INTRODUCTION:

Living organisms release reactive oxygen species (ROS) as a byproduct of regular cell metabolism. They exhibit several physiological actions at low to moderate concentrations, especially in redox signaling and growth control; nevertheless, at high concentrations, they cause harmful alterations to DNA, lipids, and proteins, among other components of cells.^1,2

As a result of an increase in ROS and a reduction in antioxidant defenses, oxidative stress plays a significant role in many health issues, including metabolic and physiological changes, as well as many illnesses in the body.³

Antioxidants are often present in the body various health supplements, and natural components. Antioxidant supplements or meals that are abundant in antioxidants might potentially enhance the body's immune system and overall defensive mechanisms, could aid in the reduction or elimination of oxidative injury and support the body's defense system.⁴phenolic compounds present in All parts of the plants, it has hydroxyl groups present on the aromatic ring. These are secondary plant metabolites from either shikimate/Phenylpropanoid pathway. These phenolic compounds, their derivatives and various subgroups of phenolic compounds contribute their major role in exhibiting antioxidant activity⁵ Polyphenols constitute a large group of bioactive phytochemicals that comprises several sub-classes including phenolic acids, flavonoids, stilbenes, and lignans⁶.The main feature of polyphenols is that they are very powerful antioxidants. Indeed, they are able to trap free radicals and activate other antioxidants in the body^7.A correlation exists between the structural characteristics of phenols and their antioxidant activity; specifically, the chemical properties of polyphenols as hydrogen- or electron-donating agents suggest their ability to function as scavengers of free radicals⁸.

The genus Allium (Family Amaryllidaceae, previously Alliaceae) With a membership exceeding 700 individuals, the organization is geographically dispersed around the world⁹, because of their similarity in organic and phytochemical compositional their great favor makes them clearly identifiable.¹⁰The polyphenolic compounds is a very high in this species, which include tannins, anthocyanins, carotenoids, flavonoids, phenolic acids, and polysaccharides. It also contains a variety of sulfuric compounds, which give it its distinct flavor and odor.¹¹ Allium plants were utilized for many years to impede and treat Multiple illnesses such as headaches, coughs, flu, asthma, inflammation, hemorrhage, arthritis, diabetes, and atherosclerosis. It has also been reported that these plants, besides having pharmacological activities as antibacterial, antiviral, antifungal, anthelmintic, and antiprotozoal, also exhibited the potency of in vitro and in vivo antimotility and antiproliferative against cancer cells. Their beneficial properties on health are due to the rich contents of bioactive phytochemicals, such as phenolic, particularly flavonoids, and some organosulfur compounds.¹² A. sphaerocephalon L. , The plant is often known as "Round-Headed Leek" and is a perennial herbaceous plant with huge, globe-shaped flower heads. A. sphaerocephalon is an edible plant that is used as a condiment and an alternative to onions (A. sativum) in several places. Additionally, other plant parts—bulb, stem, and leaves are eaten raw or cooked in Spain, Ukraine, Siberia's Lake Baikal area, and Italy.^13-14. Among the detected major phenolic compounds generally by LC-MS-MS in A. sphaerocephalon L. are gallic acid as phenolic acid and 3-O-methyl quercetin as a flavonoid¹⁴.

Many studies have been done on the chemical makeup, antioxidant capacity, and other biological characteristics of many Allium species, especially those that are cultivated as onions and garlic. However, not much study has been done on spontaneous species like A. sphaerocephalon L. especially, lack of studies on the antioxidant activities with several methods of this plant. In this research, the hydro-ethanol and aqueous extracts of Allium sphareocephalon L. flowers parts Were examined to identify and measure the antioxidant activity and phenolic substances. based on our current information, this is the first study to report the antioxidant activity specification of this species. Therefore, this research activity aimed to examine the antioxidant capacities and phenolic and flavonoid contents of hydro-ethanolic and aqueous extracts derived from wild A. sphaerocephalon L. flowers in vitro through the implementation of various methodologies.

