEREST:

The authors declare no potential conflict of interest.

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Received on 17.06.2022 Modified on 20.11.2022

Research J. Pharm. and Tech 2023; 16(10):4512-4518.

DOI: 10.52711/0974-360X.2023.00735

Concentration of extracts (mg/ml)	Zone of inhibition (mm)
Concentration of extracts (mg/ml)	*Staphylococcus aureus* (ATCC25923)	*Salmonella typhi* (ATCC29631)	*Escherichia coli* (ATCC25922)	*Klebsiella pneumonia* (Resistance in Ciprofloxacin)
200	9±0.20	11±0.17	8±0.13	6±0.00
400	15±0.12	17±0.25	16±0.26	7±0.01
600	18±0.32	19±0.12	20±0.31	8±0.02
800	21±0.34	24±0.15	22±0.36	9±0.01
DMSO 10%	--	--	--	--
Standard drug Ciprofloxacin 0.005 (5mcg/ml)	42±0.12	45±0.25	35±0.21	6 ±0.00