The Antidiabetic, Antioxidant properties in vitro of Moringa oleifera Flowers extracts grown in Sahara of Algeria

 

Amar Djemoui¹*, Djamila Djemoui², Lahcene Souli¹, Ahmed Souadia¹, Messaoud Gouamid³

¹Laboratory of Organic Chemistry and Natural Substance, Department of Chemistry,

Faculty of Exact Sciences and informatics, ZIANE Achour University of Djelfa (Algeria)

²Department of Chemistry, Faculty of Exact Sciences, Echahid Hamma Lakhdar University of El Oued, Algeria.

³Department of Chemistry, Faculty of Mathematics and Matter Sciences, Kasdi Merbah,

University of Ouargla, Algeria.

 

ABSTRACT:

Moringa oleifera Flowers extracts grown in Algerian Sahara were evaluated antidiabetic and antioxidant activity by means using divers established in vitro systems, such as α-Amylase inhibitory assay, DPPH radical scavenging assay, Phosphomolybdenum assay (PM) and Ferric reducing power assay (FRAP). Moreover, the total content of phenols, flavonoids and tannins from (MFCE) and various fractions was measured using colorimetric methods. Results demonstrated that TPC varied between 95,50 ± 0,42 and 10, 49 ± 0,053 mg GAE/g WE, while TFC was between 17,00 ± 0, 011 and 2,47 ± 0,014 mg GAE/g WE, In this study TTC ranged between 2,96± 0,016 and 1,30± 0, 014 mg GAE/g WE. All capacities of DPPH radical scavenging, Phosphomolybdenum (PM) and Ferric reducing power (FRAP) were found best in (MFEF) (IC₅₀= 0,159± 0,004mg/ml, AEAC = 42.37 ± 0.28 mM and AEAC =104.05±0.41 mM respectively). Add to this (MFEF) showed the highest α-amylase inhibitory activity (I= 38,92 %).

 

 

 

INTRODUCTION:

The family of Moringaceae is remarkable for the great variety of habit and flora morphology prevalent in tropical and subtropical regions. Moringa is the only genus of the Moringaceae family with only 13 species¹. Amongst these species, Moringa oleifera Lam (syn. M. ptreygosperma Gaertn.) is the great known and most extensively distributed, native of sub-Himalayan tracts North India and distributed in Cambodia, Philippines, Central America, North and South America and the Caribbean Islands, Pakistan, Asia Minor, Africa and Arabia².

 

Moringa oleifera tree described as among the most amazing plants is a rich of proteins, vitamins and minerals including potassium, calcium, phosphorus, iron, folic acid in addition to carotene. Its flowers include nectar and are a good source of pollen[3].they also contain sucrose, d-glucose and some flavonoids inclusive of quercetin, in addition to nine amino acids and some alkaloids and are wealthy in calcium and potassium^3,4.

 

nearly all parts of this tree were used within the indigenous medicine of South Asia, which includes the treatment of inflammation and infectious illnesses along with cardiovascular, gastrointestinal, hematological and hepatorenal disorders, such as leaf, fruit, flowers, seed, root, bark, and seed oil⁵.

 

So with this view our present study is primarily based on extraction phenolic compounds and study Antioxidant and Antdiabetic properties of Moringa oleifera flowers.

 

MATERIAL AND METHODS:

Plant materials:

Between (November – January) 2016, healthy and fresh Moringa oleifera flowers were harvested from the Tamanrasset zone in Algeria, Prof. AIDOUD Amor from (Department of Agronomy, Faculty of Nature and Life, University of Ouargla (Algeria)) described and authenticated the plant material.

 

Reagents and chemicals:

Folin-Ciocalteu reagent, sodium carbonate, Gallic acid, Aluminum chloride, quercetin, vanillin, catechin, ammonium molybdate, 2.2-diphenyl-1-picrylhydrazyl, ascorbic acid, butylated hydroxyl toluene, potassium ferricyanide, trichloroacetic acid, Enzyme α-amylase.

 

Instrumentation:

All the experiments of this work were carried out on a UV / VIS spectrophotometer (SPECTROSCAN 80 DV), the removal of solvents from plant extracts was carried out using rotary evaporators (ISOLAB GmbH).

