Biochemical and functional properties of (Moringa oleifera) drumstick leaf powder and its utilization

Introduction

Moringa oleifera, commonly known as the "drumstick tree" or "miracle tree," (Koul & Chase, 2015) [28] originated from India but has spread to various regions, including Ethiopia, the Pacific islands, Florida, Sudan, the Caribbean, the Philippines, South Africa, Asia, and Latin America (Fahey, 2005) [18] . In the scientific classification, Moringa oleifera belongs to the family Moringaceae, genus Moringa, and species oleifera (Razis et al.,2014) [52] . The moringa genus comprises a total of 13 identified species, with Moringa oleifera being one of the most prominent members of the Moringaceae family (Saini et al., 2016) [53] . Moringa oleifera is categorized as a tropical plant and is well suited to a diverse range of agro-climatic environments. It is typically grown in regions that are tropical or subtropical and semi-arid. The optimal temperature range for its growth is between 25 and 35⁰C, but it can endure temperatures as high as 48⁰C (Palada et al., 2012) [46] . Moringa oleifera is one of the most useful trees having great economic importance. Almost every plant part from roots to flowers of moringa are used for various purposes. Fresh, green and young pods, young shoots and leaves are used as vegetables (Ebert & Palada, 2017) [16,47] . Epidemiological research has demonstrated that M. oleifera leaves are a valuable nutritional source and have shown various beneficial properties, including anti-tumour, anti-inflammatory, anti-ulcer, antiatherosclerotic, and anti-convulsant activities (Chumark et al., 2008; DanMalam et al., 2001; Dahiru et al., 2006) [12,13,14] . Moringa contains large amounts of β-carotene, ascorbate (vitamin C), α-tocopherol (vitamin E), and iron, and ranking second in protein content (Freiberger et al., 1998) [19] . Moringa contains seven times more vitamin C than oranges, ten times the vitamin A International Journal of Advanced Biochemistry Research 2025; 9(7): 686-700 of carrots, higher calcium and protein content than milk, fifteen times more potassium than bananas, and a remarkable twenty five times more iron than spinach (Liu et al., 2018; Gopalkrishnan et al., 2016) [31] . Moringa is also recognized as a rich source of polyphenols and antioxidants (Mishra et al., 2011) [38] . The content of natural antioxidants, including total phenolics, and vitamins A, C, and E, ranged from 74-210 μmol/g for phenolics, 70-100 μmol/g for ascorbate (vitamin C), 1.1-2.8 μmol/g for β-carotene, and 0.7-1.1 μmol/g for α-tocopherol (vitamin E) on a dry weight basis (Yang et al., 2007) [61] . Moringa oleifera is used as an alternative to imported food supplements in developing countries to treat and combat malnutrition, especially among infants and nursing mothers, by virtue of its chemical constituents (Dhakar et al., 2011) [15] . Various extracts were derived from moringa leaves, and these extracts exhibit the ability to inhibit the growth of bacteria and fungi (Mehmood et al., 2022) [35] . Considering the significance of Moringa oleifera and the factors affecting its nutritional composition, this study aims to evaluate the biochemical and functional properties of Moringa oleifera leaf powder. The research focuses on elucidating its phytochemical makeup and nutritional benefits to optimize its utilization and enhance its application in diet formulation and medicinal use.

Material and Methodology Collection of Moringa oleifera varieties

Moringa oleifera Var. (ODC 3) grown in Marathwada region and a wild genotype were selected for research based on visual appearance of fresh, dark green leaves, suitable for making dried leaf powder.

Sample preparation

Fresh, dark green leaves of M. oleifera were air-dried until reaching a constant weight, then pulverized into fine powder using an electric blender. The resulting powder was stored in an airtight container at room temperature for biochemical analysis and further utilization, following the protocol by Stevel and Babatunde (2013) [58] with minor modifications.

Nutritional qualities of Moringa oleifera leaf powder

Moisture content 5g of moringa leaf powder subjected to oven drying at 105⁰ C for 4 hours, followed by cooling in a desiccator until a constant weight was achieved and the loss in weight was calculated as moisture content according to AOAC (2005) with minor modifications.

Ash content

A 5g sample was placed into a silica crucible over low flame and heated in a muffle furnace at 600⁰C for 4 hours.

After cooling, consistent weight measurements were obtained to determine the ash content by calculating the difference between initial and final weights, following the procedure given by AOAC (2005) with minor modifications.

Carbohydrate

The total carbohydrate content of Moringa oleifera leaf powder was determined using the Anthrone method as proposed by Jayaraman in 1981 [24] . After hydrolysing 100 mg of leaf powder in HCl, the samples were neutralized, treated with Anthrone reagent, and quantified spectrophotometrically at 620 nm.

