INTRODUCTION:

Synthetic antioxidants are used as food products spoilage inhibitors, and synthetic antimicrobials are employed to prevent microbiological deterioration¹. Therefore, the researches about natural antioxidants and antimicrobials have witnessed an increasing tendance nowadays¹ due to several studies, which was aimed at evaluation of possible adverse health effects were related to the synthetic antioxidants consumption and microbial resistance development to disposable antibiotics^2-3. The antimicrobial resistance acquired by pathogenic microbes is projected to reach 10 million by 2050⁴. As such, in food industry, some vegetables, fruits, plants species and spices are known as primary numerous therapeutic agents source^1-2-3. Although, in recent years, several studies have been undertaken to explore biological activities of Algerian medicinal plants⁵. The Erica genus, which belongs to the Ericaceae metropolitan and large family in the world and represents 4100-4250 species, spread across 124 genera^1-⁶. There are five of which are that found abundantly in the flora of Turkey and three of which are that found of Algeria flora^1-7. Erica arborea L. is a Mediterranean and indigenous flowering plant⁶, develops prevalently in Algeria and its ethnobotanical report emphasizes their significance to fight a few issues such as digestive disorders, urinary infections, renal lithiasis and insects bites⁸. Additionally, E. arborea L. is known for its astringent properties, and its aerial parts have been traditionally used for a variety of purposes such as antiulcer, antimicrobial, anti-edema, antidiarrheal, and wound healing⁶. Previous experiments have focused on the antioxidant and antimicrobial properties of E. arborea's aerial parts or leaves^8-9. However, one study specifically examined the antioxidant activities of its stem parts¹⁰. Therefore, the primary aim of this study was to investigate the antioxidant and antimicrobial capacities of E. arborea stems from Algeria using methanol, chloroform, ethyl acetate, and aqueous extracts, as well as to determine their total phenolic, flavonoid, and condensed tannin contents.

MATERIALS AND METHODS:

Plant material:

Stems of E. arborea L. were collected from Djebel of Tadergount, Bejaia, located in northern Algeria, in March 2017. The plant was identified by Professor H. Laouer from the Laboratory of Valorization of Natural Biological Resources at the University of Setif, Algeria, and a voucher specimen (015/DBEV/UFA/19) was deposited. The harvested material was dried, powdered, and stored in darkness until it was ready for use in experiments.

Preparation of methanolic extract and sub-fractions:

Briefly, One hundred grams stems powder was macerated in Erlenmeyer flasks with 1L methanol 85%. The mixture was soaked at room temperature and was left for one day. Next, the filtrate was recuperated, utilizing the filter paper and the marc was then re-macerated once with methanol/water increasing the extraction yield. Then, the methanolic extract submit to further successive fractionation through using increasing polarity of solvents, including chloroform and ethyl acetate. The solvents was evaporated by putting preweighed petri dishes containing the respective sub-fractions in an oven at 40°C. The sub-fractions were obtained and stored in a refrigerator until reconstituted for use¹¹.

Phytochemical screening:

Preliminary qualitative analysis of phytochemical compounds presence such as polyphenols, flavonoids, hydrolyzables tannins, free quinones, anthraquinones, anthocyanins, coumarins, alkaloids, terpenoids and saponins, who based on coloring and/or precipitation reactions were given according to the standard published procedure¹².

Determination of total phenolic flavonoids and condensed tannins contents:

