INTRODUCTION:

Medicinal plants are commonly adopted as traditional medicines across the world since they are an effective alternative source of pharmaceuticals¹.

In Algeria, phytotherapy has always been used in the traditional medicine sector². In recent years, many researches’ has been directed towards the valorization of traditional medicine owing to the verification of the safety and efficiency of the plants and to establish scientific rules for the use of these plants³. This is to avoid synthetic antioxidants such as BHA (butylatedhydroxyanisole) and BHT (butylatedhydroxytoluene) who’s very harmful either in their metabolism or by their accumulation in the tissues of the human organism⁴

Plants are an immense source of complex biomolecules (secondary metabolites)⁵.Among these metabolites, we distinguish terpenoids, alkaloids, flavonoids, and polyphenols⁶. The latter, mainly flavonoids, are essentially known for their numerous biological properties, particularly their anti-inflammatory, antioxidant, antimicrobial, and anticancer actions^7,8.

Antioxidants are substances that can slow or prevent lipid and other molecular oxidation and hence hinder the beginning and spread of oxidative chain reactions⁹. Furthermore, free radicals are intricate in the pathogenesis of numerous chronic disorders¹⁰. Scientific communities have returned to using bioactive compounds as a means of exploring novel biologically active compounds as an alternative to synthetic drugs^11,12, that are used to treat infections caused by microbial agents¹³, that generally induce adverse effects besides their high cost; to avoid the emergence of resistant pathogenic microorganisms^14,15; also to prevent the toxicity of synthetic products so as to find new natural antimicrobial agents¹⁶.

Hyoscyamus albus L. is a Mediterranean plant that possesses numerous health benefits as well as an abundance of secondary metabolites, including polyphenols and alkaloids^17,18. In terms of traditional uses, it is a natural parasympatholytic, widely used for general anesthesia¹⁹, as an anti-neuralgic CNS sedative, as Parkinson's disease treatment, for its antispasmodic, analgesic, anti-inflammatory, anthelmintic, antipyretic, ^18,20,21, and anti-tumor remediation especially anticancer²², in the treatment of cardiac and gastrointestinal diseases²³. The present research was undertaken to investigate the antioxidant, and antimicrobial activities of extracts from Hyoscyamus albus collected from M’sila, Algeria.

MATERIALS AND METHODS:

Plant collection and identification:

The aerial parts of Hyoscyamus albus L. were collected from M’sila, Algeria, during the flowering season in 2018. It was authenticated by Dr. Sarri Djamel: Faculty of Science of Nature and Life; University of M'sila (Algeria).

Preparation of extracts:

The four sub-fractions of H. albus L. aerial part were prepared by successive extraction using polar and non-polar solvents²⁴. 100g of dried plant material was extracted using 1 liter of 85% aqueous methanol and subjected to magnetic agitation over two days at room temperature, to acquire the crude extract (HACrE), the mixture was filtered and then evaporated under pressure to remove methanol. A portion of the residue was extracted liquid-liquid using increasing amounts of polarity solvents: hexane for defatting, chloroform, and ethyl acetate. The aqueous fraction was stained in oven while the solvents are afterwards disposed of in a rotary evaporator. At the end of the extraction process, four extracts were obtained; crude extract (CrE), chloroformic extract (ChE), ethyl acetate extract (EAE) and aqueous extract (AqE).

Extraction yield:

The extraction yield, representing the difference in weight between the extract and the processed plant material, it is usually assigned as a percentage and quantified with the formula:

Extraction yield (%) = (Weight of extract / Weight of dry plant materiel) ×100

Phenolic content determination (TPC):

Total phenolic content in H. albus extracts were assessed using the Folin-Ciocalteu reagent method²⁵. Specifically, 500μl of Folin-Ciocalteu reagent (1:10) was combined with 100μl of each plant extract or standard (gallic acid). After 4-minute incubation. Subsequently, 400μl of a solution containing 7.5% sodium carbonate was then introduced to the test tube, and the whole set was incubated at room temperature for 90min. At 760nm, the absorbance of each sample was determined. Concentrations of phenolic compounds were deduced from a calibration curve, results were shown in milligrams of Gallic acid equivalents per gram of extract (mg GAE/gE).

Determination of total flavonoids content (TFC):

The colorimetric assay using aluminum chloride was applied to establish the total flavonoid content of each extract²⁶. In summary, 1ml of each sample diluted was blended with an equal volume of 2% aluminum chloride solution (AlCl₃) prepared in the methanol. After incubation for 10minute in ordinary temperature, hereafter, the Absorbance was read at 415nm. The values were presented as milligram equivalents of Quercetin per gram of extract (mg QEq/g)

Assessment of antioxidant activity:

Total Antioxidant Capacity (TAC):

The antioxidant capacity (TAC) of samples was spectrophotometrically determined by the phosphomolybdenum assay (PM)²⁷. The PM test is a quantitative approach employed for evaluating the reduction rate influenced by oxidant, antioxidant, and molybdenum ligands. This method entails the initiation of thermal auto-oxidation over an extended duration at an elevated temperature²⁸.