Protein

Protein content was estimated using Lowry's method (Lowry et al., 1951) [32] with minor modifications. Glass test tubes were filled with varying volumes of BSA solution and test samples, then adjusted to 4 ml with distilled water. After adding reagents and incubating, absorbance at 660 nm was measured, and protein concentrations were determined using a standard curve.

Mineral composition of Moringa oleifera leaf powder

Determination of minerals was estimated by Raghuramulu et al., (1983) with minor modification.

Preparation of ash solution

Preparation of ash solution took place using the method given by Raghuramulu et al., (1983). 1 g Ash was mixed with 0.5-1 ml distilled water, followed by addition of 5 ml hydrochloric acid and evaporation to dryness twice. Then, 4 ml hydrochloric acid and water were added, heated, filtered, and made up to 100 ml in a volumetric flask used for phosphorus, iron, and calcium determination.

Iron estimation

1.5 ml of ash solution was mixed with 1 ml 30% H2SO4 and 1 ml 7% potassium persulfate. Then, 1.5 ml 40% potassium thiocyanate solution was added. Red coloration was measured at 540 nm over 20 minutes. Standard aliquots (10-50 μg) were subjected to the same treatment.

Calcium estimation

Amount of calcium was estimated using titrimetric method given by Raghuramulu et al., (1983). The ash solution was treated with ammonium oxalate and ammonia was added, centrifuged, then titrated with KMnO4 after adding H2SO4 until a faint pink colour persisted.

Phosphorous estimation

The amount of phosphorus was estimated by Fiske and Subba Raw method given by Raghuramulu et al. (1983). 2 ml of ash solution was mixed with 5 ml 10% trichloroacetic acid, centrifuged, and 3 ml of supernatant collected. To this, 0.4 ml of Aminonaphthol Sulphonic Acid (ANSA) was added. Standard aliquots (8-40 μg) were subjected to the similar treatment, volume adjusted to 10 ml, and incubated for 20 minutes before measuring color at 660 nm.

Ascorbic acid (vitamin C)

Ascorbic acid content was determined by titrating a known sample weight with 2,6-dichlorophenol indophenol dye using oxalic acid using procedure given by AOAC 2000.

Antioxidant composition in Moringa oleifera leaf powder Preparation of extracts

Mature leaves of Moringa oleifera Var. ODC-3 and wild variety subjected to washing and shade drying, followed by grinding into powder. Extracts were obtained by mixing 10 gm leaf powder with ethanol and methanol followed by centrifugation, and evaporation. The resulting filtrate was dissolved in the same solvent for further analysis. Ethanolic extract was prepared following a method outlined by Olatunde & Dikwa in 2014 [42] , while methanolic extract was prepared using a method described by (Pavithra et al., 2009) [47] .

Total phenolics

The quantification of phenolic compounds in the leaf powder extracts were determined using the Folin-Ciocalteau reagent, following the method described by El Sohaimy & Masry in 2014 [17] .

DPPH (2.2-Diphenyl-1-picrylhydrazyl) activity

The assessment of the free radical scavenging activities of Moringa oleifera leaf powder extracts were carried out using procedure given by Rakesh et al., (2010) [51] . To assess antioxidant activity, 1 ml of methanolic and ethanolic extracts were mixed with 0.5 ml of 0.15 mM DPPH solution, incubated for 30 minutes at 20⁰C, and absorbance measured at 517 nm to determine IC50 value representing the concentration needed to scavenge 50% of DPPH free radicals.

Flavonoids

Total flavonoid analysis was done by the method given by Benitez et al., (2011). The determination of the total flavonoid content was conducted through a colorimetric assay. 100 μl of methanolic and ethanolic extracts were mixed with distilled water, followed by sodium nitrite, aluminium chloride, and sodium hydroxide. After dilution, absorbance was measured at 510 nm.

Phytochemical analysis of Moringa oleifera

Quantitative analysis of phytochemicals Alkaloids: Quantitative analysis of alkaloids was done by using procedures outlined by Krishnaiah et al., in (2009) [29] . A 5 g sample was extracted with 200 ml of 10% acetic acid in ethanol for 4 hours, then concentrated. Alkaloids were precipitated with ammonium hydroxide, collected, washed, filtered, dried, and weighed.

Flavonoids

Quantitative analysis of alkaloids was done by using procedures outlined by Krishnaiah et al., in (2009) [29] . 10 gm leaf powder was subjected to repetitive extraction with 100 ml 80% aqueous methanol and ethanol at room temperature. The filtrate was evaporated to dryness in a crucible over a water bath until a constant weight was achieved.

