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Journal of Drug Delivery and Therapeutics

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Open Access   Full Text Article                                                                                                                                                                            Research Article 

Phytochemical analysis and antioxidant activity of aqueous fraction of Moringa oleifera Leaves

Muhammad Abubakar Amali*, Abubakar Muhammad Ramadan

Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria

 

 

INTRODUCTION

The World Health Organization (WHO) estimates that 4 billion people, which is 80% of the world's population, presently use herbal medicines for some aspect of primary health care in conjunction with conventional medicines¹.They have been tested and proven to be safe and efficacious with wide cultural acceptability and lesser side effects than modern pharmaceutical medicines. The chemical constituents present in them are part of the physiological functions of living flora and hence they are believed to have better compatibility with the human body^2,3. The importance of antioxidants in preventing tissue damage or cellular function cannot be overemphasized, and this has resulted in increased production of antioxidants worldwide. Plants are cheap source of varieties of antioxidants in addition to their safety and efficacy. Naturally occurring antioxidants play a major role in the prevention and development of chronic diseases such as cancer, coronary heart diseases, diabetes, obesity and hypertension and these disorders are highly linked to oxidative stress. The antioxidants compounds have the ability to neutralize free radicals, therefore play an important role in the prevention of these diseases^4,5. Large body of researches has investigated the potential role of antioxidants in the prevention of these chronic diseases⁶. The interaction between ulcers and bacteria can be stratified into four levels: contamination, colonization, critical colonization and infection. While contamination and colonization by microbes are not believed to inhibit healing, the line between colonization and infection can be difficult to define. 

The use of traditional medicine is widespread. Plants still present large source of natural antioxidants that might serve as leads for the development of novel drugs⁷. 

Moringa oleifera commonly known as horse radish tree or drumstick from the family Moringaceae is a cosmopolitan plant that grows almost everywhere even in poor soils. It is found almost everywhere especially around the tropics. Almost all the parts of this plant: root, bark, gum, leaf, fruit (pods), flowers, seed and seed oil have been used for various ailments in the indigenous medicine of South Asia, including the treatment of inflammation and infectious diseases along with cardiovascular, gastrointestinal, hematological and hepatorenal disorders⁸. Moringa oleifera Lam is a fast growing, aesthetically pleasing small tree adapted to arid, sandy conditions. The species is characterized by its long, drumstick shaped pods that contain its seeds.  It is the most widely cultivated species of the monogeneric family Moringaceae. It is native to India, Pakistan, Bangladesh and Afghanistan and was utilized by the ancient Romans, Greeks and Egyptians. Now it is available all over the tropics⁹. It was reported to possess antimicrobial action¹⁰ and anti-inflammatory¹¹, anti-diabetic¹², antioxidant¹³ and anticancer properties¹⁴. In addition, it has been confirmed that it has significant hepatoprotective action against hepatotoxic induced liver injury^15,16.

In recent years; considerable attention has been directed towards identification of plants with antioxidant ability that may be used for human consumption¹⁷.  It is in view of this that we investigated the in vitro antioxidant activity of aqueous fraction from the leaves of methanolic extract of Moringa oleifera leaves.

MATERIALS AND METHOD 

Plant collection, extraction and fractionation

The leaves of M. oleifera Lam (MO) were procured from Kara Market in the city of Sokoto, Sokoto state, Nigeria, in October, 2020. They were authenticated by a staff of Pharmacognosy department, Usmanu Danfodiyo University Sokoto. The air-dried powdered leaves of M. oleifera (500g) were extracted successively with methanol, ethanol and water (three times for each solvent to ensure complete extraction) with the help of a shaker (Lab Tech shaker, model; LSI- 3016R, Korea set at 25⁰C). The extracts were then filtered and the solvent evaporated with a rotary evaporator (Buchi, Switzerland) at room temperature to dryness under reduced pressure and further freeze dried with a freeze drier (Virtis Bench Top K, United States). The yield of each dried extracts was calculated and stored at – 20⁰C until required for further use.

Differential fractionation of the active crude methanolic extract

The methanolic crude extract was subjected further to differential fractionation using n-hexane, dichloromethane, ethyl acetate, n-butanol and water. The methanolic crude extract was dissolved in methanol: water (50:150) and partitioned against equivalent volumes each of n- hexane, dichloromethane, ethyl acetate and n-butanol. Five fractions were obtained with these respective solvents and subjected to in vitro screening to obtain the most active aqueous fraction. The aqueous fraction was freeze dried and stored at -20⁰C until when required for further study^18,19.

Phytochemical screening of methanolic crude extract and aqueous fractions

Phytochemical examinations were carried out qualitatively according to the established methods^{20,21, 22}

Test for flavonoids (alkaline reagent test).  3ml aliquot of the fraction plus 10% NaOH produced a yellow color which indicated the presence of flavonoid compounds.

