INTRODUCTION:

Phenolic compounds represent the main part of the antioxidants existing in the food. Their total dietary ingestion could be higher than 1000mg/day, which is much more important than that of all other types of phytochemicals and known dietary antioxidants¹. Several studies have shown that phenolic compounds limit the development of cardiovascular, cancers, osteoporosis and diabetes². Other studies have also shown that phenolic compounds possess biological activities such as anti-bacterial³^,⁴, anti-microbial⁵^,⁶, anti-anthelmintic⁷, anti-oxidant⁸^,⁹, anti-inflammatory¹⁰^,¹¹, anti-obesity¹²^,¹³ and anti-diabetic¹⁴^,¹⁵.

Vegetables, fruits and medicinal plants are the main sources of phenolic compounds. Moringa oleifera is among the most important medicinal plants and there has been growing attention in their nutritional value¹⁶. This tree belongs to the Moringaceae family, widely distributed in Africa, South America, and Asia, subtropical and tropical regions¹⁷. The leaves this plant can be an alternative food or natural supplement to reduce malnutrition, especially in toddlers in poor areas, because it has a high concentration in protein, potassium, iron and many kinds of nutrients such as vitamins A, B and C¹⁸.

The extraction of phenolic compounds from the plants is influenced by many factors such as their chemical nature, the size of the particles, the extraction method used and extraction temperature¹⁹. There are many methods to extract phenolic compounds like maceration²⁰, and soxhlet extraction²¹. However, in the application, they need a long time and a high amount of solvents during for extraction process²². An alternative method to increase the extraction of phenolic compounds from Moringa oleifera leaves using ultrasonic-assisted extraction (UAE). It is an economical method characterized with time-efficient and environmentally friendly. furthermore, the UAE has also shown an important effect on the extraction of phenolic compounds from other vegetal sources¹⁹.

The aim of the present study is to investigate the effects of different temperatures (15, 30 and 45°C) on the extraction of phenolic compounds and antioxidant capacity from Moringa oleifera Leaves.

MATERIALS AND METHODS:

Chemicals:

All chemicals, solvents and reagents used in this study are of the analytical category.

Plant material:

The plant material was collected by the Technical Institute for the Development of Saharan Agronomy (TIDSA) located at Ouargla-Algeria during April 2019. The leaves of Moringa oleifera. Were dried immediately after harvesting in a shady and well-aired place at 34-39 (ºC) for 12 (h). The samples were ground to their particle size through 0.5-1 (mm) sieve. Then, they were packed in paper bags and kept in this condition (dark, dry and cool place).

Plant material extraction:

Moringa oleifera Leaves samples were extracted at different temperatures (15, 30 and 45°C) using ultrasonic bath (JP Selecta. Spain) with a frequency of 40 000 (Hz), for 40 (min), in a solvent-solid ratio of 7ml/g, through mixing 10 (g) of leaves with a 70% aqueous ethanol solution. The yield of extraction was calculated according to the following equation:

W₁

Yield (%) = -------------X 100

W₂

Where, W₁= Weight of the extract residue obtained after solvent removal (g), W₂= Weight of the plant powder (g).

Determination of polyphenols content:

Determination of the Total phenolic content:

The Folin-Ciocalteu test was used to determine the total phenolic content (TPC) of Moringa oleifera leaves extracts. The reaction mixture contains 1 (ml) of diluted Folin-Ciocalteu reagent (1:10), then 0.2 (ml) of extracts is added, and after 5 (min), 0.8 (ml) of sodium bicarbonate solution (7.5%. w/v) is mixed with the above reaction. The absorbance is measured at 765 (nm) using a UV visible spectrometer (Shimadzu UV-1800. Japan). Gallic acid is utilized as a reference standard, and the total content of phenol is expressed in equivalent milligrams of Gallic acid per gram of dry weight of extract (mg GAE/g DW)²³.

Determination of the Total flavonoids content:

The total flavonoid content was determined according to the method of aluminum trichloride²⁴. Briefly, 1(ml) of 2% aluminum trichloride (AlCl₃) ethanol solution was mixed with 1(ml) of extract solution. After incubation in the dark for 30 (min) at ambient temperature. The absorbance was measured at 415(nm) on a spectrophotometer (Shimadzu UV-1800, Japan). The total flavonoid content is expressed as mg ER (equivalent of Rutin) per g of extract.