Total Tannins (TT)

Determination of tannin content was carried out through the Folin-Ciocalteu assay method. This method was in accordance with the procedure outlined by Tamilselvi et al., in 2012 [59] .

Qualitative analysis of phytochemicals

The methanol and ethanol extracts were screened for active phytochemicals (tannins, alkaloids, triterpenoids, flavonoids, saponins, anthraquinone glycosides, carbohydrates, proteins, amino acids) using methods described by Harborne JB (1998) [21] and Kokate CK (2005) [27] .

Tannins: The presence of tannins was confirmed by Ferric chloride test with the formation of a blue color indicating their presence. [3] .  Water solubility index (WSI): The water-solubility index (WSI) of the M. oleifera leaf powder determined by the method given by (Hernandez-Diaz et al., 2007 [22] .  Reconstitution Index (RI): The reconstitution index of the samples was assessed using the procedure outlined by Onwuka in 2005 [44] .  Bulk density (BD) determination: The bulk density (BD) of the samples was assessed using the procedure outlined by Onwuka in 2005 [44] .  Rehydration Ratio (RR): The rehydration ratio (RR)of the samples was assessed using the procedure outlined by Krokida &Marinos-Kouris in 2003 [30] .  Swelling index (SI): The swelling index was determined by following the method described by Ukpabi & Ndimele in 1990 [60] .

Alkaloids

 Antimicrobial activity screening of Moringa oleifera:

The stock solution was prepared for antimicrobial screening using the extract obtained from the abovementioned method. 100 mg/ml samples were subsequently prepared by diluting them with ethanol, methanol, and water.  Micro-organisms for antimicrobial activity: The microorganisms used in present study were obtained from Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani. List of microorganisms shown in Fusarium oxysporum Bacillus substilis 2.

Aspergillus niger Pseudomonas fluorescens 3.

Trichoderma viridae Escherichia coli

Antibacterial and Antifungal screening (in vitro)

The antimicrobial efficacy of Moringa oleifera compounds was evaluated using the well diffusion method. Various strains of bacteria and fungi were inoculated onto prepared culture plates, followed by the introduction of extracts into wells created on the agar surface. After 24 hours at 37±2 ºC for bacterial evaluation and 48 hours at 25±2 ºC for fungal assessment, zones of inhibition were measured, including the well diameter, to determine efficacy.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:

The GC-MS analysis of a methanolic extract of Moringa oleifera leaf powder was performed with Jeol Accut of GCv GSHRMS.

Preparation of Ready to Serve Soup from Moringa oleifera

Leaf Powder: Moringa oleifera leaves were airdried at room temperature until a constant weight was achieved, then pulverized into a fine powder using an electric blender. The powder was stored in an airtight container at room temperature for later use in soup preparation. Additionally, other ingredients listed in Table 2 were cleaned, chopped into small pieces, and sun-dried for 5-6 hours before being utilized in soup preparation. Different formulations were prepared by mixing sugar, cornstarch, cumin, black pepper, salt, coriander, garlic, onion, tomato, lentil, sodium benzoate with 3 treatment including control sample. Only the most preferable of that combination was selected through sensory evaluation.

Result and Discussion

Nutritional qualities of Moringa oleifera leaf powder

The nutritional profiling of two varieties of Moringa oleifera was analysed comprehensively to evaluate their quality. Ash, moisture, carbohydrate, and protein content were estimated and reported in Table 3. The high amount of moisture content was observed in ODC 3 leaf powder (11.54%), than in wild variety (7.94%). Ash content in leaf powder of ODC 3 and wild variety was (25%) and (33.6%) respectively. Similar result for moisture and ash content was reported by Olusanya et al., (2019) [43] and Madukwe et al.

(2013) [33] in Moringa oleifera leaf powder. Looking on the carbohydrate, reveals a significant higher content present in ODC 3 and wild were 8.1gm/100gm and 8.0gm/100gm respectively. In addition, protein content in ODC 3 is 25.1 gm/100 gm and 23.2 gm/100 gm in the wild variety. Similar findings for carbohydrate and protein content in Moringa oleifera leaf powder were reported by (Gopalakrishnan et al., 2016) [20] .

