Nutritional and phytochemical screening of Moringa oleifera leaf powder in aqueous and ethanol extract

ABSTRACT Moringa oleifera has gained tremendous popularity in recent time due to its unlimited nutraceutical and functional properties. Present study was intended to investigate bioactive compounds present in Moringa oleifera leaf powder (MoLP). Moringa leaves were plucked, dried, ground, and were preliminarily analyzed for their proximate composition and mineral profile. Standard tests were conducted to evaluate flavonoids, alkaloids, saponins, proteins, amino acids, fats, and oils. Total phenolic content (TPC), total flavonoid content (TFC), total saponins content (TSC), total tannins content (TTC), FRAP assay, and DPPH radical scavenging property was also analyzed. Phytochemical investigation of Moringa oleifera leaf powder and its extract depicted the existence of rich bioactive compounds including amino acids, phenols, flavonoids, and saponins. The study illustrated that the phytochemicals in Moringa oleifera exhibit higher antioxidant activity against free radicals, revealing its strong nutritional worth.


Introduction
Moringa oleifera due to its abundant vitamins, minerals, and protein content is considered as a promising food plant, promoting several health benefits.M. oleifera is enriched in multiple therapeutically active chemicals: a good applicant supplemented with effect enhancing and neutralizing side-effects combinations.It belongs to the genus Moringa, family Moringaceae, and has 14 species, also called miracle tree, nature's gold, and sohanjna. [1]The nutrient profile of M. oleifera mostly depends on the soil composition, climatic characteristics, cultivating conditions, processing, and storage quality. [1]It is often recommended as a healthy plant to cure malnutrition; its leaves and other products are promoted as nutritional supplements for its strong vitamin profile. [2]M. oleifera is extensively consumed as food, medicines, cosmetic oil, and feed for livestock.It has been used as a medicinal plant to provide nutrients, promote health, decrease stress, and enhance food efficiency.Its leaves are especially beneficial in addressing chronic inflammations, including hypertension, diabetes, insulin resistance, cancer, and hypercholesterolemia. [3] M. oleifera leaves are an appreciable source of alkaloids, carotenoids, saponins, isothiocyanates, phenolic acids, glucosinolates, and flavonoids.They present more quantity of these compounds as compared to fruits, vegetables, and other food plants consumed as human nutrition.This high bioactive profile is famous to boost immune systems and defeat reactive oxygen species.They may also be used as additives on animal feed, an alternative to supplements, and a remedy to many inflammations due to its relative low cost. [4]M. oleifera leaves are enriched with tannins, the water soluble phenolic compounds that can bind to gelatin, proteins, and alkaloids, and have antibacterial and anti-inflammatory activity. [4]Saponins are anticancerous in nature, ranges between 64-81 g/kg of dry weight, lower the plasma cholesterol, and decrease enterohepatic circulation of bile acids by increasing their fecal excretion. [5]iological activites of M. oleifera leaves and phenolic compounds depend upon the solvent method used for the extraction of these bioactive compounds. [6]The flavonoid composition of Moringa leaves differs significantly when collected from different varieties.The concentration of flavonoids is dependent upon growing environment, treatment method, leaf maturity, and cultivar.A significant positive interaction (ƿ < 0.001) is found between extraction temperature and ethanol concentration.Total phenols decrease with the rise of temperature. [7]Polyphenols are the most vital antioxidants present in human diet.Several researches have shown a close relationship between many diseases such as diabetes, cardiovascular, neurodegenerative disorders, and consumption of polyphenol-rich diet. [8]Moringa is a highly nutritious medicinal plant that has the ability to cure infertility, obesity, hypertension, hyperlipidemia, bacterial infections, diabetes, inflammation, and oxidative stress.Preventive medication has been greatly improved by the use of natural plant.These plants contain free radical scavenging molecules such as phenols, alkaloids, flavonoids, amines, and turpines with a great tendency of antioxidant activity. [9]Previous studies reported that these polyphenols and antioxidants further minimizes the damages reported by many tissues during physiological process. [10]. oleifera tree has an unusual variety of valuable properties saddle in nutrition, medicines, and for many other industrial purposes. [11]M. oleifera has gained popularity in recent times due to its phytochemical, antioxidant profile and pharmacological properties.Additionally, the enriched phytochemical and antioxidant components of M. oleifera leaves also attributed to show anti-apoptotic and anti-inflammatory effects. [12]Moringa oleifera is a chief source of minerals, proteins, folates, phenols, and unsaturated fats, considered as a superb extract with valuable properties and hence can be integrated into the diet as a fortifier of any kind of food. [13]oringa oleifera is recounted to improve a wide series of biological activities including antiinflammatory, antioxidant, neuroprotective, hepatoprotective, anticancer, and antidiabetic.M. oleifera has been broadly preferred as a healthy food supplement due to its strong protection against numerous diseases and environmental toxins. [14]The high phytochemical profile of M. oleifera supports the usage of food plant as nutraceutical and pharmaceutical purpose to mitigate inflammatory and oxidative stress in human beings at a low cost and easy access.The present research emphasizes the health benefits and bioavailability of Moringa oleifera leaf powder and extract to treat oxidative stress-related diseases.In the present study, we planned to study the proximate and mineral composition of Moringa oleifera powder; the qualitative phytochemical grading in M. oleifera leaf powder and extract; and free radical scavenging activity and total antioxidant capacity of aqueous and 70% ethanol extract of Moringa oleifera leaf.