Test for tannins. 5% ferric chloride solution was added drop by drop to each of the 2-3ml the fraction and a dark green color was observed which indicated the presence of tannins. 

Test for Saponins. 5ml of each of the fraction was shaken strongly with 5ml of water for about 15mins; formation of a layer of foam that lasted for several minutes indicated the presence of saponins. 

Test for Glycosides. 2.5ml of 50% H₂SO₄ was added to 5ml of the fraction and heated in boiling water with NaOH. 5ml of Fehling’s solution was then added and the mixture was boiled. A brick red precipitate formed indicated the presence of glycosides.

Test for alkaloids. 2ml of each of the fractions was stirred in 2ml of 10% HCl. 1 ml was stirred with few drops of Wagner’s reagent and another 1ml with a few drops of Meyer’s reagent. Turbidity or precipitation with either of these reagents was taken as preliminary evidence for the presence of alkaloids.

Test for cardiac glycosides (Keller-Killiani’s test). 3.5% ferric chloride solution was added to the fraction and allowed to stand for a minute. 1ml of conc. H₂SO₄ was carefully poured on the walls of the tube to form a lower layer. A reddish brown ring at the interface indicated the presence of glycosides. 

Test for steroids (Salkowski). 0.5ml of the fraction was dissolved in 5ml of chloroform. 2ml of conc. H₂SO₄ carefully added to form a lower layer. A reddish brown color at the interface indicated the presence of a steroidal ring.

Test for saponin glycosides. 2.5ml of Fehling’s solution A and B was added to each extract and fraction. A bluish green precipitate showed the presence of saponin glycosides. 

Test for volatile oils. 1ml of the fraction was mixed with dil. HCl. A white precipitate was observed which indicated the presence of volatile oils.

Test for anthraquinones. 0.5ml of the fraction was shaken with 10ml benzene and 5ml of 10% ammonia solution. There was no presence of any color change which indicated the absence of Anthraquinones.

 Antioxidant assay of bioactive aqueous fraction

Determination of total antioxidant capacity of the aqueous fraction

For sample preparation, 1mg of the freeze dried aqueous fraction sample was measured and dissolved completely in 1 ml of methanol and a 1mg/ml of sample solution was obtained. Serial dilutions of 50, 100, 150, 200, 250 and 300µg/ml were made from the stock solution of the aqueous fraction sample. For the preparation of the standard solutions, ascorbic acid was used as a standard.  1mg of the ascorbic acid was measured and dissolved completely in 1ml of methanol and a 1mg/ml standard solution was obtained. Serial dilutions 50, 100, 150, 200, 250 and 300µg/ml were made. 

Phosphomolybdenum Assay

The method described by²³ with slight modification was adopted to measure the total antioxidant capacity of the aqueous fraction of M. oleifera. Briefly, to a known aliquot of the aqueous fraction sample, 0.4ml was placed in a vial; 4ml of the reagent solution    (containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was added and incubated in a water bath at 95^oC for 90 mins. The samples were cooled down to room temperature and absorbance was measured at 630nm. The blank solution contained 4 ml of reagent solution and 0.4 ml of methanol; the control solution consisted of 4ml of reagent and 0.4 ml of various concentration of ascorbic acid (50 – 300µg/ml). The control and blank solutions were treated in the same procedure as that of sample solution. A calibration curve was prepared by using the standard solution of ascorbic 

acid and the antioxidant activity was expressed as µg of ascorbic acid equivalent antioxidant capacity (AAEAC) per gram of extract.

Ferric reducing antioxidant power (FRAP Assay)

The typical FRAP assay method²⁴ was employed using 96-well microplates. Briefly, the FRAP reagent was prepared immediately by mixing 10 ml of acetate buffer (pH 3.6) with 1 ml of ferric chloride hexahydrate (20 mM in distilled water) and 1 ml of 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM in HCl 0.04 N) and placed in a water bath at 37 °C.   Then, 25µL of each sample were pipetted into the wells in triplicates and 175µl of FRAP reagent was added in each well. Microplates were incubated in darkness at 37°C for 30 min and the absorbance was measured using microplate reader at 595 nm. The blank solution contained 175µl of FRAP reagent and 25µl of methanol; the control solution consisted of 175µl of reagent and 25µl of various concentrations of ascorbic acid (50 – 300µg/ml).  A calibration curve was prepared by using standard solutions of ascorbic acid. The results were expressed as µg of ascorbic acid equivalent antioxidant capacity (AAEAC) [FRAP value] per gram of aqueous fraction.