The different extract were subjected to the total phenolic and flavonoids determination, using the colorimetric methods, according to our previous publication¹³. Briefly, 100µL of the extract was mixed with 500µL of Folin-Ciocalteu’s reagent (1/10). After 4 min, 400µL of a 7.5% Na₂CO₃ solution was added to the mixture. After 1.5 hours of incubation, the absorbance of the samples was measured at 765 nm to determine the total flavonoid content. Specifically, 1mL of each sample was mixed with 1mL of 2% aluminum chloride (AlCl₃) solution, and after 10 minutes of further incubation, the absorbance was recorded at 430 nm against a blank. The condensed tannins in the extracts were quantified using the vanillin method, as described by reference¹⁴. For this, 1mL of extract was combined with 1.5mL of 4% vanillin/methanol solution, followed by the addition of 750μL of concentrated hydrochloric acid (HCl). After allowing the mixture to stand in the dark at 20°C for 20 minutes, the absorbance was measured at 500nm against a prepared calibration curve. The results of polyphenols, flavonoids and tannins were expressed as µg Gallic Acid Equivalent/mg dry weight (DW), µg Quercetin Equivalent/mg DW, and µg Catechin Equivalent/mg DW, respectively. All analyses were performed in triplicate (n = 3).

Antioxidant capacity:

DPPH scavenging and reducing power assay:

The stem extracts of E. arborea were evaluated for antioxidant activity using the DPPH radical scavenging assay and ferric reducing power assay¹⁵. For the DPPH assay, a 0.4mM DPPH radical solution was freshly prepared in methanol and protected from light. Various concentrations of the E. arborea stem extracts were prepared by serial dilution in methanol. Each serial dilution (50µL) was mixed with 1.25mL of the DPPH solution and incubated in darkness for 30 minutes. A control solution containing 1.25mL of DPPH solution mixed with 50µL of methanol was also prepared. The absorbance of all samples was measured at 517nm after incubation, and the percentage of DPPH radical inhibition was calculated using the formula: [(Ac - As)/Ac]*100, where Ac is the absorbance of the control and As is the absorbance of the extract.

For the ferric reducing power assay¹⁶, 400µL of extract was mixed with an equal volume of phosphate buffer (0.2M, pH 6.6) and potassium ferricyanide (1%). After incubation at 50°C for 20 minutes, the reaction was stopped with 400µL of trichloroacetic acid (10%) and then centrifuged. To the supernatant (400µL), distilled water (400µL) and 80µL of 0.1% ferric chloride were added. The absorbance of the resulting mixture was measured at 700nm after 10 minutes of incubation. Butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls. Both the DPPH radical inhibition and ferric reducing power were calculated to assess the antioxidant activity of the E. arborea stem extracts.

Antimicrobial activity:

Antimicrobial activity was determined using the disk diffusion method¹⁷ The microorganisms used in the assay include three Gram positive and three Gram negative bacterial strains and three fungal strains. The Bacterial strains; Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 27141), Bacillus cereus (ATCC 10987), Escherichia coli (ATCC 11303), Pseudomonas aeruginosa (ATCC 27853), Salmonella gallinarum (ATCC 700623) fungal strain Aspergillus brasiliensis (0392 ATCC) and yeast Candida albicans (0443 ATCC) belonging to American Type Culture (ATCC). Aspergillus niger and Aspergillus flavus were isolated from grains of wheat and supplied by Dr. O. Benserradj (Laboratory of mycology, biotechnology and the microbial activity, University Constantine1, Algeria). The bacterial were routinely sub-cultured into nutrient broth at 37°C for 24h, with the exception of fungal strains and Candida albicans for which the growth temperature was set to 30° on sabouraud dextrose agar for 7days and 48h, respectively.

Under aseptic conditions, the bacterial suspension was prepared in saline solution (0.9 % NaCl) and adjusted to 0.5 McFarland standard turbidity (equivalent to DO=0.08-0.1/λ= 620nm) for bacteria. Fungi strains were suspended in sterile physiological solution and the spore suspension was adjusted to a concentration of 1-5 * 10⁶ spores/mL corresponding to 0.12 to 0.15 absorption at 625nm. Subsequently, the extracts were dissolved in DMSO (dimethyl sulfoxide) to final concentrations of 100mg/mL. Uniformly, all Mueller hinton agar petri dishes were spreaded with microbial inoculum, using cotton swab and the wattman paper discs N°3 of 6mm diameter were then impregnated with 10µL of each extract equal to 2mg by disc. Gentamycin disk (0.120 mg/mL) and Verten powder (1mg/mL) served as positive controls for antibacterial and antifungal assays respectively. The petri dishes were incubated in an incubator at 37°C for 24 h for bacteria and at b30°C for three-seven day for yeast and fungi strains, respectively. Finely, the antimicrobial activity was determined by measuring growth inhibition zones diameters around each disk.