MATERIALS AND METHODS:

Vegetative matter:

A. sphaerocephalon, L. naturally growing plant, was obtained in July 2018 at Megress district of Setif. Professor Smain Amira of the Department of Animal Biology and Physiology at University Setif 1, Algeria, carried out the taxonomic identification of the plant. A voucher number 81 A.S. 02/07/18 set/SA/ was deposed at the laboratory of Phytotherapy Applied to Chronic Diseases, Faculty of Nature and Life Sciences, University of Setif 1, Algeria.

Methods of extraction:

Hydro-ethanolic extract:

We macerated 500 grams of flowers in 1000 milliliters of 80% ethanol for three days at room temperature while being constantly agitated to produce the hydro-ethanolic extract of flowers (EOH).¹⁵. The mixture underwent filtering, condensing, and oven drying at 38°C in a vacuum evaporator.

Aqueous extract:

The aqueous extract (AQE) was prepared using the methodology outlined in¹⁶, Ten grams of powdered flower parts were boiled for twenty minutes with agitating magnetically in 200 milliliters of purified water. Subsequently, the blend was dried at 45°C and filtered using Wattman filter paper N°1. The resulting dried extract (AQE) was next examined for pharmacological characteristics.

Extraction yield:

The weight difference between the extract's and the treated plant's weight is known as the plant extract yield. it is represented as a percentage, and was computed using the following Eq.:

Extraction yield (%) = ---------------------- x 100

Were, m₁- sample extract weight, g

m₂- sample weight, g;

Phenolic compounds:

Quantification of total phenol content:

Folin–Ciocalteu reagent (FCR) was utilized to evaluate total phenolic content (TPC) in accordance with the method described by¹⁷. A well microplate containing 100μL of diluted FCR (1:10) was filled with a volume of 20μL of extract or different concentrations of standard. A further addition of 75μL of 7.5% sodium carbonate was made. Two hours later, in low light conditions and at a stable ambient temperature. A Perkin Elmer 96-well microplate reader (USA) was then used to measure the absorbance at 765nm. The TPC was represented as μg gallic acid equivalent for every milligramme of extract.

Determination of the overall amount of phenols flavonoid:

The aluminium nitrate (Al (NO₃)₃) reagent was used to measure the total flavonoid content (TFC) according to the¹⁸. A 96-well microplate was filled with a volume of 50μL of every extract, 130μL of methanol, 10μL of potassium acetate, and 10μL of aluminium nitrate. After 40 minutes of incubation, the mixture's absorbance at 415nm was measured. The TFC was represented as μg QE/mg E, or quercetin equivalent per milligramme of extract.

Assessment of the anti-oxidant capacity:

DPPH radical scavenging test:

The approach outlined by¹⁹ was used to determine this activity. In a 96-well microplate, 160μL of DPPH reagent (0.006%) was combined with 40μL of various extract concentrations or the standard solution. Following that, the mixture was allowed to remain at room temperature in the dark for 30minutes, at which point the absorbance at 517nm was measured. Eq. (2) was used to compute the percent inhibition.

A_control - A_sample

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

A_control

(IC₅₀ refers to the concentrations at which 50% of the DPPH radical was scavenged)

Ferrous ion chelating activity:

To summarise, 50µL of FeCl2 (0.6mM) and 450µL of methanol were combined with 250µL of various extract concentrations or an EDTA solution. After five minutes, 50μL of ferrozine (5 mM) was added to begin the reaction. Let the mixture to sit for 10 minutes at room temperature. The produced Fe2+-ferrozine complex's absorbance was measured at 562 nm, and Eq. (3) was used to report the chelating activity as a percentage of inhibition²⁰.

Where, AS and AC represent the absorbance values of the control and test sample, respectively.

