G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, India.

Corresponding author email: krishnashanti@gmail.com

Article Publishing History

INTRODUCTION

Moringa oleifera (Moringaceae family) is native to the Indian subcontinent and Africa and is also known as the Miracle tree. It is multipurpose tree species with multifarious pharmacological and nutraceutical property (Thurber and Fahey, 2009). Each and every part of Moringa oleifera possesses potential phytopharmacological properties. Apart from its use as a food product it also possesses Medicinal and Industrial application (Moyo et al., 2011). The leaf material of this plant is well-known for its high mineral content as it is rich in essential amino acid, vitamins and minerals (Tahiliani and Kar, 2000; Amabye and Tadesse, 2016). Owing to the multipurpose properties of M. oleifera, this plant is also rich in important antioxidants that can quench free radicals (Rockwood et al., 2013). It was also reported that M. oleifera is rich in important phytocompounds such as phenolics, flavonoids, tannins, and saponins with superior bioactivity (Mishra et al.,2011; Patel et al., 2014; Zainab et al., 2020).

The incidence of UTI (Urinary tract Infections) infections and Staphylococcal infection is still high in many developing countries and this is because of the lack of information and knowledge regarding multi-drug resistance (Arumugam et al., 2011). To overcome the problems associated with synthetic drugs, researchers are now focusing on plant-based materials as they are readily available with no known side effects (Ncube et al., 2008; Al_husnan and Alkahtani,2016). Various portions of this plant including gum, pod extract, flowers and roots, have shown potential effects as a source of indigenous medicine (Odebiyi and Sofowora, 1978; Anwar et al., 2007; Sandeep et al., 2019). Therefore, the objective of this study is to evaluate the antibacterial efficacy and to screen Moringa oleifera’s substantial phytochemicals to imply a natural plant-based system as an alternative to the synthetic drug (medicine) system.

MATERIAL AND METHODS

Moringa oleifera accessions were collected from different regions of Gujarat such as Navsari, Anand, Bardoli and Vadodara (Table I) and were maintained at study farm of Uka Tarsadia University, Bardoli, Gujarat, India. The voucher specimens were deposited at herbarium of C.G.Bhakta Institute of Biotechnology, Uka Tarsadia University. Disease free and healthy Moringa oleifera leaves of all the accessions were thoroughly washed under running tap water and dried separately at room temperature for three weeks until completely dried. The powdered samples  (5g)  were  extracted by double distilled water (150 ml) using soxhlet apparatus and were dried and preserved at 4 °C prior to further use (Patel et al., 2020). Preliminary phytochemical screening was carried out by following standard protocols as defined by (Harborne, 1984; Patel et al., 2020)

The quantitative estimation of phenolic and flavonoid contents of Moringa oleifera accessions was determined by following Folin–Ciocalteu and Aluminum chloride colorimetric method respectively as reported by (Patel et al., 2020). The  presence of total tannins was determined by using the method reported by (Patel et al., 2019). The bacterial strains used in the study were procured from National Collection of Industrial Microorganisms (NCIM), Pune, India (Table 2). The standard bacterial strains were maintained on nutrient agar medium at 37°C prior to further use (Balouiri et al., 2016; Patel et al., 2019).

Agar well diffusion method was employed for monitoring the antibacterial activity of Moringa oleifera extracts where streptomycin (10µg/ml) was used as positive control and zone of inhibition was recorded in mm (Balouiri et al., 2016). Broth Macro-dilution method was employed in investigating the Minimum inhibitory concentration (MIC) of Moringa oleifera extracts (Rahman et al., 2009; Adamczak, et al., 2020).

Table 1. Moringa oleifera accessions collected from different regions of Gujarat (Tables were prepared by us according to the area of collection)

Sr. no	Accession code	Area of Collection
1	MONV	Navsari
2	MOBL	Bardoli
3	MOVR	Vadodara
4	MOAN	Anand

Table 2. Standard bacterial strains used in the study

Sr.no	Bacterial strain	NCIM Accession no
1	Escherichia coli	NCIM 2931
2	Vibrio cholerae	NCIM  5316
3	Bacillus subtilis	NCIM 2921
4	Staphylococcus aureus	NCIM 5345

RESULTS AND DISCUSSION

The present study was carried out to analyze the preliminary phytochemicals present in Moringa oleifera accessions collected from different regions of Gujarat. Preliminary phytochemical screening revealed the presence of Alkaloids, Phenols, Flavonoids, Tannins, Carbohydrates and Amino acids in all the accessions except alkaloids, it was absent in the accession collected from Bardoli and Valsad (Table III). The quantitative analysis of phenolic, flavonoids and tannin content are as shown in (Table IV). The total phenolic content ranged from 0.0076 mg/g to 2.98mg/g GAE equivalent in the order of MONV>MOAN>MOVR>MOBL. The total flavonoids ranged from 1.12mg/g to 1.59mg/g QE equivalent in the order of MONV>MOAN>MOVR>MOBL. Total tannins were found in the range of 0.66mg/g to 1.35mg/g tannic acid equivalent in the order of MOBL>MOVR>MOAN>MONV.

