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Phytochemical analysis of Moringa Oleifera leaves extracts by GC-MS and free radical scavenging potency for industrial applications

Natural extracts have been of very high interest since ancient time due to their enormous medicinal use and researcher’s attention have further gone up recently to explore their phytochemical compositions, properties, potential applications in the areas such as, cosmetics, foods etc. In this present study phytochemical analysis have been done on the aqueous and methanolic Moringa leaves extracts using Gas Chromatography-Mass spectrometry (GCMS) and their free radical scavenging potency (FRSP) studied using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical for further applications. GCMS analysis revealed an extraction of range of phytochemicals in aqueous and methanolic extracts. In aqueous, extract constituents found with high percent peak area are Carbonic acid, butyl 2-pentyl ester (20.64%), 2-Isopropoxyethyl propionate (16.87%), Butanedioic acid, 2-hydroxy-2-methyl-, (3.14%) (also known as Citramalic acid that has been rarely detected in plant extracts) and many other phytochemicals were detected. Similarly, fifty-four bio components detected in methanolic extract of Moringa leaves, which were relatively higher than the aqueous extract. Few major compounds found with high percent peak area are 1,3-Propanediol, 2-ethyl-2- (hydroxymethyl)- (21.19%), Propionic acid, 2-methyl-, octyl ester (15.02%), Ethanamine, N-ethyl-N-nitroso- (5.21%), and 9,12,15-Octadecatrienoic acid etc. FRSP for methanolic extract was also recorded much higher than aqueous extract. The half-maximal inhibitory concentration (IC50) of Moringa aqueous extract observed is 4.65 µl/ml and for methanolic extract 1.83 µl/ml. These extracts can act as very powerful antioxidants, anti-inflammatory ingredient for various applications in diverse field of food, cosmetics, medicine etc.


Introduction
Plants and extracts of their various sections have been used for their medical characteristics and to cure specific ailments as well as general tonics, meals, and other methods to increase the body's immunity and vigor since ancient times (Ullah et al., 2020;Ageel et al., 1986;Gamal et al., 2010;Purena et al., 2018). However, since last few decades the interest of researchers has gone up dramatically to understand their detailed compositions and also to explore and establish their potential applications in diverse areas. In fact, it is the need of the hour to leverage the vital power of the nature to combat proliferating diseases like cancer, heart attacks, diabetes, rapid skin aging etc. and upcoming varieties of new alarming health concerns like recent concerns of Coronavirus disease in 2019 , which affects the respiratory system acutely (Varahachalam et al., 2021;Paliwal et al., 2020).
Different parts like seeds, roots, stem, bark, leaves, flower and fruits of the plant have their own phytochemical compositions and potential medicinal properties. Moringa have various species across the globe which are known for their variety of usages few examples of Moringa species are Moringa longituba, Moringa drouhardii, Moringa ovalifolia etc. (Leone et al., 2015). Moringa Oleifera is one of the magical plants considered in India due to its high medicinal properties. However, there is still a lot to unleash the potential of Moringa Oleifera by understanding their phytocomponents and variation in extraction due to solvents, understanding their potential properties and to establish their applications in var-ious fields. The present study is focused to investigate the phytochemical composition of the Moringa Oleifera leave's aqueous and alcoholic extracts by GC-MS and their free radical scavenging potencies, which make them useful for their further applications in cosmetic products to prevent skin damage due to free radicals generated because of pollution, UV from sunlight, smoke, also for animal feed etc.

Materials
Methanol of 99.8% purity, HPLC grade from SD fine chemical limited, distilled water having pH 5.3-7 and conductivity 2mS/cm. Raw leaves powder of Moringa Oleifera of India origin grown in the climate of tropics and sub tropics were used for the investigation. Extra pure 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) from SRL Pvt. Ltd was used for the free radical scavenging studies.

Preparation of aqueous and Methanolic extracts
The suspension of 5% Moringa Oleifera leaves were prepared in distilled water and in Methanol separately and were subjected to continuous stirring at 45-50°C for 8 h respectively. The resultant samples were filtered through Whatman filter paper 1 to extract the phytochemicals. The filtrates were concentrated by evaporating the solvents and were used to prepare the samples for further analysis.