(As – Ac)

Fe2 chelating effect (%) = ------------------- x 100 … (3)

Method of bleaching β-carotene:

The β-carotene bleaching technique as outlined by ²¹was used to calculate how well the molecules of antioxidants in the extract blocked the oxidation of β-carotene. The β-carotene/linoleic acid emulsion stock solution was prepared by first combining 25 µL of linoleic acid, 200 mg of Tween 40, and β-carotene solution (containing 0.5 mg of β-carotene dissolved in 1 mL of chloroform). Following this, chloroform was extracted via vacuum evaporation. The residue was combined with 100 mL of oxygenated distilled water for 30 minutes. After a vigorous shake of the mixture, 2.5 mL of this blend was incorporated and thoroughly mixed with 350 μL of BHT or flowers' extract (2 mg/mL). The absorbance was calculated at 490 nm at zero, one, two, four, six, and after day. (Shimadzu1601). This formula (Eq. (4) has been used to calculate the antioxidant activity (AA):

(AS₀- AS₁)

AA = 1- ----------------------------------------- ………(4)

(AC₀- Ac_t)

Where AS₀ and AC₀ are, In the order mentioned, the absorbance levels of the test sample and the control after zero minutes of incubation. The absorbance of the test sample A_St and the control A_Ct are calculated after incubation for 24 hours.

Reducing power assay:

Method²² was used to determine the extract's reducing power. 10μL of various concentrations of extracts or the standard solution was added to her a 40μL of 0.2M phosphate buffer (pH 6.6) and 50μL of potassium ferricyanide (1%). After 20 minutes of incubation at 50 °C, the solution was supplemented with 50μL of TCA (10%), combined with 40μL of distilled water and 10 μL of ferric chloride (0.1%). The absorbance at 700 nm was then measured.

Cupric reducing antioxidant capacity:

The extracts' cupric-reducing capacity was determined using the procedure described in²³. 50μL of Cu2+ solution (10mM) was combined with 60μL of ammonium acetate buffer (1 M, pH 7.0) and 50 μL of neocuproine solution (7.5mM). The mixture was added to 40μL of the sample solution of each extract at different concentrations. The absorbance at 450nm was measured using a 96-well microplate reader after 60 minutes in the absence of light, with a reagent blank as reference. The positive control utilized was BHT. The findings were reported as the EC₅₀ value. (μg/mL) corresponding to the concentration indicating 50% absorbance.

Statistical analysis:

The data was shown as means ± standard deviation (SD). In three determinations (n=3), every measurement was performed. Using Graph Pad Prism 8.00, we conducted an analysis of variance using the student’s and one-way ANOVA tests. Statistical significance was assumed at the 0.05 level.

RESULT AND DISCUSSION:

Phytochemical analysis:

The results of extraction yield, TPC, and TFC of A. sphaerocephalon L. EOH and AQE were summarized in table 01.

Table 1: Extract yields total phenolic and total flavonoid content of EOH and AQE

	EOH	AQE
Extract yield (%)	18.30	11.80
TPC (µg GAE/mg DW)	12.73±0.91	9.71±0.53
TFC (µg QE/mg DW)	7.67±0.24	5.68±0.90

The yield percent from the EOH was higher (18.30%) than the AQE (11.80%). The results show that EOH had the highest TPC level (2.73±0.91µg GAE/mg) than AQE (9.71±0.5391µg GAE/mg). In addition, EOH had higher TFC level (9.71±0.53µg QE/mg) than AQE (5.68 ±0.90µg QE/mg).

In the present study, the percentage yield of hydroethanolic extract was approximately comparable with the flowers of A. atroviolaceum Bois¹¹ and A. paniculatum subsp. Villosulum²⁴ with 19.44% and 19.12%, respectively. The present values were higher than the previous findings in the flowers of A.pallens (from different localities in Turkey) which were 11.65% and 14.75%, respectively²⁵, and A. paniculatum subsp Paniculatum (15.43%)²⁴ and lower than those of A. ampeloprasum L. (27.31%)²⁶, and A. stylosum O. Schwarz (26.25%)²⁷. AQE extract was comparable with A.pallens (11.65%)²⁵ and Allium stylosum O. Schwarz (12.36%). In this comparison, there is a difference in allium species, collected regions, and solvent of extraction. According to^28-29, the nature of the extracting solvent and its polarity, nature and polarity of the extracting solvent as well as variety diversity, growth conditions, ripening degree, and climate, can all have an impact on the extract yields of plant materials.