Materials and methods
The M. oleifera leaves were collected from the lawn of botanical garden, University of Sargodha.The leaves were washed with fresh water, and the inedible parts were removed. [15]After that, the experimental work was performed at the soil and water testing laboratory, Institute of Food Science and Nutrition, University of Sargodha, Pakistan.

Aqueous and ethanol (70%) extract formation
M. oleifera leaf extracts (MoLE) aqueous and ethanol 70% were prepared according to the process discussed in detail in the previous manuscript. [15,16]

Determination of yield %
The Yield % of MoLE was calculated by following formula:

Proximate analyses of Moringa oleifera leaf powder (MoLP)
The proximate analysis of MoLP was done to find the moisture content (%), crude proteins, crude fats, crude fiber, ash carbohydrates, and nitrogen free extract (NFE).The proximate analysis was performed by following the procedure of Association of Official Analytic Chemist. [17]

Estimation of Minerals of Moringa oleifera Leaf Powder
The analyses of MoLP for N, P, K, Ca, Mg, Zn, Cu, Mn, Cd, Fe, Ni, and Pb were performed by following the methods of digestion, filtration, dilution, and reading according to the procedure of Association of Official Analytic Chemist. [17]N by Kjeldahl's method (Fahey, 2005), P by Colorimeter, Phospho-Molybdate Vonadate, K by Flame Photometer, and Ca, Mg, Cd, Ni, Pb, Zn, Cu, Fe, and Mn by Atomic Absorption Spectrophotometer. [18]ytochemical screening of Moringa oleifera leaf extract Phytochemical examinations of flavonoid, phenols, alkaloid, tannins, saponins, glycosides, steroids, triterpenoids, phytosterols, reduced sugars, carbohydrates, proteins, amino acids, fats, and oil present in the MoLE were carried out as per standard tests.[19,20] Presence of flavonoids.Flavonoids were detected by Ferric Chloride test.The formation of dark brown or blackish red colored precipitates depicted flavonoids.

Presence of phenols.
Phenols were detected by Folin-Ciocalteu test.The formation of gray or blackish colored precipitates showed phenols.

Presence of alkaloids.
Wagner's test was carried out to confirm alkaloids in M. oleifera leaf extract.Reddish brown precipitates developed when 2 ml extract was mixed thoroughly in 1% HCl solution with 0.5 ml of Wagner's reagent.
Presence of saponins.Extract (0.5 g) was taken in 2 ml water and continuously shaken; foaming occurred for 10 min, which indicated the saponins' concentration.
Presence of tannins.Add 1% gelatin reagent containing few drops of NaCl in 1 ml of extract; white precipitates appear to confirm tannins.

Presence of glycosides.
MoLE and dilute HCl were mixed and legal's reagent was used to dissolve sodium nitropruside in pyridine and NaOH.Glycosides were confirmed due to the appearance of blood-red color.