Determination of Total flavanols content:

Total flavanols content was determined using the method of aluminum trichloride²⁵. A volume of 1(ml) of the plant extract was mixed with 1(ml) of AlCl₃ prepared in ethanol and 1.5(ml) of sodium acetate solution (50g/L). The mixture was incubated at ambient temperature for 2.5 (h). The absorbance was measured at 440(nm) on a spectrophotometer (Shimadzu UV-1800, Japan). The total flavanols content is expressed as mg EQ (the equivalent of quercetin) per g of extract.

Evaluation of antioxidant activity:

Determination of total antioxidant activity:

The total antioxidant activity of the extracts was estimated by the phosphomolybdenum method²⁶. An aliquot of 0.1(ml) of the sample solution was mixed with 1(ml) of the reagent solution (28 mM sodium phosphate, 600 mM sulfuric acid and 4 mM ammonium molybdate). The reaction mixture was incubated at 95(°C) for 1 (h) and absorbance was measured at 695(nm) against a blank contained 1(ml) of reagent solution. Total antioxidant activity was expressed equivalent to as mg EGA (the equivalent of Gallic) per g of extract.

Determination of Ferric Reducing Antioxidant Power (FRAP):

To determine the antioxidant capacity we used the FRAP method²⁵, the FRAP reagent was freshly prepared by mixing 2.5(ml) of TPTZ (10mM), 25 (ml) of a solution buffer of acetate buffer (3mM, pH 3.6), 3(ml) of distilled water and 2.5(ml) of chloride ferric (20 mM). 0.03(ml) of the extract was added to 970(μl) of the reagent FRAP. Absorbance was read at 593(nm) after 30 (min) incubation at ambient temperature, against a blank contained 970(μl) of the reagent FRAP with 30(μl) of solvent. The results obtained are expressed in mg equivalent of FeSO₄ per g of dry matter (mg FeSO₄E /mg DW).

Determination of DPPH Radical Scavenging Activity:

To evaluate the antioxidant capacity we used the DPPH radical scavenging method²⁵. 1(ml) of a methanolic solution of DPPH (0.1 mM) was mixed with 0.5(ml) of the extract. The resulting mixture is then kept in the dark at ambient temperature for 30 (min). The absorbance is measured at 517(nm) against a blank contains pure methanol. The effective concentration to reach a 50% inhibition of DPPH radical absorption, IC₅₀(µg/l) was calculated according to the formula next.

A_c - A_e

% Inhibition = --------------------- x 100

A_e

Where, A_c: Absorbance of the control (containing no antioxidant) after 30 (min), A_e : Absorbance of extracts measured after 30 (min).

Statistical analysis:

All results obtained in this study were expressed as the mean of three replicate determinations plus or minus the standard deviation (SD).

RESULTS AND DISCUSSION:

Temperature is a very essential factor in studies related to the food industry because it intervenes in the chain of several manufacturing processes, conservation and extraction of the foodstuffs¹. In this experiment, the effects of different temperature (15, 30 and 45°C) were investigated on the extraction of phenolic compounds and antioxidant capacity from Moringa oleifera Leaves.

Effect of extraction temperatures on the extraction yield:

The results indicate that the extraction output increases with an increase in extraction temperature, the improvements recorded were from 14.26% to 24.25% with 15 to 30 (°C), respectively, whereas an extraction temperature of 45(°C) resulted in a decreased yield to 20.7%, as shown in Fig 1. These results were in accordance with those reported by Tchiegang et al²⁷. Having explained that the reduction of the extraction yield would be at high temperature. An increase in temperature reduces the plant extract kinematic viscosity with the increasing mobility of biopolymers in cellular walls²⁸. Based on this result, we can conclude that 30 °C is the optimum value of extraction temperature to obtain the maximum of extraction yield.

Fig.1: Effect of extraction temperatures on the extraction yield.