The phenolic compounds are secondary metabolites with miscellaneous protective roles; their synthesis is carefully controlled through many stress signals and environmental factors.³⁰ They are aromatic hydroxylated compounds that have one or more hydroxyl groups on their aromatic rings, Polyphenols possess the ability to function as antioxidants due to their capacity to undergo robust hydrogen or electron donor reactions, stabilize chain-breaking reactions, and terminate Fenton reactions.³¹

The TPC of hydroethanolic and aqueous extracts of A. sphaerocephalon flowers was approximately similar to those of flowers of A. sphaerocephalon subsp. Sphaerocephalon, A. sphaerocephalon subsp. Trachypus ¹⁴. A. paniculatum subsp. Villosulum and A. Paniculatum subsp. Paniculatum²⁴ with (14.83mg GAE/g, 11.44mg GAE/g, 15.19mg GAE/g, 10.97mg GAE/g) respectively. However, our results were higher than those of flowers of A. roseum var. odoratissimum (135 mg GAE/100 g)³², and lower than A. atrovioleceum Boiss. (33.72mg GAE /gand 25.81mg GAE /g)¹¹. Collected from two different localities in İzmir (Turkey).

TFC value was approximately similar to those of flowers of A. atrovioleceum Boiss (8.63 mg QE/gand 6.93mg QE/g)¹¹, and higher than flowers of A. sphaerocephalon subsp. Sphaerocephalon, A. sphaerocephalon subsp. Trachypus (1.56mg QE/g 2.07mg QE/g, respectively)¹⁴, A. ampeloprasum L (1.78 mg QE/g 1.14mg QE/g²⁵ and A. roseum var. odoratissimum) (74.3mg QE/100 g)³².

The investigation by³³ indicates that the properties and polarity of the solvent have a significant impact on phenolic and antioxidant extraction.

Transport and storage conditions, alongside genetic and environmental factors, exerted a substantial impact on the concentrations of plant metabolites. In addition to growth-influencing elements including light, temperature, humidity, soil type, fertilizer applications, damage brought about by microorganisms and insects, UV radiation stress, heavy metal exposure, as well as pesticide application, alter the metabolite profile of plants.³⁴

Antioxidant activity:

The use of antioxidant therapy in the treatment of illness has become increasingly important. Antioxidant compounds interfere with oxidative stress by a variety of mechanisms including interactions with free radicals, chelating, catalytic metals, and also by oxygen scavenger activity.³⁵ Soin the present work, the antioxidant activity of A. sphaerocephalon L. flowers were determined using five different assays: DPPH radical scavenging assay, reducing power, cupric reducing antioxidant capacity, ferrous ion chelating, and β-carotene bleaching test.

Activity of radical DPPH scavenging:

DPPH is a free radical distinguished by its purple hue; when interacting with an antioxidant, it generates a compound that is persistently yellow in color, the quantity of the reaction is ascertained by the hydrogen-donating capacity of the antioxidant.³⁶ As seen in Table 2, EOH. had the strongest radical-scavenging effect (IC_{50 =}0.28±2.02mg/mL) compared to AQE (IC₅₀˃0.8 mg/mL).

ETOH extract of this study was higher than flowers’ methanolic extract of A. roseum var. odoratissimum³² and lower than flowers of methanolic extract of A. atrovioleceum Boiss¹¹ and A. nevsehirense, A. sivasicum, A. dictyoprosum, and A. scrodoprosum subsp. Rotundum³⁷. But the AQE extract was lower than all species

There is a difference in the capacity of radical scavenging between the species of Allium. Consequently, through these comparisons, whenever the extracts were rich in total phenolics, it was a stronger scavenging ability on DPPH.³⁸ Proved a strong correlation between total phenolics and antioxidant activity.