 

Phytochemical test:

The crude methanolic extract of Moringa oleifera flowers was tested to identify diverse classes of bioactive chemical constituents using standard procedure^6,8

 

Preparation of extracts:

The flowers of Moringa oleifera were cleaned and air dried under shade for many days then pulverized. The Powder flowers material (100g) was macerated at room temperature with (MeOH–H₂O) (70:30, v/v) for (48 h, two times).The extracts were subsequently filtered and evaporated to dryness under reduced pressure. In order to separate the active compounds dissolved in the methanolic extracts as a function of polarities, we have made a choice for fractionation of the liquid-liquid extraction which makes it feasible to transfer the molecules according to physico-chemical properties between two immiscible phases. In this technique solvents of increasing polarity were chosen, namely: petroleum ether (for extracting lipids and chlorophyll), chloroform, ethyl acetate and n-butanol. The organic extracts were then grouped together. After evaporation of the extracts under reduced pressure at a temperature of (50°C), the residues obtained were solubilized in methanol and stored. The extraction yield was calculated relative to the total weight of the materiel powder. The result of the procedure was represented respectively: Moringa flowers crude extract (MFCE), Moringa flowers chloroform fraction (MFCF), Moringa flowers ethyl acetate fraction (MFEF) and Moringa flowers butanol fraction (MFBF)⁶.

 

Quantitative assessment:

Assessment of total phenolic content (TPC):

Folin-Ciocalteu reagent was used to assess the total phenolic content (TPC) of MFCE and all fractions and the results were expressed as mg Gallic acid equivalent per gram extract weight (mg GAE/g EW)⁹. Briefly, A volume of (0.5 ml) of the plant extract was mixed with (1.5 ml) of Folin-Ciocalteu reagent (diluted 10 fold with distilled water) and had been neutralized with (3 ml) of sodium carbonate solution (7.5%, w/v). The combination was shaken and reacted for 30 min at room temperature inside the dark. The absorbance was measured at 765nm.

 

Assessment of total flavonoid content (TFC):

Aluminum chloride colorimetric method was used to estimate the total flavonoid (TFC) of MFCE and all fractions¹⁰ (2 ml) of (2%) AlCl₃ ethanol solution was added to (2ml) of extract. After 30 min incubation at room temperature, the absorbance’s become measured at 430 nm and the results were expressed as mg quercetin equivalent per gram extract weight (mg QE /g EW).

 

Assessment of total tannin content (TTC):

Colorimetric method was used to calculate the total tannin content (TTC) of MFCE and all fractions¹¹ (3ml) of (4%) methanol vanillin solution and (1.5ml) of concentrated hydrochloric acid have been added to (0.4 ml) of extract. Combination was allowed to stand for 15 min, and the absorbance become measured at 500 nm expressed as mg catechin equivalent per gram extract weight (mg CE/g EW).

 

Antioxidant capacity assays:

DPPH radical scavenging assay:

The free radical scavenging activity of MFCE and all fractions was evaluated using a procedure described by^{12, 13} with slight modifications.(10 µl) of diluted plant extract was added to (190 µl) of a (0.250 mmol/l) DPPH^• ethanol solution. The solutions were placed in the dark at room temperature for 30 min. The absorbance of the resulting solution was then measured at 517 nm by the usage a UV spectrophotometer. Inhibition of DPPH radical was calculated as follows:

                                                A₀   - A₁

DPPH scavenging effect =  –––––––––  × 100

                                                    A₀

Where;

A1 and A2 are the absorbance at 517 nm of the control and the sample, respectively.

 

Phosphomolybdenum assay:

The different fractions and MFCE also were used to determine their antioxidants capacities by phosphomolybdenum assay according to the procedure¹⁴ of  A (0.2 ml) of diluted plant extract was mixed with (2ml) of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a water bath at 95°C for 90 min, then cooled to room temperature and the absorption of the solution was measured at 695 nm against a blank. Ascorbic acid was used as a standard and the results were expressed as mM equivalent ascorbic acid.

 

Reducing power assay:

The reducing power of MFCE and all fractions was calculated to the reported method¹⁵ of (1ml) of various concentrations of the extracts were mixed with (2.5 ml) phosphate buffer solution (0.2 mol/l, pH 6.6) and (2.5 ml) of potassium ferricyanide [K₃Fe (CN) 6] (1%).The resulting solutions were incubated at 50ºC for 20 minutes. the reaction mixture mixed with (2.5 ml) of 10% trichloroacetic acid (TCA), (2.5 ml) of the supernatant was taken and (2.5 ml) distilled water and (0.5 ml) of ferric chloride (0.1%) were added to it. The absorbance was measured at 700 nm, using ascorbic acid as a positive control, and the results were expressed as mM equivalent ascorbic acid.