Determination of minimum inhibitory concentration:

The minimum inhibitory concentration was evaluated by serial dilution microplates procedure with some modifications¹⁸. Bacterial cultures were prepared overnight in sterile nutrient broth and adjusted at density of (0.08–0.10 /625nm).Using the 96-well microplates, 50mL of extracts (100mg/mL) was added to the first well, followed by serially dilution from 1st well to the 12th. The bacterial suspensions (100μL) were also added to each well and Gentamicin and buffer was employed as a positive and negative control, respectively. The microplates were incubated overnight at 37°C and the absorbance was measured at 625nm after 24h of incubation. Each experiment was replicated at least three times.

Statistical analysis:

Statistical analysis was performed by using the Graph Pad Prism (version 5.03 for Windows). In this study, statistical analysis was analyzed by one-way analysis of ANOVA. All determinations were carried in triplicate, and all results were estimated as the mean±standard deviation (SD). Tests of significant differences were determined by multiple range tests at p< 0.05.

RESULTS:

Phytochemicals screening:

The extract phytochemical characters in methanol, chloroform, ethyl acetate and aqueous were investigated and represented in Table 1. The observed phytochemical analysis of stems revealed the existence of vary metabolites such as polyphenols, hydrolyzable tannins, quinones and terpenoids, while alkaloids and anthocyanins were absent in all extracts. Flavonoids are found to be present only in methanol and ethyl acetate extracts.

Phytochemicals compounds quantification

At last, a total yield of methanol (16.15%), chloroform (0.99%), ethyl acetate (2.60%) and aqueous extracts (5.63%). The results reported in Table 2 showed that the distribution of total phenolic compounds (TPC) total flavonoids (TFC), and condensed tannins (TC), where depends on the polarity of the extraction solvent used. The total polyphenol and flavonoids contents of the different sub-fractions vary between 171.83-973.52µg GAE/mg extract and 8.24-19.35µg QE/mg extract, respectively. The highest concentration of them was recorded in the ethyl acetate extract, compared to the methanolic, chloroform and aqueous extracts. The aqueous extract records a maximum of condensed tannins (275.53µg CatE/mg extract).

Antioxidant activity:

Figure 1 presents the results of DPPH scavenging and reducing power inhibition percentages of stems extracts. All the results showed the increase of percentages inhibition with the concentration increase. In DPPH scavenging activity method, the IC₅₀ of ethyl acetate extract of stems E. arborea. L was found to be 0.02± 0.00μg/mL, which having potent antioxidant as compared to aqueous, methanolic and chloroform extracts. In reducing power method, the IC₅₀ of stems methanolic extract E. arborea. L was found to be 0.006 ±0.00mg/mL which having potent antioxidant as compared to ethyle acetate, aqueous and chloroform extracts. Comparative studies with standards have shown that the IC₅₀of extractsvalues are more less than BHT and ascorbic acid employed as positive control, which proved that the stems of E. arborea. L have a good antioxidant activity.

Table 2: Total phenolic, flavonoids and condensed tannins contents of stems extracts.

Extracts	TPC	TFC	TC
Methanol	688.34 ± 1.46	13.54 ± 1.60	218.93 ± 0.23
Chloroform	171.83 ± 0.73	7.09 ± 0.30	3.68 ± 1.30
Ethyl acetate	973.52 ± 1.95	19.35 ± 0.06	115.87 ± 0.47
Aqueous	582.27± 1.46	8.24 ± 0.04	275.53 ± 0.94

The data represent the mean ± SD of three determinants. TPC : total phenols content (µg GAE/mg extract) ; TFC : total flavonoids content (µg QE/mg extract); TC: total condensed tannins (µg CatE/mg extract). Data are presented as the mean±SD (n = 3).