Bioactive compounds
The bioactive compounds were determined from MoLE by using the different methods.For measuring the bioactive compounds in MoL extract, different tests were performed including total polyphenols, total flavonoids, total saponins, total tannins, DPPH, FRAP, and ABTS.
Total polyphenols.TPC (Total polyphenols content) of MoLE was estimated by Folin-Ciocalteu methodology as described by. [11,21]About 3 ml distilled water and 50 µl MoLE were mixed in test tube and put on vortex for 15 min.Then, 750 µl of Na 2 CO 3 and 950 µl of distilled water was added, followed by incubation for 30 min at 25°C.UV-visible spectrophotometer was used to measure absorbance at 765 nm.Total polyphenols content was represented as mg Gallic acid equivalent per 100 ml. [11]tal flavonoids.TFC (Total flavonoid content) was calculated by following AlCl 3 method as described by. [11,21]About 1 ml MoLE and 4 ml distilled water were filled in test tube and added to 5% NaNO 2 (300 µl), put aside for 15 min, then a methanol solution of 10% AlCl 3 (300 µl) by vortex was added. 1 M NaOH (2 ml) was mixed with distilled water to make 10 ml of total volume.UV-visible spectrophotometer was used to measure absorbance at 415 nm.TFC was represented as mg quercetin equivalent per 100 ml. [11]tal saponins.TSC (Total saponins content) was found as described by. [11,22]A sample of MoLE (250 µl) and 8% vanillin reagent (250 µl) were mixed with 72% H 2 SO 4 (2.5 ml) for 10 min, and absorbance at 544 nm was measured by UV-visible spectrophotometer.Total saponin content was represented as mg diosgenin equivalent per 100 ml. [11]otal tannins.TSC (Total tannins content) was calculated by following the method as described by. [11,23]A sample of MoLE (0.5 ml), 4% vanillin reagent (3 ml) and 36% HCl (1.5 ml) were mixed thoroughly and kept for 15 min in darkness at room temperature.Absorbance at 500 nm was calculated by UV-visible spectrophotometer.Total tannins content was represented as mg catechin equivalent per 100 ml. [11]dical scavenging assay DPPH (1,1-diphenyl-2-picryl-hydrazyl) assay and ABTS (2,2-azinobis-3-ethylbennzothiazoline-6-sulphonnnic acid) assays were conducted to assess the antioxidant activity of MoLE by the method of. [11,23]The absorbance was noted at 515 nm for decline in DPPH and 734 nm for ABTS.The graduation curve for DPPH and ABTS was set by following Trolox (standard); data were represented as mM Trolox equivalents/100 ml. [11]rric reducing antioxidant power assay Ferric Reducing Antioxidant Power (FRAP) was assayed to calculate the antioxidant activity of MoLE by the method of.[24] .About 1 ml extract and varying amounts of FeSO 4 were taken, 6 ml of FRAP reagent was added, and kept for 30 min in a water bath at 37°C.The absorbance was determined at 593 nm, and a decline in values was found.[25] Data analysis and statistical application All results were statistically analyzed by applying One-way analysis of variance (ANOVA),and data were organized as mean ± SEM.Statistical analysis was performed by using MiniTab software 18.1.