In addition, several publications proved the existence of a relationship between the greater TPC which has stronger radical scavenging activities^{37,38, -24-11}. But the AQE extract was lower than all species mentioned. In this case, it appears that the conflicting results are very likely because of differences in methodology and experimental conditions used.

Ferrous ion chelating activity:

Due to its potent reactivity with transition metals, iron is regarded as the most significant lipid pro-oxidant.³⁹. Lipid peroxidation is induced via the Fenton and Haber-Weiss reaction, and the lipid hydroxide is decomposed into peroxyl and alkoxyl radicals, which have the potential to sustain the chain reactions. ⁴⁰The metal chelating capacity is crucial since it reduces the concentration of metals, which in turn has a catalytic impact on lipid peroxidation. Metal chelating compounds function as secondary antioxidants by lowering the redox potential and so providing stability to the oxidized molecules.⁴¹

AQE. displayed a chelating activity to Fe²⁺ ions (IC₅₀=0.076±0.006mg/mL), higher than EOH (IC₅₀=0.161±0.012mg/mL), but it appeared to be weaker than the positive control EDTA (IC₅₀=0.005±0.0002) in this test (Table 02). These extracts and standard prevented the production of a ferrous-ferrozine combination, indicating that they possessed chelating properties and absorbed iron ions before ferrozine.

Phenolic compounds' antioxidant capacity is additionally ascribed to their capacity to chelate metal ions that participate in the generation of free radicals.⁴², More precisely, the flavonoids that shown the ability to bind and remove excessive metal ions in the human body.⁴¹ The antioxidant efficacy of flavonoids is affected by the quantity and positioning of their aromatic hydroxyl groups.⁴³

Our investigation revealed that the existence of polyphenols and flavonoids in these extracts is accountable for their capacity to chelate metals. Nevertheless, the efficacy of this action is not contingent upon the amount of these chemicals. In fact, AQE extract had a lower amount of polyphenols and flavonoids but had good metal-chelating activity.

β-carotene bleaching method:

The β-carotene bleaching assay measures the capacity of antioxidants to shield target molecules from the effects of free radicals through the prevention of lipids oxidation. When compared with the negative control (methanol or distilled water), the flowers’ extracts of A. sphaerocephalon L. and standard at 2mg/demonstrated a substantial inhibition. After 24hours, EOH showed high inhibition percent (67±0.009%) than AQE (60±0.037%). These values still lower than BHT as a positive control (96±0.005%) (Table 2).³⁶ evaluated the antioxidant activity of five Allium species from Turkey and demonstrated the inhibiting action of lionolic acid oxidation, where the ethanol extract had lower activity than A. atroviolaceum and. dictyoprosum (71.2±2.20% and 72.3±1.20%, respectively). These values were close to those of A. nevsehirense and A.scrodoprosum subsp. Rotundum, but higher than those of A. sivasicumand A. tuncelianum bulbs (51.1±5.5%)⁴⁴. The AQE was greater than the latter and approximately similar to A. sivasicum and less than the rest of the species. This activity can be related to the difference in phenolic content, or some compounds other than phenols, such as sulfur compounds.

Table 02: DPPH scavenging assay metal chelating and β-carotene bleaching of A.sphaerocephalon L. flowers’ hydroethanolic and aqueous extracts.