Table. 3  Preliminary Phytochemical Screening of Aqueous extract of Moringa oleifera accessions + indicates present; – indicates Absent

Phytochemical Constituent	Accessions
	MONV	MOBL	MOVR	MOAN
Alkaloid	+	–	–	+
Phenol	+	+	+	+
Flavonoids	+	+	+	+
Tannins	+	+	+	+
Carbohydrates	+	+	+	+
Amino acid	+	+	+	+

Table 4. Quantitative Phytochemical Analysis of Aqueous extract of Moringa oleifera accessions

Phytochemical Constituent mg/g	Accessions
	MONV	MOBL	MOVR	MOAN
Phenol	2.98±0.02	0.0076±0.003	0.14±0.031	0.35±0.09
Flavonoids	1.59±0.29	1.12±0.088	1.43±0.18	1.54±0.29
Tannins	0.66±0.008	1.35±0.16	0.97±0.013	0.74±0.03

n=3, Value indicates Mean ±SEM

Antibacterial activity of Moringa oleifera accession MONV, MOVR and MOAN showed potent inhibitory activity against all the test bacteria in dose dependent manner except Vibrio cholerae (Table V and Table VI). Previous studies also reported good antibacterial activity of Moringa oleifera leaf extracts against various human pathogens (Rahman et al., 2009; Amabye and Tadesse, 2016).

Table 5. Antibacterial activity of Moringa oleifera accessions against test organism at the concentration of 50 mg/ml

Test Organisms	Accessions (zone of inhibition in mm) Concentration (50mg/ml)				Streptomycin
	MONV	MOBL	MOVR	MOAN
E.coli	0.66±0.33	–	–	4.66±0.33	8.6±0.33
S.aureus	7.66±0.33	–	–	–	10.66±0.33
V. cholerae	–	–	–	–	7.25±0.08
B. subtilis	1.66±0.33	–	4.66±0.33	–	9.34±1.3

n=3, Value indicates Mean ±SEM

Table 6.  Antibacterial activity of Moringa oleifera accessions against test organism at the concentration of 100 mg/ml

Test Organisms	Accessions (zone of inhibition in mm) Concentration (100mg/ml)				Streptomycin
	MONV	MOBL	MOVR	MOAN
E.coli	4.33±0.33	–	–	10.33±0.33	8.6±0.33
S.aureus	12.33±0.33	–	–	–	10.66±0.33
V. cholerae	–	–	–	–	7.25±0.08
B. subtilis	3.33±0.66	–	9.33±0.33	–	9.34±1.3

n=3, Value indicates Mean ±SEM

The antibacterial property attributed to any plant material is because of the presence of important phytochemicals such as Phenols, Flavonoids, Alkaloids, Triterpenoids and Saponins (Chandra et al., 2014; Patel et al., 2020). Phytochemicals play a major role in preventing various diseases. They possess immunomodulating and cardioprotective properties. Several researchers have reported that flavonoids and phenols play a major role in showing inhibitory activity by modifying or preparing complex with bacterial cell wall (Olowosulu and Ibrahim, 2006).  In a study conducted on antibacterial activity of different extracts of Moringa oleifera leaf against pyogenic bacteria the ethanolic extract showed highest bioactivity when compared to hot water extract (Fouad et al., 2019).

However, the present study showed good antibacterial activity of aqueous extract of Moringa oleifera against the pathogenic bacteria. The minimum inhibitory activity of the extracts ranged from 9.37mg/ml to 75mg/ml against E. coli, S. aureus and B. subtilis (Table VII). Thus, in the present study it was revealed that there is a varietal response in bioactivity of Moringa oleifera accessions because of broad spectrum of antibiotics present in these extracts.  Hence, Moringa oleifera can serve as good and natural immunobooster to treat various ailments (Fouad et al., 2019).

Table 7. Minimum Inhibitory Concentration against test organism

Test Organisms	Accessions
	MONV	MOVR	MOAN
E.coli	37.5mg/ml	–	75mg/ml
S.aureus	18.75mg/ml	–	–
B. subtilis	9.37mg/ml	18.75mg/ml	–

CONCLUSION

Owing to the issue of drug resistance caused by microbial mutation over the years, certain antibiotics have become almost ineffective. Thus, it can be inferred from the present analysis that there is a wide variability in phytochemical constituents of Moringa oleifera accessions collected from different regions of Gujarat and in order to establish the relationship between the MIC’s obtained in this study and the active doses at which the herbs can be used in conventional practice these accessions could be further explored for in vivo studies with pure extracts for enhanced applications in pharmaceutical as well as herbal industries.

ACKNOWLEDGEMENTS

We are thankful to the Management of Uka Tarsadia University, Gujarat, India, for providing necessary research facilities to conduct the study and supporting NP through Shri B. U.  Patel Research Fellowship.

Conflict of Interest: The authors have no conflict of interest to declare.

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