INTRODUCTION:

The constituents of our organism (lipids, proteins, DNA) can be oxidized by reactive oxygen species (ROS) including free radicals (^•OH, ROO^•, RO^•). The ROS can be produced by normal aerobic metabolism by phagocytic cells in response to bacteria invasion or by exposure to ionizing radiation and antitumoral agent¹. The oxidative damage caused by ROS is called Stress Oxidant which causes many of chronic and degenerative diseases. Under normal physiological conditions, our cells are equipped with antioxidant defense systems, which allow them to neutralize the ROS to keep them at a low level in these cells, and therefore prevent the onset of oxidative stress.

The plants are source of most natural antioxidants. The expression of medicinal plants comprise a different kind of plants and several of these plants possess a medicinal activity². Medicinal flora are the “backbone” of old medicine, which signify more than 3.3 billion person in the poor countries who use medicinal plants a lot³.

These medicinal plants count being a rich of ingredients, which can help in developing the drug. However, these plants play a high role in the evolution of human cultures around the whole world⁴. Many plants contain compounds that are responsible to antioxidant activity. Polyphenols constitute the largest class of phytochemicals and dietary polyphenols have been manifest to play critical roles in human health. They are natural antioxidants present in plants, conferring protection against free radicals when consumed, thus they are important in maintaining good health⁵. Therefore, evaluation of plants for their antioxidant and lipid lowering capacity is a good approach to finding safer and cheaper ways of managing chronic diseases^{6, 7}.

The milkweed family (Asclepiadaceae) is very rich in latex and Pergularia tomentosa L. is plant belonging to the milkweed family. The synonyms for this plant include Daemia tomentosa, Telosma tomentosa and Daemia cordata⁸. P. tomentosa is well known in the Algeria as Ghalaka. This plant is an evergreen permanent shrub and has an exotic odor⁹. This shrubby plant species possess less than one meter of height.

Etymologically, tomentosa means as hairy, because the plant enveloped by many small hairs which causes a greenish color. This plant is vastly spread in horn of Africa (north Sudan, Egypt, Ethiopia, Algeria, Niger and Kenya) and Middle East (Iran, Oman, Pakistan, Afghanistan, and Saudi Arabia)¹⁰.

Pergularia tomentosa L. is used in traditional treatment as laxative, an antirheumatic, abortive and treatment of asthma and bronchitis and some skin diseases¹¹. This plant also used for treatment of helminthiases, chills, constipation and allergies¹⁰.

Diabetes mellitus is a metabolic disease characterized by a disorder in the regulation of carbohydrate metabolism resulting in hyperglycemia is caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced, another consequence of diabetes is increase production of free radicals^12,13,14. The number of people with diabetes has increased dramatically in recent years. The WHO estimates that diabetes will be the 7^th primary cause of fatality by 2030¹⁵. Many plants are used for the treatment of diabetes mellitus.

In Algeria, the leaves and flowers of Pergularia tomentosa are used as treatment for angina, ringworm, dermatitis¹⁶ and hypoglycemic¹⁷.

Until now, little work on the biological effects of this plant in vitro has been cited in the literature. It is therefore important that this study aimed to provide the phytochemical tests of principal polyphenols extracted from leaves of Pergularia tomentosa L., the antioxidant capacity of leaves is examined in vitro using reducing power assay as well as scavenging of DPPH^·. In addition, this study is about evaluating of antidiabet activity of different extracts.

MATERIALS AND METHODS:

Materials:

Chemicals and reagents:

Folin-Ciocalteu reagent, Ascorbic acid, Gallic acid, Catechin, Vanillin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sulfuric acid, Butylated hydroxytoluene (BHT) were purchased from sigma Aldrich, Ammonium molybdate, Sodium carbonate, Hydrochloric acid, Sodium dihydrogen phosphate, were provide by Biochem chempharma. All other chemicals and solvents used in this research were of analytical grade.

Instrumentation:

The proposed work was carried out on a UV/VIS Spectrophotometer (SPECTROSCAN 80 DV). All weighing was done on electronic balance. Plant extracts preparation was carried out by using Rotary evaporators (ISOLAB GmbH) for degassing solvent.