 

α -Amylase inhibitory assay:

Anti diabetic activity of Three fractions of Moringa oleifera  flowers was examined in vitro by method proposed by¹⁶. The in vitro anti-diabetic activity was determined by assaying the inhibitory activity of the enzyme α-amylase which includes in the breakdown of starch to produce glucose. In this method a starch solution (1% w/v) was prepared by stirring (1g) starch in (100 ml) of (20 mM) of phosphate buffer (pH 6.9).The enzyme solution was prepared by mixing (27.5 mg) of porcine pancreatic amylase α-amylase (PPA) in (100 ml) of (20 mM) of phosphate buffer.(0.5ml) of (1%) starch solution was added to (50 μl) of plant extract and 0.5ml porcine pancreatic amylase .the mixture was incubated at 37 ºC for 30 min. The reaction was stopped by appending (1 ml) DNSA (1g of 3,5 dinitro salicylic acid, 30g of sodium potassium tartrate and 20 ml of 2N sodium hydroxide was added and made up to a final volume of 100 ml with distilled water) and kept it in a boiling water bath for 10 minutes. 10 ml of distilled water was added to the reaction mixture and the absorbance was read at 540 nm. For each extract, blank tubes were prepared by replacing the enzyme solution with (0.5 ml) in distilled water. Control, representing 100% enzyme activity was prepared in a similar manner, without extract.

 

The percent inhibition of α-amylase activity was determined by using the following equation:

 

𝐼 (%) = [A₀-A₁/A₀] ×100

 

Where;

A₀ was the absorbance of the control and A₁ was the absorbance of the sample, respectively

 

RESULTS AND DISCUSSION:

Phytochemical test:

The results of preliminary phytochemical test of Methanolic extract Moringa oleifera flowers were summarized in the (table 01)

 

Table 01: Results of phytochemical test of Methanolic extract Moringa oleifera flowers

Note: ‘+’ indicates presence and ‘-’ absence

 

Table (01) shows the results of the qualitative analysis tests of Methanolic extract Moringa oleifera flowers subjected to a phytochemical survey of 09 chemical families, which clearly shows their ability to synthesize various effective natural products, like Phenols, flavonoids, coumarins, tannins, carbohydrates. These effective herbal products are natural products of plant metabolism have various biological activities, antitumor, antimicrobial, and antioxidant activity are the beneficial properties of phenols^16,17,While flavonoids contribute to the plant's growth and reproduction and protect it from the damaging effects of ultraviolet rays¹⁸, The antiviral efficacy of tannins is also due to the fact that the tannin molecules form a complex with the virus-enveloping protein and thus prevent the penetration of the virus^19,20 Moreover, coumarins have biological activity, As a treatment for vaginal candidiasis, anticoagulant, and antimicrobial properties^21-23.

 

Proportion extraction yield (%):

The proportion extraction yield of the Moringa flowers crude extract (MFCE) and its fractions was decided as following the equivalent:

 

Yield (%) = (W _extract * 100)/ W _{dry plant material}

 

Where W _extract, W _{dry plant material} are the weight of the extract after evaporation of the solvent and the weight of the dry plant material successively.

 

Table 02: Proportion extraction yield

 

Results of proportion extraction yield of Moringa oleifera  crude extract (MFCE) and its fraction Table (02) Arrange the ratios between (0.30 % - 41,33 %),It registered the highest yield at MFCE (41,33 %) while registered the lowest yield at MFCF (0.30 %),The yield of chemical extraction is commonly known to depend on the form of solvent with varying polarities, time and temperature of extraction, sample-to-solvent ratio, as well as the chemical composition and physical characteristics of the samples²⁴.

 

Phytochemical Contents assessment:

The proportion of total phenolic content (TPC) from Moringa oleifera  flowers extracts varied between 95,50 ± 0,42 and 10,49 ± 0,053 mg GAE/g WE (MFCF < MFBF< MFCE< MFEF), while the amount of total flavonoids content (TFC) from Moringa oleifera  flowers extracts was between 17,00 ± 0,011 and 2,47± 0,014 mg GAE/g WE (MFCF < MFBF< MFCE< MFEF).In this study, the quantum of total tannins content (TTC) from Moringa oleifera  flowers extracts varied between 2,96± 0,016 and 1,30± 0, 014 mg GAE/g (MFBF< MFCE< MFEF< MFCF) (table 03)(figure 01).

 

Figure 01: Total phenolic, Flavonoid and Tannin contents of Moringa oleifera flowers.

 

Table 03:  Total phenolic, Flavonoid and Tannin contents of Moringa oleifera flowers.

 

Antioxidant capacity:

DPPH radical scavenging activity:

DPPH free radical scavenging activity is an easy and widely used method for testing in-vitro antioxidant activity of natural compounds or plant extracts. (2-diphenyl-2-picrylhydrazyl hydrate) is purple-colored stable organic nitrogen centered free radical, and is the most common synthetic used for study of scavenging activity which particular becomes yellow color when reduced to its non-radical form by AH. Its reduction capability to accept an electron or a hydrogen radical from antioxidants is determined by measuring decrease in its absorbance values at 517 nm^25,26.