Free radical scavenging activity assay

Preparation of sample solution: 1mg of the freeze dried sample was measured and dissolved completely in 1ml of methanol and 1mg/ml sample solution was obtained. Serial dilutions of 10, 50, 100, 200, 300, 400 and 500µg/ml were prepared and used. For the preparation of the

standard solution: ascorbic acid was used as standard. 1mg of the ascorbic acid was measured and dissolved completely in 1ml of methanol and 1mg/ml standard solution was obtained. Serial dilutions of 10, 50, 100, 200, 300, 400 and 500µg/ml respectively were prepared and used.

DPPH radical scavenging activity assay

DPPH radical scavenging activity of plant extract was determined according to the method of²⁵ with slight modification using 96-well microplates. Briefly, 150 µl of extract was added to 50 µl of DPPH (1, 1-diphenyl-2-picrylhydrazyl) solution (0.2 mM in methanol) as the free radical source. The mixture was shaken and kept for 30minutes at room temperature. The decrease in absorbance due to proton donating activity of components of each extract was determined at 540nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. Methanol was used as blank and added to 50µl of DPPH (control). Ascorbic acid was used as the standard. The DPPH radical scavenging activity was calculated using the following formula; DPPH Radical Scavenging Activity (%) = [(A0 – AI /A0) ×100], where A0 is the absorbance of the control, and AI is the absorbance of extract or standard sample. The IC50 values of the extract and standard were determined by plotting DPPH Radical Scavenging Activity (%) vs. Concentration.

Nitric oxide radical scavenging activity assay

Nitric oxide radical scavenging activity of plant extract was determined according to²⁶. The reaction mixture (6 ml) containing sodium nitroprusside (10 mM, 4 ml), phosphate buffer saline (PBS, pH 7.4, 1 ml) and extract in methanol at various concentrations or standard was incubated at 25^oC for 150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite ion was removed, 1 ml of sulphanilic acid reagent was added, mixed well and allowed to stand for 5 min for completion of diazotization. Then, 1 ml of napthylethylenediamine dihydrochloride (NEDD) was added, mixed and allowed to stand for 30 min in diffused light. A pink coloured chromophore was formed. The blank solution was performed by replacing the sample with methanol. The absorbance of these solutions was measured at 540nm. The nitric oxide radical scavenging activity was calculated using the following formula: Nitric oxide Radical Scavenging Activity (%) = [(A0 – AI /A0) ×100], where A0 is the absorbance of the control, and A1 is the absorbance of extract or standard sample. The IC₅₀ values of the extract and standard were determined by plotting nitric oxide radical Scavenging Activity (%) vs. Concentration.

 

RESULTS AND DISCUSSION

Phytochemical analysis

The results of qualitative phytochemical analysis revealed the presence of tannins, flavonoids, saponins, glycosides, alkaloids, cardiac glycosides, saponin glycosides, steroids and volatile oils while anthraquinones were absent. The presence or absence of phytochemicals is presented in Table 1.

Table 1: Phytochemical composition of methanolic crude extract and aqueous fraction

Key: (-): Absent.  (+): Present.

 

Antioxidant activity 

Phosphomolybdenum and Ferric reducing potential of aqueous fraction.

The results of Phosphomolybdenum assay and ferric reducing capacity of aqueous fraction of M. oleifera is presented in Table 4.4. The results indicated that, aqueous fraction demonstrated a good antioxidant activity by reducing phosphate molybdenum (VI) to phosphate molybdenum (V) at concentration of 56.35 + 0.29 mg ascorbic acid equivalent/g of 

aqueous fraction of M. oleifera and reduction of ferric ion at concentration of 124.16 + 0.6 mg ascorbic acid equivalent/g of aqueous fraction of M. oleifera. This is represented in table 2.

 

Table 2: Ferric reducing antioxidant capacity (FRAP) and Total antioxidant capacity of aqueous fraction of M. oleifera by Phosphomolybdenum assay.

Sample	(mg) of ascorbic acid equivalent by phosphomolybdenum assay	mg ascorbic acid equivalent by FRAP assay
Aqueous fraction	56.35 + 0.29	124.16 + 0.6

Data are represented as mean ± SD of triplicate values (mg of ascorbic acid /g of aqueous fraction of M. oleifera.

 

 

 

 

 

DPPH radical scavenging capacity

The radical scavenging activity of aqueous fraction of M. oleifera following DPPH assay was observed to increase with increasing concentration. The minimum scavenging activity of 10% was obtained at lower concentration of 10 µg/mL, and a maximum scavenging activity of about 70% was obtained at concentration of 500 µg/mL. Similarly, maximum activity of 70% was also obtained from standard ascorbic acid. The IC₅₀ of aqueous fraction was 179µg/ml as shown in figure 1

 

 

 

Figure 1: Graph of DPPH radical scavenging activity of aqueous fraction of M. oleifera by DPPH compared with standard ascorbic acid. Values are expressed as mean ± S.D of three independent experiments.