Plant material:

Pergularia tomentosa was collected from the region of Zelfana (east of Ghardaia, Algeria, arid climate 32° 23′ 50″ N, 4° 13′ 34″ E). The collection was in morning in the beginning of November 2017. AIDOUD Amor botanist in University of Ouargla (Algeria) identifies plant. Leaves were dried and cut into small parts.

Preparation of the extracts:

(88.28g of leaves) was defatted with petroleum ether and extracted 3 times at 25°C with MeOH/H₂O (80/20) for 48 h. After filtration, the filtrate was evaporated until dryness, diluted with distilled water and extracted using chloroform, ethyl acetate and n-butanol. The extracts were concentrated under reduced pressure to calculate the yield. Then, they were re-dissolved with minimum of methanol and kept at 4°C. We obtained five extracts respectively: crude extract, chloroform extract, ethyl acetate extract, butanol extract and aqueous extract.

Phytochemical Screening:

10.22g of the leaves was macerated in a 70/30 system (MeOH/H₂O) for 24 hours, after filtration, the crude extract retained for carrying out the phytochemical tests according the methods explained by Trease and Evans¹⁸ and Yadav et al¹⁹ to identify the different constituents in P. tomentosa.

Determination of total phenolic content (TPC):

Folin-Ciocalteu reagent was used to measure the total phenolic content TPC of the various extracts²⁰. Gallic acid (0.03-0.25g/L) was used as a reference standard for plotting calibration curve. A volume of 0.1mL of the plant extract was mixed with 0.5mL of Folin-Ciocalteu reagent 10%; after 2 min, 2mL of Na₂CO₃ solution (20%) were added. The reaction mixture was kept in dark at 25°C for 30 min. The absorbance of blue color was measured by UV/Vis spectrophotometer at fixed wavelength of 760nm. The TPC were calculated using linear regression equation obtained from the standard curve of gallic acid. The content of total phenolic compounds was calculated as mean±SD (n=3) and expressed as (mg gallic acid equivalent GAE/g DW).

Determination of total flavonoid content (TFC):

Total flavonoid content was estimated according to a literature procedure²¹. To 1.5mL of each sample, 1.5mL of 2% AlCl₃ ethanol solution was added. After 30 min at room temperature, the absorbance was measured at 430 nm. The content of total flavonoids was calculated as mean±SD (n=3) and expressed as (mg quercetin equivalent QE/g DW).

Determination of total tannin content (TTC):

The total tannin content of the leaves extracts of Pergularia tomentosa was estimated by colorimetric method²². 3 mL of 4% ethanol vanillin solution were added to 0.4 mL of extract and 1.5mL of concentrated hydrochloric acid. The mixture is left for 15 min, and the absorbance was measured at 500nm. The content of total tannin was calculated as mean±SD (n=3) and the results were expressed as (mg catechin equivalent CE/g DW).

Antioxidant Activity:

Determination of reducing power assay by phosphomolybdenum assay PM:

The reducing power of the various leaves extracts of Pergularia tomentosa L. was estimated by the phosphomolybdenum assay²³. 0.3 mL of different concentrations of each extract was mixed with 3 mL of reagent solution (8mM sodium phosphate, 0.6 M sulphuric acid, and 4mM ammonium molybdate). The obtained solutions were kept in a water bath at 95°C for 90 min. The mixture was left to cool at 25°C, the absorbance was read at 695nm, using ascorbic acid as a positive control, and the results were expressed in mM as the ascorbic acid equivalent antioxidant capacity AEAC.

DPPH radical scavenging activity:

The radical scavenging assay of extracts was measured as equivalent of hydrogen donating, according to DPPH method^24,25 with some modifications. Briefly, 1.5mL of DPPH solution (250µM) was added to 1.5mL of sample solution at various concentrations. The mixture was incubated for 30 min in dark, and then the absorbance was measured at 517nm against a control. The antioxidant capacity of the extract was expressed as an IC₅₀ value defined as the concentration (g/L) of the extract that inhibited 50% of DPPH^· radicals.

a-amylase inhibition assay:

Preparation of DNSA (3,5-dinitrosalicylic acid reagent :

0.5 g of DNSA was dissolved in 20 ml of distilled water. Then, 15 g of double sodium and potassium tartrate was added to this solution with stirring. The obtained solution was opaque yellow. After that, its color changed to orange due to addition of 10 ml of NaOH solution (2N). The volume of the solution obtained was adjusted to 100 ml by adding distilled water. The reagent obtained was stored away from light and at 4 C°.