Results and discussion
Weight of MoLP.Weight of Moringa oleifera Leaves (MoL) fresh, dried, and powder form were measured and a considerable decrease in the weight was found.
Extraction rates of MoLP.MoL were dried and powdered @ 11.50%.The extraction rate of MoLP in distilled water and 70% ethanol was 13.23, and 12.65%, respectively.
Proximate analysis of Moringa oleifera leaf powder.Proximate composition of MoLP is showed in Table 1.The moisture, crude protein, fat, fiber, ash, and NFE of M. oleifera leaves powder were 6.30, 26.23, 2.82, 10.25, 8.24, and 46.14, respectively.The moisture content of current study was similar to the previous study. [26]Another study showed the similar results of moisture to our outcomes. [27]esearch data suggested that the low content of moisture in MoLP that can reduce the microbial activity and enhance storage value.However, the discrepancies are augmented by processing and different storage methods. [28]he protein contents of MoLP as fall in the same range as of the previous study (22.99-29.36. [27]ats content of MoLP is less than form from different plant leaves of M. oleifera. [26,27]However, the fat value of the present research is also parallel to the Kenaf leaves. [29]High proteins and fats highlighted the energy value of MoLP and its' efficacy as a main source of energy for human body. [28]iber contents of MoLP is more than the previous study. [26,27]The ash content in MoLP is similar to the soybean and corn meal. [30]The high value of ash indicated that MoLP is a good source of mineral elements.NFE is similar to M. oleifera leaves of different plants. [26]. [31]The little bit variation in carbohydrates values noted in this work to previous study due to variation in soil composition and climatic conditions. [28]neral composition of Moringa oleifera leaf powder.The mineral profile of MoLP are showed in Table 2.The contents of Cd, Ni,, Pb, Mg, Zn, and Cu (±1.0) are 0.1, 0.5, 10, 0.6, 25 and 8.0, respectively.The current outcomes are similar to the range of the previous study. [32]However, the slight variations in the mineral content may be due to soil nature, weather conditions, and growth pattern of leaves.
The outcome of K is similar to previous result (1.3-2.1). [25]The Fe value of MoLP is more as compared to Acer pictum thumb. [33]The current research agreed with the findings of Kumar et al. [34] that the leaves of the particular plants are a rich source of minerals.The study suggested that plants contain freely accessible phytonutrients and restocked nutrients.Though, the nutrients of the plants may be influenced owing to the growth level of leaves and by the abiotic and biotic settings of the environment. [28]ytochemical profiling of MoLE.In the present study, the phytochemical analysis of MoLP was conducted by using an aqueous and organic solvent (70% ethanol).Results depicted that phenolic acids are frequently miscible in nonpolar solvents. [34]Table 3 depicted the results of the basic phytochemicals of the aqueous and 70% ethanol of MoLE.
The aqueous extract showed the moderate abundance of flavonoids, steroids, carbohydrates, and amino acids, while least availability of phenols, alkaloid, saponins, glycosides, proteins sugars, fats, and oils was detected.Triterpenoids, tannins and phytosterol were not detected in aqueous MoLE.The data are in accordance with results mentioned by Sudha et al. [19] More abundance of flavonoids, phenols, alkaloids, and carbohydrates were reported in 70% ethanol extract.Moderate abundance of proteins, glycosides, steroids, amino acids, triterpenoids, and tannins and least availability of phytosterols, reducing sugars, and fats were found in 70% ethanol extract.The phenols and flavonoids found in MoLE might be active as usual antioxidant supplements. [34]eone et al. [4] attributed the medicinal worth of M. oleifera to the higher yield of phenolic acids, saponins, alkaloids, flavonoids, polyphenols, and tannins.
The ferric chloride test for flavonoids indicated its moderate availability in aqueous while more abundant availability in ethanol extract.The Folin-Ciocalteu test showed the presence of grayish black precipitates to indicate the more abundance of phenols in alcoholic solvent than water.The less concentration of alkaloid in aqueous MoLE and higher in 70% ethanol MoLE was proved by reddish brown precipitates appeared by using Wagner's reagent.Less foaming appears in aqueous whereas rich foaming was observed in organic solvent that revealed the presence of saponins in MoLE.All phytochemicals are supported by the previous study conducted by Sudha et al.. [19] Gelatin test conducted for tannins was negative in aqueous and positive in 70% ethanol extract.Legal's test showed pink to red colored precipitates for glycosides existence.Moderate abundance of steroids in aqueous and more in ethanol extract was confirmed by the appearance of brown ring with Salkowski's reagent.Proteins and amino acids gave yellow and blue precipitates by using reagents Xanthoproteic and ninhydrin, respectively, in both extracts of M. oleifera.Fehling's reagent, Molicsh's reagent, and filter paper pressing was used to perform decisive tests for the indication of reducing sugars, carbohydrates, and fats and oils, respectively, discussed in Table 3.The Salkowski's test was only positive in 70% MoLE but negative in aqueous MoLE.Phytosterols were detected in ethanol extract by performing Liebermann-Burchard's test, while they were absent in aqueous extract.The observations noticed in the current research were similar to the previous research Sudha et al., [19] and emphasized the nutritional worth of M. oleifera being enriched with carbohydrates, proteins, and amino acids.Data in our study revealed the high yield of some phytochemicals including amino acids, proteins, alkaloids, saponins, steroids, and flavonoids, which is quite related to the study of Okiki et al., [35] phytochemical screening of MoL from Nigeria showed more amount of flavonoids, alkaloids and saponins.4 represents total flavonoid contents, total phenolic contents, total saponin content,s and total tannin contents in aqueous and 70% ethanol MoLE.TPC was measured in aqueous and 70% ethanol extract are similar to the aqueous and 50% ethanol extract of Moringa oleifera. [11,36]urther, MoLE outcomes showed TPC concentration similar to the leaf extract [37, whereas higher concentrations in moringa infusion and methanolic extract. [38]Du Toit et al. [36] reported that moderately reaped moringa leaves contain high amount of TPC from spring to summer climates.Food plant contains physiologically and therapeutically active flavonoid, tannins, and saponins along with phenolic acids. [14]In this research, TFC was found in aqueous and 70% ethanol MoLE as 3.91 and 4.02 mg QE/100 ml, respectively; the values are in accordance with aqueous and 50% ethanol extract of Moringa oleifera. [11]otal tannins content of aqueous and 70% ethanol was 10.09 and 12.58 mg CE/100 ml, respectively.The outcome of current study is similar to the aqueous and 50% MoLE. [11]The TSC of liquid samples showed highest extraction in aqueous and alcoholic extract. [39,40]The current results of tanning are similar to previous study. [41]Total Saponin content was calculated in the range from 36.26 to 25.14 mg DE/100 ml.