 

 

Nitric oxide scavenging activity

Following nitric oxide (NO) scavenging assay, the scavenging activity of NO by the aqueous fraction was seen to increase as concentration of aqueous fraction increases. At concentration of 10, 50, 100, 200, 300, 400 and 500 µg/mL, the NO scavenging activity was found to be; 35, 50, 52, 53, 54, 55 and 57% inhibition. However, the scavenging activity of standard ascorbic acid was found to be 60 % inhibition. The aqueous fraction with maximum activity of 57% inhibition and an IC₅₀ of 46µg/ml. This is shown in figure 2.

Figure 2: Graph of nitric oxide scavenging activity of aqueous fraction of M. oleifera compared with standard ascorbic acid. Values are expressed as mean ± S.D of independent experiments. 

Hydrogen peroxide scavenging activity

Following hydrogen peroxide assay, the aqueous fraction of M. oleifera demonstrated a good scavenging activity. The fraction exhibited a concentration dependent scavenging activity with a minimum scavenging activity at 40% and a maximum scavenging of 86% inhibition while that of the standard ascorbic acid was 90% inhibition. The result of the hydrogen peroxide scavenging activity is illustrated in figure 3.

Figure 3: Graph of hydrogen peroxide scavenging activity of aqueous fraction of M. oleifera compared with standard ascorbic acid. Values are expressed as mean ± S.D of three independent experiments.

 

DISCUSSION

In the present study, antioxidant properties of aqueous fraction of Moringa oleifera was evaluated for potential in wound healing applications. Preliminary qualitative phytochemical screening of the aqueous fraction revealed the presence of some useful phytochemical compounds that included tannins, flavonoids, saponins, glycosides, alkaloids, cardiac glycosides, saponin glycosides, steroids and volatile oil while anthraquinones were absent. The antibacterial and antioxidant properties exhibited by aqueous fraction of M. oleifera may be linked to presence of some phytochemical compounds revealed in this study. These activities could be the result of an individual or additive effect of these compounds. This finding is in accordance with other previous studies that reported, constituents like alkaloids, triterpenoids and tannins found in Vinca rosea may play a major role in the process of wound healing in diabetic rats due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and increased rate of epithelialization²⁷. 

Another study also reported that, the wound healing effects of the chloroform, methanol, and aqueous extracts of H. indicum may be attributed to the presence of phytoconstituents like alkaloids, triterpenoids, tannins and flavonoids in the extracts which are known to promote the wound healing process mainly due to their antimicrobial property²⁸.As widely reported, a strong relationship does exist between wound healing and oxidative stress. Oxidative stress plays a pivotal role in the development of diabetes complications, both microvascular and cardiovascular²⁹. The reactive oxygen species (ROS) generation by inflammatory cells within the dermis plays an integral role in mediating wound healing³⁰. Over production of ROS can affect proliferative and cell survival signaling to alter apoptotic pathways in the skin diseases and this contributes significantly to pathogenesis of impaired skin wound healing³¹. In our antioxidant assays, aqueous fraction of M. oleifera was shown to possess significant antioxidant properties comparable to those of various standard ascorbic acid used. The antioxidant properties of our aqueous fraction could be linked to the presence of phenolic compounds which scavenge oxygen free radicals, thus help in prevention of tissue damage associated with delayed healing of wounds, thus, aqueous fraction of Moringa oleifera may help facilitate wound healing through its antioxidant capacity. The bioactive aqueous fraction of M. oleifera was obtained from crude methanolic extract which could be one of the reasons associated with its high antioxidant ability. This is in agreement with the previously reported study that reported methanolic extracts having higher antioxidant activity compared to other solvent extracts from plant derived food using various study models. The high antioxidant nature of methanol solvent extracts may be linked to high chemical polarity of methanol when compared to other solvent system³². The phytochemical compounds such as flavonoids revealed aqueous fraction of M. oleifera which are poly phenolic which further justifies that, the major active compounds could be responsible for the antioxidant actions and the compounds are polar compounds that could be useful in promoting wound healing. The presence of some of these phytochemical compounds in the aqueous fraction of M. oleifera makes it a potential wound healing agent.

CONCLUSION

The ability to modulate production and quenching of free radicals may contribute significantly to the demonstrated property of bioactive aqueous fraction of M. oleifera to help in enhancing wound healing especially in chronic wounds. The results of this study are encouraging, proposing further investigation of the wound healing properties of bioactive aqueous fraction of M. oleifera that could have value clinically. 

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