Procedure:

The activity of α-amylase is carried out according to Rahmani Zineb protocol²⁵. Briefly; an aliquot of 0.5mL of 1% (w/v) starch solution (prepared in 0.02M sodium phosphate buffer pH 6.9) was mixed with 0.5mL of 1.3UI/mL α amylase of Aspergillus oryzae (Enz-no:3.2.1.1) prepared in the same buffer solution. 50μL of the plant extract at 0.5mg/mL was added; then the mixture was incubated at 37°C for 30minutes. 1mL of 3,5-dinitrosalicylic acid reagent (DNSA) was added to reaction mixture. Same procedure was performed on a negative control that did not contain extract. The absorbance was read at 540 nm against a blank which containing buffer solution.

RESULTS:

The medicinal properties of Pergularia tomentosa L. are may be due to the presence of different secondary metabolites like cardenolides, alkaloids, glycosides, flavonoids, steroids, saponins, tannins etc. The Phytochemical tests of the leaves extracts of Pergularia tomentosa L. is shown in (Table 1).

Table 1. Phytochemical analysis of Pergularia tomentosa L. extract

Phytochemical components	*Pergularia tomentosa* Leaves
Flavonoids	++
Alkaloids	++
Tannins	++
Glycosides	+
Cardenolides	+++
Terpenoids	++
Steroids	++
Saponins	++
Quinones	++
Protein	-

+ = Present, - = Absent

The results of phytochemical tests revealed the presence of cardenolieds, flavonoids, terpenoids, alkaloids, steroids, tannins, glycosides, and quinones in leaves of Pergularia tomentosa.

The richness of the plant Pergularia tomentosa by cardenolides explains the toxicity of this plant, which certainly explains its use in the treatment of diseases. Another specie of Pergularia is Pergularia daemia and one of its uses is inhibition against bacteria²⁶. In addition, alkaloids are used as analgesics and antibacterial²⁷.

Preliminary tests have shown the presence of phenolic compounds. Phenolic compounds have an enormous range of biological activities^28,29. Therefore, our choice focused on this family of compounds that were obtained by the methods of extraction of polyphenols. The results are shown in (Table 2).

The TPC were calculated using the following linear regression equation derived from the standard curve of gallic acid: y= 3.326x, r²=0.992. Where y is absorbance and x is the concentration of gallic acid in g/L. Total phenolics: ranged from 2.599± 0.49 (mg GAE/g DW) to

0.173±0.00 (mg GAE/g DW) (Table 2). The highest content was found in aqueous extract; the lowest content was registered in chloroform extract (Table 2). Tylophora asthmatica, a plant, belongs to the family asclepidaceae their TPC was 5.68 ± 0.06 mg GAE / g extract³⁰.

The TFC were calculated using the following linear regression equation obtained from the standard plot of quercetin: y = 36.37x, r²=0.999. Where y is absorbance and x is the concentration of quercetin in g/L. TFC of the various extract of Pergularia tomentosa was between 0.48± 0.01 and 0.025 ± 0.00 (mg QE/g DW). The highest content was found in butanol extract; the lowest content was registered in chloroform extract.

The TTC were calculated using the following linear regression equation obtained from the standard plot of catechin y = 4.378x, r²=0.997. Where y is absorbance and x is the concentration of catechin in g/L. TTC in the extracts of Pergularia tomentosa, varied between 0.436 ± 0.00 and 0.069± 0.00 (mg CE/g DW). The highest content was found in aqueous extract, while the lowest was found in ethyl acetate extract.