Free radical scavenging and total antioxidant capacity in MoLE.
Reactive oxygen species accumulated in biological systems and cause various degenerative disorders due to oxidative damage.There is an equilibrium required for the scavenging activity of antioxidants and production of free radicals and oxidative stress due to minor changes in this equilibrium. [42]Ahmed et al. [34] justified that antioxidant constituents present in dietary sources are comparatively safe.They suggested that M. oleifera leaf extracts contribute antioxidant property against free radicals and provide defense against oxidative mutilation.
Table 4 revealed that MoLE (70% ethanol) proved to be more functioning than aqueous MoLE.This is perhaps due to high content of phenols, flavonoids, tannins, and saponins in MoLE.MoLE is blessed with more free radical scavenging activity, reducing power, and anti-oxidant capacity. [43,44]It was mentioned that polyphenols contain phenol units considered to be a class of biologically active compounds in MoLE, further represent a group of secondary metabolites efficacious in curing chronic diseases owing to their strong anti-oxidant potential. [34]he anti-oxidative activity of MoLE varied with different solvents used for extraction.The MoLE aqueous has more anti-oxidative potential with the ABTS assay than the MoLE 70% ethanol.The results are in agreement with the values in water and ethanol extract respectively. [11]he anti-oxidant activity estimated with the DPPH assay was higher in 70% ethanol MoLE (28.48 mM trolox/100 mL) and aqueous (33.42 mM trolox/100 mL).Moreover, DPPH inhibition in methyl and aqueous extracts of M. oleifera are similar to the current study.Ahmed et al. [34] investigated that M. oleifera leaf extract significantly decrease DPPH radicals.They narrated that anti-oxidants donate an electron or hydrogen to DPPH, hence balances its free radical property.Data provided in this research suggested the relationship between phenolic acids and anti-oxidative response.70% ethanol MoLE contain more phenolic acids due to organic in nature justified the high anti-oxidative action as compare to aqueous MoLE.This particular behavior was also supported by the study in the leaves of Morus nigra and Morus alba L.. [45] Ferric Reducing Antioxidant Power (FRAP) assay was performed to evaluating the total antioxidative activity of MoLE in aqueous and 70% ethanol extract.The solution of FeSO 4 was used and concentration of extract was noted as 0.87 and 1.24 mmol FeSO 4 in aqueous and 70% ethanol MoLE respectively.These results clearly indicate that M. oleifera leaves are potentially more beneficial than seeds. [43]

Conclusion
It is concluded that 70% ethanol extract of Moringa oleifera leaves are contained appreciable amount of flavonoids, alkaloids, glycosides, steroids, phenols, and proteins.Based upon radical scavenging activities, total reducing power and total antioxidant capacity measured by ABTS, DPPH, and FRAP assayed in Moringa oleifera leaves depicts its worth as a natural effective.However further research is required to isolate individual components to develop antioxidants for pharmaceutical and food industries.
Presence of steroids.Steroids were detected by Salkowski's test in which MoLE was dissolved in chloroform and filtered, mixed with concentrated H 2 SO 4 .Brown color appeared to indicate the presence of steroids.Protein was confirmed by following Xanthoproteic test.MoLE was treated with few drops of concentrated HNO 3, which resulted in the formation of yellow colored precipitates.Amino acids were indicated by Ninhydrin test.Extract was mixed with 0.25% Ninhydrin reagent and boiled for few minutes; blue colored precipitates appeared.Brick red colored precipitates appeared to show the availability of reducing sugars in MoLE when 1 ml Fehling's reagent was added in 2 ml of each extract.The aqueous solution and 70% ethanol MoLE was treated with Molicsh's reagent.After dissolving in 5 ml distilled water, a purple ring appeared.Now mix few drops of alcoholic α naphthol solution in a test tube to show the presence of carbohydrates.
Presence of protein and amino acids.Detection of fats and oils.Extracts were pressed under the folds of filter paper and results were observed.Detection of reducing sugars.Presence of phytosterols.Liebermann-Burchard's test was effective to indicate phytosterols in MoLE.Add Chloroform in MoLE and filter.A few drops of acetic anhydride was mixed, boiled, and cooled rapidly.Finally, H 2 SO 4 was mixedtill brownish ring appeared.Detection of triterpenoids.Triterpenoids were detected by Salkowski's test in which MoLE was dissolved in chloroform.H 2 SO 4 (few drops) was added after filteration and mixed thoroughly.Golden color appeared to indicate the presence of triterpenoids.

Table 4 .
Free radical scavenging activity and total antioxidant capacity of aqueous and 70% ethanol mole.