Effect of extraction temperatures on the polyphenols:

The phenolic compounds one of the contents of beverages, foods and plants. Plant polyphenols consisted of most plant parts, which played a significant role in plant physiology. The plant polyphenols can be extracted by many methods, each with their own disadvantages and advantages. An ultrasonic-assisted method has been proposed for extraction polyphenols from Morinaga Oleifera Leaves using ethanol, which was used as a solvent because altogether fitting to a green chemistry approach. The results shown in Fig 2 indicate that the extraction of compounds phenolic increases with an increase in extraction temperature from 20.7±2.1 (mg GAE/100mg DW), 46.42±0.6 (mg RE/g DW) and 7.26 ± 0.49(mg QE/g DW) to 23.68±1.1(mg GAE/100mg DW), 58.65±2.4 (mg RE/g DW) and 10.04 ± 0.21 (mg QE/g DW) with 15 to 30 (°C), However, extraction temperature 45 (°C), resulted in a decreased the yield to 21.01±1.9 (mg GAE/100mg DW), 50.06±1,6 (mg RE/g DW) and 7.78±0.14 (mg QE/g DW) for the phenolic, flavonoid and flavanols content respectively. This phenomenon can be explained by the fact that high temperatures increased the polyphenol's solubility and facilitated their diffusion out of the cells²⁹. However, a higher temperature may destroy the construction of polyphenols, and lead to breakdown and oxidation of phenolic compounds³⁰.

Fig. 2: Effect of extraction temperatures on the phenolic, flavonoid and flavanols content.

Effect of extraction temperatures on the antioxidant activity:

In recent years, phenolic compounds have received considerable attention, which was imperative in a human diet. Furthermore, the phenolic compounds possessed tremendous benefits, like the antioxidant property, which was associated with the ability of free radical scavenging and broke its series reaction³¹. The results listed in Fig 3 and 4 indicate that antioxidant activity increases with an increase in extraction temperature with 15 to 30 (°C), from 244.68±0.2 (mg FeSO₄ E/mg DW) and 62.97 1.5 (mg GAE/g DW) to 236.37±0.5 (mg FeSO₄ E/mg DW) and 72.26±1.2 (mg GAE/g DW) for FRAP and Antioxidant Total respectively and decreased half-maximum inhibitory concentration (IC₅₀) from 76.92± 1.9 (µg/ml) to 45.04±1.1(µg/ml). A temperature increment to 45 (°C) resulted in a decrease in both FRAP and Antioxidant Total to 240.52±0.5 (mg FeSO₄ E/mg DW) and 66.8±1.7 (mg GAE/g DW) respectively and increment in half-maximum inhibitory concentration (IC₅₀) to 59.78± 1.7 (µg/ml). This phenomenon could be explained by the appearance of new molecules endowed with a strong antioxidant activity formed after hydrolysis under the heat treatment. However, it should be noted that increasing the temperature beyond certain values leads to a decrease in antioxidant activity is caused by a decrease in the ability of antioxidants to interact with free radicals at higher temperatures. Hence, it makes sense that the less oxidizing antioxidants will lose their antioxidant activity sooner³².

Fig.3: Effect of extraction temperatures on the FRAP and Antioxidant Total.

Fig.4: DPPH radical scavenging activities of Moringa oleifera Leaves.

Generally, it should be noted that increasing the temperature beyond some values may promoting possible concurrent decomposition of phenolic compounds which were already mobilized at lower temperatures or even the break-down of phenolic that are still remained in the plant matrix, additionally, high temperatures may encourage solvents loss through vaporization and increase the cost for the extraction process from the industrialization point of view. Normally, extraction performance increases at higher extraction temperatures, but the working temperature affects the stability of the phenolic compounds, which also depends on their chemical composition³³.

CONCLUSION:

Our study proved that Moringa oleifera leaves is a good source of major medically active compounds like phenolic, flavonoids, flavanols. The results reveal higher volumes of phenolic compounds and good antioxidant activities if ideal extraction conditions are present, which justify its use in traditional medicine all over the world.

ACKNOWLEDGMENTS:

All thanks and gratitude to the ‎Process Engineering Department of El-Oued University-Algeria for the efforts and contribution to achieving this study.

CONFLICT OF INTEREST:

The authors declare that there is no inconsistency for interests regarding the publication from this paper.

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Received on 24.07.2020 Modified on 07.08.2020

Asian J. Research Chem. 2021; 14(2):120-124.

DOI: 10.5958/0974-4150.2021.00022.5