The function of an antioxidant is to scavenge free radicals. One mechanism through which this is achieved involves donating hydrogen to a free radical and consequently converts it to an unreactive species³¹. Addition of hydrogen eliminates the single electron, which is responsible for radical reactivity. The antiradical activity of extracts of Pergularia tomentosa leaves were investigated by used radical scavenging methods such as DPPH.

The scavenging effect of different concentration of Pergularia tomentosa leaves extracts on the DPPH free radical was compared with standard antioxidant (ascorbic acid and BHT). The results were expressed as IC₅₀ g/L shown in (Table 2).

The method of DPPH is based on the reduction of the stable radical DPPH with a violet color to non-radical DPPH-H with a yellow color. The disappearance of the violet color can be monitored spectrophotometrically at 517 nm. The values of IC₅₀ varied between 0.94±0.32 and 0.05±0.02 mg/mL (Table 2). The best activity was found in the ethyl acetate extract. Ascorbic acid and BHT showed a good antiradical activity and better than all extracts.

The reducing power of the different leaves extracts of Pergularia tomentosa was measured by the phosphomolybdenum method, which is based on the reduction of Mo (VI) to Mo (V).

The formation of green phosphate/Mo (V) compounds measured at 695nm. Reducing power of all extracts varied between 265.85 ± 8.09 and 21.80 ± 4.28 mM. Aqueous extract had a strong antioxidant activity with a value of 265.85 ± 8.09 mM followed by butanol extract with a value of 221.58 ± 7.09 mM. The lowest antioxidant activity was recorded in ethyl acetate extract with a value of 51.45 ±2.91 mM.

Plants are important source of medicinal uses and therapeutic agents. One of the major advantages of using plants is that they serve as effective anti-diabetic agents. This study investigated the ability of four extracts (dichloromethane, ethyl acetate, butanol and aqueous extracts) of Pergularia temontosa to inhibit a-amylase enzyme. The results of this test present that extracts have different capacity to inhibit the a-amylase (Table 3).

Table 3. Percentage of inhibition of a-amylase

Extract of leaves	Dichloromethane	Ethyl Acetate	Butanol	Aqueous
Percentage of inhibition I%	18.01	11.22	8.68	2.74

DISCUSSION:

Comparing our results with previous studies we note that the extraction yields obtained by Hassan S. W. et al.³²are higher by way of example, for the leaves, the chloroform phase and the aqueous phase give 7% and 15.25% respectively. We recall that the extraction system used in this study is ethanol: water (1:1).

We have remarked that phenolic and flavonoid contents are higher in the polar extracts (aqueous, ethyl acetate and n-butanol extract), that may indicate that these polyphenol compounds are more hydroxylated and/or glycosydated. The content of phenolic or flavonoid compounds in extracts was affected by their solubility in solvent used for extraction. Polar extracts had more polyphenols than non-polar extracts.

These results of our study concurred with the findings of I. Lahmar et al.³³ who reported that leaves had higher contents of phenolic compounds (including rosmarinic acid, gallic acid and chlorogenic acid) and flavonoids (including Isorhamnetin-3-O-glucoside, apigenin, myricetin, malonylglycosides of kaempferol, isoramnethin and quercetin)³⁴ than roots and stems.

Tannins are phenolic compounds that soluble in water, that which explains the richness of the aqueous phase in condensed tannins.

Antioxidant capacity assays may be in general classified as single electron transfer (SET) and hydrogen atom transfer (HAT) based assays. SET assays measure the capacity of an antioxidant in the reduction of an oxidant. Phosphomolybdenum Assay method is based on the redox antioxidant reaction.

In fact, electron-donating groups such as (OH, OCH₃, and alkyl) minimize the redox potential of polyphenols and increase their antioxidant capacities. Among the flavanols glycosides isolated from Pergularia tomentosa are 3-O-galactoside and 3-O-glucosides of quercetin, we recall that the oxidation potential of quercetin is of the order 0.25 V (Ag / AgCl)³⁵, therefore, the low potential value of oxidation greatly facilitates electron donation.

Consequently, quercetin glycosides are better antioxidants than kaempferol glycosides, independent on the relative capacities to donate electrons or hydrogen atoms³⁶. These results suggest that the antioxidant activities of these extracts are probably due to their reducing capacities, since these extracts contain some compounds like tannins³⁸, which are electron donors, and can reduce the oxidized intermediates of lipid peroxidation process.