 

The results of DPPH inhibition by different extracts of Moringa oleifera flowers are reported in (table 04). Highest value of IC₅₀ (0,159± 0,004mg/ml) was detected in MFEF Followed by MFBF (0.417±0.03mg/ml) While the lowest value of IC₅₀ (2.56± 0.08mg/ml) was detected in MFCF. While there was greater scavenging activity for standard antioxidants. Based on the findings, a positive correlation between the quantity of phenolic compounds in the extracts and the antiradical potential was found, Phenolic compounds are well recognized for their anti-oxidative activities, they scavenge free radicals due to the mobility of an electron or an hydrogen²⁷.

 

Phosphomolybdenum capacity (PM):

The antioxidant capacity of Phosphomolybdenum method depends on the reduction of Mo (VI) to Mo (V) by the sample and subsequent formation of a green phosphate/Mo (V) complex at acid pH, which the formation of the complex was measured²⁸ at 695 nm. The results of total antioxidants capacities from all extracts of Moringa oleifera flowers (table 04) showed that MFEF had the strongest antioxidant capacity with a value 42.37 ± 0.28 mM, while the MFCF had the weakest antioxidant capacity with a value 13.05 ± 0.28mM. After comparing, the results showed that the total antioxidant potential of Moringa oleifera flower extracts was higher than that of BHA and BHT (0.868± 0.12 and 0.772±0.101mM, respectively).

 

The antioxidant capacity by this test is due to the presence of biologically active compounds represented by phenolic compounds and flavonoids that are characterized by the presence of aromatic benzene rings and hydroxyl groups capable of transferring an electron to the oxidizing complex. This transition depends at the redox potential, the pH of the medium and the antioxidant compound structure^29,30.

 

Ferric reducing power activity (FRAP):

This examination measures the antioxidant activity by measuring the reduction of iron (Fe⁺³) in ferric cyanide complex [FeCl₃/K₃Fe(CN)₆] to ferrous (Fe⁺²) form by donating an electron³¹. The AEAC value of reducing power of Moringa oleifera flowers extracts is shown in (table 04), Results showed that the AEAC of MFEF was the highest with a value 104.05 mM whereas the AEAC of MFCF was the lowest with a value 10.97 mM. All extracts of Moringa oleifera flowers better than BHA and BHT (0.772±0.101 and 0.576±0.019 mM) with high reductive power. During this examination, the reductive power is related to the presence of the B-ring catechol group in the chemical structure of the phenolic compounds it is affected by the presence of electron donor substitutes such as: CH₃, C₂H₅, or by the presence of donor aliphatic substituents, these substitutes reduce the oxidation potential of the phenolic compounds and thus increase the ability of these compounds to give electrons. For example, quercetin, which is one of the compounds isolated from the Moringa plant, has an oxidation potential Small at 0.25V, making it a powerful and well-known antioxidant^32,33.

 

Table 04: Total antioxidant activity of Moringa oleifera flowers.

 

 

Figure 02: α -Amylase inhibitory of Moringa oleifera flowers.

 

In Vitro Antdiabetic activity:

The activity of α-amylase is technique primarily based on the dedication of the aldehyde and ketone groups free of the reducing sugars released during the hydrolysis of starch by the 'α-amylase^34,37. The results of α-amylase activity of Moringa oleifera flowers extracts is proven in (Figure 02), Results confirmed that the highest inhibition % was in MFEF with a value 38,92 % then MFBF with a value 26.14 % While the lowest inhibition % was in MFCF with a value 17.37%.

 

Several Studies indicated that polyphenols have an action on α-amylase such as tannins which are able to bind to digestive enzymes and to inhibit them thanks to its richness in hydroxyl groups. Tannins bind to α-amylases, the binding can take place at the active site of the enzyme, these bonds generate complexes of the enzyme-inhibitor type, since tannins act as competitive inhibitors of α-amylases^38,41..

 

CONCLUSION:

The various extracts of Moringa oleifera flowers were examined for their phytochemical contents and biological activities including antioxidant and antidiabetic properties. The results obtained from this present work showed that Moringa oleifera flowers have high antioxidant and α-Amylase inhibitory capacity. This study encourages further studies such as isolation and identification of active compounds present in these extracts.

 

ACKNOWLEDGEMENT:

The authors wish to thanks The Pr Mohamed Hadjadj Director of Laboratory of Valorization and Promotion of Saharan Resources (LVPRS), Department of Chemistry, Faculty of Mathematics and Matter Sciences, Kasdi Merbah University of Ouargla, Algeria, for providing necessary laboratory facilities to carry the research work smoothly.

 

CONFLICT OF 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.

 

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Received on 28.10.2020          Modified on 29.11.2020

Accepted on 14.01.2021          ©AJRC All right reserved