Antimicrobial Activity:

In the present finding (Table 3, Figure 2), the stems methanol extract had antibacterial activity against all bacteria strains than other sub-fractions, in the range of 11.03 to 12.98. The ethyl acetate stems extract displayed 17.60mm, the highest inhibition zone against P. aeruginosa, which indicates very good activity of the extract, while no activity was observed by chloroform and aqueous fractions. The results obtained indicated that no one of methanolic extract and their resulting sub-fractions inhibited the growth of tested yeast and fungal strains.

Table 3: Antimicrobial activity of methanolic extracts and sub-fractions of E.arborea stems en mm.

Extracts/diameter of inhibition zone (mm)
Isolates		Extract-fraction		Standard
Bacterial strains	Methanol	Chloroform	Ethyl acetate	Aqueous	Gentamycin
*B.cereus*	12.20 ± 0.88^***	-	-	-	27.96 ± 1.71
E. *coli*	12.98 ± 0.89^***	-	-	-	27.34 ± 0.29
M. *luteus*	11.91 ± 0.54^***	-	-	-	26.48 ± 2.00
P. *aeruginosa*	11.04 ± 0.09^***	-	17.60 ± 0.85^***	-	27.28 ± 0.50
S *.gallinarum*	11.03 ± 1.18^***	-	-	-	26.35 ± 1.21
S. *aureus*	12.66 ± 0.98^***	-	-	-	27.28 ± 0.27
Fungal strains	Methanol	Chloroform	Ethyl acetate	Aqueous	Verten
*A.niger*	-	-	-	-	17.17 ± 1.84
A. *flavus*	-	-	-	-	12.76 ± 1.32
A. *brasiliensis*	-	-	-	-	29.32 ± 4.75
C. *albicans*	-	-	-	-	8.81 ± 0.08

-: no inhibition zone. Data are presented as the mean ± SD (n =3). *** : p < 0.001.

Figure 2: Antibiogram results of E.arborea. L extracts. (a): B. cereus, methanolic extract; (b): E. coli, methanolic extract; (c): M. luteus, methanolic extract; (d): P. aeruginosa, methanolic extract; (e): S. aureus, methanolic extract; (f): S. gallinarum, methanolic extract; (g): P. aeruginosa, ethyl acetate extract.

Table 4 : MIC/MBC stems extracts of E. arborea. L

MIC/MBC (mg/mL)
Extract-fraction					Standard
Bacterial strains	Methanol		Ethyl acetate		Gentamycin
Bacterial strains	MIC	MBC	MIC	MBC	MIC	MBC
*B.cereus*	2.51 ± 0.25^***	3.52 ± 0.34^***	-	-	0.02 ± 0.00	0.13 ± 0.03
E. *coli*	16.94 ± 1.64^***	19.26 ± 1.98^***	-	-	0.05 ± 0.00	0.1 ± 0.00
M. *luteus*	2.41 ± 0.22^**	3.50 ± 0.26^***	-	-	1.01 ± 0.17	0.40 ± 0.01
P. *aeruginosa*	23.63 ± 1.16^***	29.10 ± 1.20^***	1.56 ± 0.25^***	2.51 ±0.27^***	0.01 ± 0.00	0.08 ± 0.06
S *.gallinarum*	7.39 ± 0.05^***	8.47 ± 0.06^***	-	-	0.04 ± 0.00	0.09 ± 0.01
S. *aureus*	5.03 ± 2.38^***	6.11 ± 2.55^***	-	-	0.01 ± 0.00	0.1 ± 0.00

MIC : Minimum inhibitory concentration, MBC : minimum bactericidal concentration. Data are presented as the mean ± SD (n = 6). ***: p < 0.001, ** : p < 0.01.