Mineral composition of Moringa oleifera leaf powder

The results revealed that Moringa oleifera leaf powder was found to be significantly rich in minerals viz., iron, calcium, phosphorus and data regarding mineral composition was mentioned in Table 4. Estimated iron content was 80 mg/100gm and 70mg/100gm in ODC-3 and wild varieties respectively. Similarly, Mikore and Mulugeta (2017) [37] reported iron contents of 80.03±2.50 mg/100gm on the basis their study. Calcium content was found to be higher in ODC -3 880.16mg/100gm whereas wild variety contains 760.12mg/100gm of calcium. The results of this study were less than the findings of (Raghavendra et al., (2016) [49] ; Abuye et al., (2003); and Melesse (2011) [2,36] reported Ca contents of 971 mg/100gm. In this study, phosphorus content in Moringa oleifera leaf powder was found to be significantly higher in the ODC-3 variety (330 mg/100g) compared to the wild variety (290 mg/100g). This result findings were similar with Penalver et al. (2022). [9,26] . Vitamin-C (mg/100gm) 625 500

Antioxidant composition of Moringa oleifera leaf powder

Antioxidants like total phenolics, DPPH, flavonoids, and total tannins were calculated and mentioned in Table 6. In this study, significantly higher antioxidant activity was observed in ethanolic extract than in methanol. The result of this study showed that the total phenolics in ethanolic extract of ODC-3 and wild variety were 30 mg/g and 31 mg/g respectively, whereas total phenolics in methanolic extracts were 32 mg/g and 29mg/g in ODC-3 and wild variety respectively. All these findings were similar to the TPC value (45.4 mg /g) given by Sreelatha & Padma (2009) [57] . The DPPH scavenging activity of the ethanolic extract of ODC 3 variety of moringa was (71.14%) and in wild variety (60.69%) scavenging activity was observed which was higher than the methanolic extract of ODC-3 (46.26%) and wild (52.23%) respectively. These results were in a harmony with (Abdulkadir et al.,2015) [1] who mentioned (58.62%) DPPH scavenging assay in the case of methanol solvent. In another study, ethanol extract was found to exhibit the highest DPPH activity (53.30% -71.10%) reported by (Nobossé et al.,2018) [39] . Flavonoid content present in the ethanolic extract of ODC 3 and wild variety were (30.7 mg/g and 30.6 mg/g) respectively which was higher than the methanolic extract of ODC 3 and wild were (30.4mg/100g and 30.8mg/100g). Similar findings were reported by Shanmugavel et al. (2018) [54] , showing a total flavonoid content of 22.16 mg/g in ethanolic extract, consistent with previous studies (Pakade et al., 2012) [45] .

The ethanolic extracts of ODC-3 and wild varieties of Moringa oleifera leaf powder contained 12 mg/g and 10 mg/g of total tannin, respectively, higher than their methanolic extracts, which contained 10 mg/g and 8 mg/g of total tannin, respectively. Shakeela et al. (2023) reported 10.09 mg/g total tannin in methanolic extract and 13.08 mg/g in ethanolic extract which was similar to our current findings. (2020) [4] . Ethanolic extracts of wild and ODC-3 Moringa oleifera had the highest flavonoid content (28 mg/g and 22 mg/g, respectively), while methanolic extracts showed 20 mg/g (wild) and 18 mg/g (ODC-3). Results were similar to Ajayi & Fadeyi (2015) [6] . Ethanolic extracts of ODC-3 and wild Moringa oleifera had higher saponin content (9 mg/g and 8 mg/g) compared to methanolic extracts (7 mg/g) in both wild and ODC-3, similar to findings by Ogbe & Affiku (2011) [40] . Fats and fixed oils --+++ +++ +++ +++ *The yield obtained was graded as high (+++); moderate (++); low (+); nil (--) based on the intensity of the color the test compared to control in each case.

Reconstitution properties of Moringa oleifera leaf powder:

The study examined various physicochemical properties of ODC-3 and wild varieties of Moringa oleifera as result mentioned in Table 9. The result of this study showed that ODC 3 had a higher water absorption capacity (59%) compared to the wild variety (57%). The findings aligned with previous studies, such as those reported by Singh et al. (2010) [56] . ODC 3 variety exhibited a higher water solubility index (10%) compared to the wild variety (8%), similar to the study conducted by (Singh et al., 2012) [55] . The reconstitution index provides valuable insights into the ability of a substance to form a concentrated solution upon reconstitution. Slightly higher reconstitution index in ODC 3 (7g/ml) compared to the wild variety (6.9g/ml). Additionally, ODC-3 had a slightly higher bulk density (0.33 g/ml) compared to the wild variety (0.32 g/ml). Whereas higher swelling index in ODC-3 (3.6%) compared to the wild variety (3.3%) implies that ODC-3 could absorb and retain more water. Reconstitution Index (g/ml) 7 6.9 4.

Bulk Density (g/ml) 0.33 0.32 5.