This can be explained by the extracts of this plant may possess different secondary metabolites like alkaloid, cardenolides and phenolic molecules which may have reacted against the enzyme, and that the phenomenon of inhibition is caused by a synergy between several molecules^37,38.

In this paper, the obtained results have shown that inhibitory activity was proportional to the content of phenolic compounds (r²=0.72), which is not accord with those published by Mahfoudi F. et al³⁹.

Relationships among antioxidant capacity estimates and TPC, TFC, TTC:

In the present study, the extracts of leaves give a medium correlation between TPC and TFC, TCC with (r²= 0.63 and r²=0.61 respectively). Moreover, the TFC and TPC do not correlate with the antioxidant activity (especially with IC₅₀).

On the contrary, the extracts give an excellent correlation between TFC and the reduction capacity of Mo (VI) to Mo (V). On the other side, according to some studies, the antioxidant capacity depends on the structural conformation of phenolic compounds.

The latter is generally influenced by the phenolic compounds in the samples and by the different mechanisms involved in the radical–antioxidant reactions. These compounds may have a vast set of chemical structures that could react with radicals by hydrogen donation and/or by electron transfer.

There are no correlations between the two methods of antioxidant activity (PM and DPPH), these differences are attributed to different mechanism of action of antioxidant compounds in the assay.

In PM assay, antioxidants donate electrons to reduce Mo (VI) to Mo (V) by the extracts at the acidic pH. Due to different mechanism of action of antioxidants, the correlation of DPPH and PM was very weak.

However, employing a method dependent on one mechanism may not reflect the true antioxidant capacity. Hence, in the current research two types of antioxidant assays were performed to check the antioxidant potential of this plant.

CONCLUSION:

In the present study the extract of P. tomentosa exhibited appreciable content of secondary metabolites, what proves that P. tomentosa plant is the source of the secondary metabolites i.e. cardenolides, alkaloids, flavonoids, terpenoids, steroids, tannins, reducing sugars and saponins. The antidiuretic, anti-inflammatory, antibacterial, antiviral, anti-analgesic, antioxidant proprieties of the various parts of plants are due to the presence of the above-mentioned secondary metabolites. In addition, this study indicated that the aqueous extract is the highest phenolic and tannic content than the other extracts it exhibited strong antioxidant capacity in PM assay. Our results suggest that Pergulation tomentosa could be a potential source of antioxidant compounds and anti-diabetic drug. The isolation and identification of compounds from Pergulation tomentosa could be an interesting future study.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors gratefully acknowledge the support provided by Mr BELFAR Mohamed Lakhdar director of VPRS Laboratory, University of Ouargla, Algeria.

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Received on 09.11.2021 Modified on 16.12.2021

Research J. Pharm. and Tech 2022; 15(9):3941-3946.

DOI: 10.52711/0974-360X.2022.00660

Extracts and standards	Yield (%)	TPC (mg GAE/g DW)	TFC (mg QE/g DW)	TTC (mg CE/ g DW)	IC₅₀ (g/L)	AEAC (mM)
Crude	4.07	0.412 ±0.01	0.066 ± 0.00	0.136±0.01	0.175 ±0.01	84.76 ±0.05
Chloroform	0.52	0.173 ±0.00	0.025 ± 0.00	0.085± 0.00	0.605± 0.03	70.36 ±7.71
Ethyl Acetate	0.13	0.451±0.01	0.043± 0.00	0.069± 0.00	0.05 ±0.02	51.45 ±2.91
Butanol	1.68	2.255±0.25	0.486± 0.01	0.149 ±0.01	0.07 ±0.01	221.58± 7.09
Aqueous	8.64	2.490±0.49	0.205±0.00	0.436±0.00	0.94 ±0.32	265.85 ± 8.09
BHT	/	/	/	/	0.002± 0.0	4.96± 1.51
BHA	/	/	/	/	/	11.1± 7.55
Vitamine C	/	/	/	/	0.0045 ±0.0	/