Minimum inhibitory concentration and minimum bactericidal concentration:

The bacteriostatic and bactericidal activities of extracts are denoted by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), respectively. As shown in Table 4. The MIC and MBC of ethyl acetate extract of stems E.arborea. L against the tested organisms are 1.56 and 02.51mg/mL, respectively. The MIC and MBC of methanolic extract varied between 2.51-23.63 and 3.50-29.10mg/mL, respectively. While that of Gentamycin varied between 0.01-1.01 and 0.08-0.13mg/mL, respectively. Two extracts (ethyl acetate and methanolic) presented the MIC values superior to antibiotic standard.

DISCUSSION:

Medicinal plants are frequently studied attributed to their chemical constituents and possiblility of therapeutic uses¹⁹. Extraction is a very important step for the discovery of bioactive compounds from plant powder. Various parameters like extraction techniques and type of extracting solvents are affected the extraction yield²⁰. The yield extraction is generally tended to increase with the increasing solvents polarities used for plant extraction as suggested by^21-22. The final sub-fraction exhibited the second higher extractive yield value, which may be due to the higher polarity of water, according to²³. Ericaceae family species have nutritional value containing certain secondary compounds, including polyphenols, flavonoids, tannins, terpenes, and some other phytochemicals^1-23. Although, no previous was work realized on stems extracts of E.arborea. L phytochemical structure. The presence of the most metabolites such as polyphenols, flavonoids hydrolyzable tannins, anthraquinones, quinones, and terpenoids in all stems methanolic extracts of E.arborea. L as compared to other extracts like chloroform and ethyl acetate was also reported in many research of ^24-25, which, methanol solvent is considred as more polar solvent for extracting many of the active phytochemical constituents. The secondary metabolites derived by medicinal plants such phenolic compounds are essentiel for the antioxidant potential and prevent the food deterioration^8-26. Its widely distributed and may exert other beneficial health effects to humans, animals, including anti-inflammatory, analgesic antibacterial and antifungal activity^26-27-28.

In general, stems have the highest amount of phenolic compounds. This result is similar to that obtained by¹⁰and is in a good agreement with the literature⁹. Maximum total phenolic content found in ethyle acetate extract of aerial part of E.arborea. L was 875.5 (mg gallic acid equivalent/g extract)⁹. Environmental conditions, extracts type and extraction procedure can affected the composition and quantity of presented phenolic compound in extracts^23-29.Extracts of E. arborea stems may will be a good alternative for the treatment of many diseases since it contains a number of phytochemical compounds which possess medical properties such as phenolic compounds, hydrolyables tannins and flavonoids.

DPPH assay is tremendous technique to evaluate the free radical scavenging activity ¹⁹. Similar results have been reported by³⁰ using Hypsizygus ulmarius (Bull.) sub-fractions, in which the ethyl acetate fraction possesses the highest scavenging activity than methanolic extract and petroleum ether fraction³⁰. Indeed, Melakhessou et al. ⁽³¹⁾ indicated that the excellent capacity of ethyl acetate fraction to trap the DPPH radical could be due to the complex interaction of major biomolecules isolated in the ethyl acetate extract of Atractylis Flava Desf such as quercetin and apigenin or is the result of bioactive component impact just alone³¹. Thus, the reducing activity was found higher in methanol extract and is followed by ethyl acetate, aqueous and chloroform stems extracts. These findings are agreeable with those obtained in some previous studies ^29-32. Dileep et al. ³²and Marref et al. ²⁹ suggested thatthe reducing power of alcoholic extracts of Polyalthia longifolia Thw ripe pericarp. (Annonaceae) and Gladiolus segetum aerial parts (Iridaceae) may be correlated to high reductants in them, which cause the reduction of the Fe³⁺ to Fe²⁺. Hence, the methanolic extract of E.arborea. L stems perfomed the high amount of reductants, including polyphenols and flavonoids.