Swelling Index (%) 3.6 3.3

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis:

The methanolic extract of Moringa oleifera leaf powder was used for chromatography separation. The GC-MS Chromatograph with various peaks of compounds detected in the methanolic extract of Moringa oleifera leaf powder is shown in 10. Table 11 presents the compounds, their common name, molecular formula, molecular weight, retention time, compound type and their biological activity. A total of 40 bioactive compounds were identified, showing the diverse chemical profile of the extract. These findings align with Karthika et al. (2016) [25] , who identified 28 bioactive compounds in the ethyl acetate extract of moringa leaf powder. 1.

Antifungal activity of different leaf extracts Moringa oleifera:

Leaf extract of Moringa oleifera showed antifungal activity against Fusarium oxysporum, Aspergillus flavus, and Aspergillus niger as mentioned in Table 12. In case of aqueous leaf extract of Moringa oleifera, the results showed a similar zone of inhibition, measuring 3mm, for both Aspergillus flavus and Fusarium oxysporum, as illustrated in Fig. ,3. However, aqueous extract did not exhibit any zone of inhibition against the Aspergillus niger, suggesting a lack of antifungal activity. The result obtained by (Bagheri et al., 2020) [10] was also similar to this findings. He ethanolic extract of Moringa oleifera leaf powder showed varied zones of inhibitions as shown in Fig 3.

against different strains. both Aspergillus flavus and Aspergillus niger exhibited a similar zone of inhibition, measuring 3.5 mm at a concentration of 100mg/ml of the ethanolic extract and 3mm zone of inhibition was observed for Fusarium oxysporum. Oladeji et al. (2019) [41] reported a similar zone of inhibition for Aspergillus flavus (5. [34] . They observed the maximum zone of inhibition of (5.3 ± 0.57 mm) at 100 mg/ml concentration of methanolic extract for Aspergillus niger. [5] reported 0.6mm inhibition zone with aqueous extract for E. coli and Issa et al. (2021) [23] (2021) [23] , showed ethanolic extract exhibited 10.00±1.41mm inhibition against Escherichia coli and 8.00±1.21mm against Pseudomonas aeruginosa at 100mg/ml concentration. In the methanolic extract of Moringa oleifera leaf powder, Escherichia coli, Pseudomonas fluorescens, and Bacillus substilis showed a similar zone of inhibition i.e. 3mm. This finding underscores the uniform inhibitory effect of the methanolic extract against the tested bacterial strains.

Nutritional composition of ready to serve soup of Moringa oleifera:

The nutritional analysis of ready-to-serve soup with various treatments of moringa leaf powder (Treatment 1, Treatment 2, and Treatment 3) was conducted and levels of ash, moisture, carbohydrate, and protein content were determined and reported in Table 14. Treatment 1, Treatment 2, and Treatment 3 showed varying ash content levels at (10%, 10%, and 12%), respectively. In contrast, control showed a relatively high ash content of (15%). Based on the findings of the current investigation, it was determined that the moisture content rises proportionally with the increasing quantity of moringa powder in the soup mixture. Treatment 3, containing (22%) moringa leaf powder (MLP), exhibited the highest moisture content of (16%). In contrast, Treatment 2, Treatment 1, and the Control showed decreasing moisture content of (14%, 12%, and 10%), respectively. The study revealed that an increase in moisture content in the soup mix corresponds to an increase in the amount of carbohydrates. The highest carbohydrate content was observed in Treatment 3, amounting to 38gm/100gm. Treatment 2, Treatment 1, and the control showed carbohydrate levels of 37.5gm/100gm, 37gm/100gm, and 30gm/100gm, respectively. Protein content increased with higher amounts of moringa leaf powder in the soup mix: Treatment 3 had the highest at 36gm/100gm, followed by Treatment 2 (35gm/100gm), Treatment 1 (33gm/100gm), and the control (29gm/100gm).

Conclusion

Moringa oleifera, widely known as the "drumstick tree" or "miracle tree," is a remarkable plant with a broad global distribution and a significant impact on nutrition and health.

Originating from India and now grown in various tropical and subtropical regions, Moringa oleifera thrives in diverse agro-climatic environments. Its leaves, rich in essential nutrients and bioactive compounds, offer numerous health benefits, including anti-tumour, anti-inflammatory, and antioxidant properties.

This study highlights the biochemical and functional properties of Moringa oleifera leaf powder, emphasizing its potential as a valuable resource for combating malnutrition, especially in developing countries. The findings underscore the plant's role as a natural source of vitamins, minerals, and antioxidants, making it a viable alternative to conventional food supplements.

Overall, the research supports the optimization of Moringa oleifera's utilization in dietary formulations and medicinal applications, contributing to improved health outcomes and food security.

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