IC₅₀ (mg/mL)			Inhibition (%)
	DPPH	Fe²⁺ ion chelating	β-carotene bleaching
ETOH	0.28±2.02	0.161±0.012	67±0.009
AQE	˃0.80	0.076±0.006	60±0.037
BHA	0.0061±0.41
BHT	0.012±0.41		96±0.005
α-Tocopherol	0.01±5.17
EDTA		0.005±0.0002

Reducing power assay:

The reducing power and CUPRAC assays are commonly employed to evaluate the electron transfer capability of antioxidants in the presence of radicals. The reducing capacity method is commonly associated with the presence of reductons, whose antioxidant properties have been established through the disruption of the free radical chain and the donation of a hydrogen atom.⁴⁵Within this research, each extract demonstrated a perceived Decreasing power capacity. AQE had higher activity (EC₅₀=0.29±0.00mg/mL) than EOH (EC₅₀=0.36±6.66mg/mL). However, their effect lower than that of α-Tocopherol (EC₅₀=0.03±2.38mg/mL) and acid ascorbic (EC₅₀=0.0067±1.15mg/mL) (Table 03).

According to the literature, the ariel parts and bulbs of A.scabriscapum⁴⁶ and A. triquetrum L.⁴⁷ had a good activity, but not comparable with the control as was the case with our samples.

In addition, the reducing power of the extract increases in a concentration dependant manner.⁴⁸ It was said that the blooms and leaves of A. roseum var odoratissimum have the highest antioxidat activity, while the bulbs, showed the lowest antioxidant activity, which was linked to the presence of specific phenolic components as well as their structures, in addition to the sugar moieties, also known as glycosylated phenolics, are present and have a rather low antioxidant activity. Antioxidants transform Fe3+ ferricyanide complexes into the ferrous (Fe2+) form³⁸, This conversion is facilitated by the reducing capacity of the antioxidants, which is likely due to the presence of a hydroxyl group that may donate electrons⁴⁹.

CUPRAC reducing antioxidant capacity:

The methodology of this assay involves the reduction of Cu (II) to Cu(I) through the collective activity of antioxidants (reducing agents) present in a sample containing neocuproine (2,9-dimethyl-1,10-phenanthroline)³⁹Antioxidants that are both hydrophilic and lipophilic can be quantified using the CUPRAC method. In this study, there is no significative difference between the two extracts, where the ability of reducing Cu (II) to Cu(I) was almost equal EOH (EC₅₀= 0.39±6.35mg/mL) and AQE (EC₅₀=0.35±9.07mg/mL), but it was lower than the standards BHA (EC₅₀=0.005±0.71mg/mL) and BHT (EC₅₀=0.008±3.94 mg/mL). It is hard to compare our results to other results, because of the difference in the expression of results, but according to the following species A. ampeloprasum, A.atrovioleceum Boiss. A.pallens L., it has an activity that is attributed to the presence of phenolic and flavonoid components.

Table 03. Reducing power assay and Cupric reducing antioxidant capacity of EOH and AQE

EC₅₀mg/mL
	Reducing power assay	Cupric reducing capacity
ETHO	0.36±6.66	0.39±6.35
AQE	0.29±0.00	0.35±9.07
BHA		0.005±0,71
BHT		0.008±3.94
Ascorbic acid	0.0067±1.15
α-Tocopherol	0.03±2.38

CONCLUSION:

In this investigation, the polyphenol and flavonoid content, and the in vitro antioxidant activities of the hydroethanolic and aqueous extracts of the flowers of Allium sphaerocephalon L. were investigated. The results demonstrated that this natural plant has a significantly higher yield in both extracts and a moderate total phenol and flavonoid content, with the hydroethanolic extract containing a higher concentration. The flowers’ extracts have demonstrated acceptable antioxidant activities, which are not all in line with the amount of phenolic compounds. The antioxidant activities of A. sphaerocephalon L. are comparableto the previously studied species. Therefore, more studies are needed to explore in-depth the relation between the chemicals of this wild edible species and their biological activities.

CONFLICT OF INTEREST:

The writers do not have any conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Algerian Ministry of Higher Education and Scientific Research funding was secured to support this investigation.

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Received on 10.10.2023 Modified on 21.11.2023

Research J. Pharm. and Tech. 2024; 17(2):903-909.

DOI: 10.52711/0974-360X.2024.00140