Overall, It was reported that phenolic compounds and flavonoids are classified as plant constituents with strong redox properties and scavenging radicals inhibition, which are contributed for antioxidant effect³³. Polyphenols, considred as tremendous contributors, which have diverse chemical structures with different polarities and make them solubles in a specific extraction solvents²⁰. As a result, many hydroxyl groups present, especially in their B ring and act as a good electron and hydrogen donors, which trap reactive oxygen species ^19-29-31-34.

Generally, the effectiveness of the extracts largely depended on the type of extraction solvent used^21-35. The recent report indicated antibacterial properties of Erica arborea methanolic extracts of leaves and flowers of E.arborea. L against all bacterial strains¹⁷. Ramakrishnan et al.³⁶ reported that the antibacterial potential of Glossocardia bosvallia whole plant against tested pathogens was also identified in the methanol extract, due to their active secondary metabolites abondance. Thus, The research concluded by³⁵ indicated that the methanolic extract of Sargassum wightii was effective against various infectious bacteria of urinary tract. Despite the presence of complex cell wall, that reduces the entry of herbal extract into the bacterial cells, Erica arborea. L stems methanolic extract moderately inhibited the growth of E. coli, S. gallinarum and P. aeruginosa, proving the capability of herbal antimicrobial agents of stems extract to penetr into bacterial cells of Gram negative bacteria tested ³⁷ The highest antibacterial activity of ethyl acetate fraction compared to crude extract and/or other fractions were also reported in previous studies ^38-39-40. As a result, the ethyle acetate fraction of Adathoda vasica showed prominent antibacterial activity against S. aureus compared to the other extracts³⁸.The promising antibacterial effect of ethyl acetate fraction could be attributed to the action of its chemical components^39-41. Flavonoids were proved to be the predominant components in ethyl acetate extract of A. chrysostachys roots and ethyl acetate fraction of Astragalus membranaceus aerial parts⁴¹. Ethyl acetate extracts proved to be riche source of fatty acids, which the authors speculated to be responsible for the observed antibacterial activity and probably account for the high activity of ethyl acetate extract observed in this work⁴³. The results obtained showed a good correlation between the reported uses of E. arborea. L plant in traditional medicine against infectious diseases caused by a broad spectrum of pathogens.

CONCLUSION:

The present study aimed to investigate the quantification of phytochemicals compounds by spectrophotometry, antioxidant and antimicrobial activities of dry stems extracts of the Algerian E. arborea. L. The outcome of the research establish that the stem extracts possess bioactive compounds against the various diseases with pharmacological value. The total phenolic content, flavonoids and condensed tannins contents in extracts can be renforced this statement. The methanolic extract and its sub-fractions possess a good antioxidant activity in which ethyl acetate fraction and methanolic extract showed the highest scavenging activity and ferric reducing power, respectively. The chance to find antimicrobial activity was more apparent in ethyl acetate and methanol than chloroform and aqueous extracts of the same plant. It is concluded that the methanolic extract of the plant possess moderate antimicrobial activity, against all the human pathogenic bacteria except against yeast and fungal strains tested. However, further investigation is more required for their antioxidant and antibacterial effects in order to the discovery of high value of new pharmaceuticals drugs.

ACKNOWLEDGEMENT:

This work is supported by DGRSDT, Algeria.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 27.07.2024 Revised on 15.10.2024

Accepted on 22.12.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):757-764.

DOI: 10.52711/0974-360X.2025.00112

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Secondary compounds	Methanol	Chloroform	Ethyl acetate	Aqueous
Polyphenols	+	+	+	+
Flavonoids	+	-	+	-
Hydrolyzable tannins	+	+	+	+
Saponins	-	-	-	-
Anthraquinones	+	-	+	+
Quinones	+	+	+	+
Anthocyananins	-	-	-	-
Terpenoids	+	+	+	+
Alkaloids	-	-	-	-