Phytocomponents identification
The phytochemicals were identified based on their retention time, percentage of peak area and pattern of mass spectra and its comparison with the data of library of NIST11.LIB of National Institute of Standards and Technology (NIST).
2.5. Anti-oxidant/free radical scavenging potency (FRSP) using diphenyl picrylhydrazyl (DPPH) The Anti-oxidant efficacy and in other words free radical scavenging potency (FRSP) of the aqueous and methanolic extract of the Moringa leaves powder were determined by using DPPH. Methanolic solution of 1.52*10 -4 M of DPPH was used in 1:1 ratio with Moringa aqueous and methanolic extracts at varying concentrations, studied the scavenging activity as a function of time. FRSP percentage rate was studied after incubating the mixture of DPPH and extracts for 30 min and recording the UV absorption at 517 nm. The same was used to calculate the concentration of antioxidant required to reduce the concentration of DPPH to 50% (IC 50 ). Higher value of IC 50 indicates the lower antioxidant activity and vice a versa. The control samples were prepared using DPPH solution and mixing only with respective solvents (Aqua/Methanol) at 1:1 ratio and measured at 517 nm. FRSP% = (Control abs -Sample abs ) x100/Control abs 3. Results

Discussion
This study is among the very few reports where the phytochemical profile of Moringa extracts with their free radical scavenging potency studies have been reported. In this study, higher number of phytochemicals in methanolic extract (Table 3) of Moringa extract in comparison to aqueous (Table 1) could be due to difference in their polarity, which could have led to difference in the extraction of phytochemicals. These, phytochemicals have various industrial applications and medicinal properties like anti-tumor, anti-cancer, Insulin regulation, anti-oxidant etc. which makes it really a magical plant for food, medicine and suitable natural ingredient to explore its further applications in diverse areas like in cosmetics, personal care products etc.
Natural antioxidants are always of very high importance for health as a part of food and as a part of cosmetics for topical applications to combat the detrimental affects of free radicals on internal and external organs like skin aging etc. Both aqueous and methanolic Moringa extracts have excellent anti-oxidant/FRSP. However, in relative terms Methanolic extract found to have lower IC 50 (1.83 ml/ml) Fig. 4 reflecting higher free radical scavenging ability in comparison to of aqueous extract having higher IC 50 (4.65 ml/ml) Fig. 3. This could be mainly due to higher number of polyphenolic component extracted in Methanolic extracts and relatively at higher percentages, which is contributing towards higher scavenging potency at lower concentration. It has been found that Methanolic extract at 5 ml/ml gives the 88.5% FRSP and does not change significantly over the period of time and reaches maximum level of 92.8% FRSP. Whereas aqueous extract at 5 ml/ml show 35.8% FRSP at the initial time point and which further scavenge the DPPH free radical significantly up to 68.4% in 160 min Fig. 6, however still less FRSP of Moringa Aqueous extract than the Methanolic extract could be due to the same reason as explained above of having more polyphenolic components in Methanolic extract of Moringa as reflected in GC-MS results.

Conclusions
This study has investigated the Moringa aqueous extract and identified twenty-five phytochemicals and similarly in Methanolic extract fifty-four phytochemical components were identified. The higher number of these constituents in methanol may be due to polarity difference, which contributed to solubilize the various molecules. Few of phytochemicals reported here are rarely  detected in plant extracts like 3,3 0 -Iminobispropylamine, Butanedioic acid, 2-hydroxy-2-methyl-etc. and have potential applications. In further studies these extracts have shown substantial free radical scavenging potency, IC 50 of Moringa aqueous extract observed is 4.65 ml/ml and for methanolic extract 1.83 ml/ml. Also, found that Methanolic extract at 5 ml gives the 88.5% FRSP at initial time point and does not change significantly over the period of time and reaches maximum level of 92.8% FRSP. Whereas aqueous extract at 5 ml show 35.8% FRSP at the initial time point and which further scavenge the DPPH free radical significantly up to 68.4% in 160 min. These high FRSP make them very suitable ingredients for various applications like food, animal feed medicines, cosmetic etc. We are in the process of establishing the same in products of topical application to have skin benefits like prevention of skin damage, aging etc.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.