A volume of 100μl of extract samples and standards at different dilutions was mingled with 1 mL of the reaction which contains: sulphuric acid (0.6mM), sodium phosphate (28mM), and ammonium molybdate (4mM). The test tubes were capped and incubated in a water bath for 90min at 95°C using an iron rack. After the samples are cooled to ambient temperature, the mixture was measured at 765nm. The standard used was ascorbic acid. The increase in absorption corresponds to an enhancement in molybdenum Mo (VI) reduction by an antioxidant present in the tested extracts. Results were quoted as EC₅₀: the effective concentration for which absorbance values were found at 0.5; the lower value of EC₅₀ means the higher potency in the reduction of Mo⁺⁶ to Mo⁺⁵.

DPPH radical scavenging:

The free radical scavenging ability of the extracts against the DPPH stable radical was assessed by monitoring the diminution of the DPPH absorbance at 517nm²⁹, this method consists of a total of 50µL of various extract dilutions were added to 1250µL of DPPH methanolic solution (0,004%). The absorbance was taken after a 30-minute incubation time at room temperature in the dark. The positive control was butylatedhydroxytoluene (BHT). The ability to scavenge the DPPH radical was computed using the equation:

Scavenging capacity % = (Ac–As)/Ac ×100. Where Ac: the DPPH solution absorbance without the tested sample, and As: absorbance in the presence of extracts. The IC₅₀ values were calculated to compare the antioxidant effects of samples (the concentration necessary to neutralize 50% of DPPH radicals).

Hydroxyl radical scavenging potency:

Assessment of OH radical scavengability was carried out through the hydroxyl radical neutralization method by generating OH radicals using the Fenton reaction (involving the reaction of H₂O₂ and Fe²⁺)³⁰. Briefly, a 500µL volume of FeSO₄ (1.5mM) was combined with 350μL of H₂O₂ (6 mM). Then, the mixture was supplemented with 100 µL of extracts at varying concentrations or BHT (standard). Next, 150 µL of sodium salicylate (20mM) was then introduced, and the combined solution was incubated for 1 hour at 37°C. Subsequently, the absorbance of the resultant mixture was measured at 562 nm. The trapping capacity of the extract was evaluated with the next equation:

I % = [(AC - AE) / AC] × 100

AC: control absorbance and AE: Absorption of the extract

β-Carotene bleaching assay:

In this evaluation, the antioxidant efficacy of the extract was detected by measurement of its inhibitory effect on conjugated diene hydroperoxides³¹, stemming from the oxidation of linoleic acid. A stock solution for preparing a mixture comprising β-carotene and linoleic acid was formed by melting 0.5mg of β-carotene in 1ml of chloroform, followed by the addition of 25μL of linoleic acid and 200mg of Tween 40 in a flask. Then the chloroform was completely evaporated at a temperature of 40°C using rotary steam. An emulsion was obtained through the addition of 100mL of oxygen-saturated distilled water with vigorous shaking. After this, 2.5mL of this emulsion was reacted with 350μL of the extracts dissolved in methanol or distilled water, together with BHT (positive standard) at a concentration of 2mg/mL. Negative controls consisted of 350μL of distilled water and methanol. The emulsion's bleaching kinetics in the presence and absence of antioxidants were monitored at 490nm after incubation periods (0 h, 1 h, 2 h, 4 h, 6 h, 12 h and 24 h), in the dark at room temperature

Antioxidant efficiency was assessed by calculating the percentage of β-carotene bleaching with the formula:

AA % = (AE / AE₀) × 100

Where: AE: Absorbance when reading in the presence of extract; AE₀: Absorbance of extracts in time 0 (0 h)

The percentages of inhibition used for comparison were those calculated after 24 hours.

Antimicrobial screening:

The evaluation of antimicrobial behavior was performed using the agar diffusion well method³²; it is largely used to evaluate the antimicrobial efficacy of plant extracts or antibiotics. Since this method is easy to implement and reproducible, it does not require expensive equipment and increases the amount of contact between extract and bacteria³³.

Strains studied:

The four plant extracts are individually tested against 8 groups of ATCC bacteria strains from the American Type Culture Collection (ATCC) standards: Gram negative: Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Acinetobacter baumannii (ATCC 17978), Klebsiella pneumoniae (ATCC 70603). Moreover, Gram positive; Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 1247); plus 2 Clinical bacteria: Serratia marcescens and Proteus mirabilis; as well with a yeast Candida albicans (ATCC 10231), at three different concentrations, all the strains were obtained from the Laboratory of Bacteriology at Sétif hospital.

Preparation of inoculum:

Bacterial strains were subcultured for 18hours on Mueller Hinton Agar (MHA) at 37°C, while the yeast was grown for 48hrs. The different bacterial species were first transplanted by the striae method onto the mid-point Mueller Hinton then placed in a 37°C-controlled incubator for 24hours. One or more colonies of each pure culture are collected and transferred into tubes containing physiological water (NaCl 0.9%) to have microbial suspensions with turbidity equivalent to 0.5 McFarland, determined spectrophotometrically by an optical density between 0.08 and 0.10 at 625-630nm. From these young culture suspensions, bacterial strains are swabbed onto MHA media in Petri dishes³⁴.

Preparation of extracts dilutions:

After spreading the strains on the MHA plates, 06 mm diameter wells are cut from the inoculated agar and filled with 50μl of solubilized extracts of H. albus (CrE/ChE/EAE/AqE) dissolved in sterile distilled water and 10 % DMSO with three concentrations (150, 250, and 500 mg/ml). Distilled water and DMSO were considered as the negative control, while ketoconazole and Gentamicin were served as the positive control. After 24–48 hours of incubation period at 37°C, the diameters of the inhibition zones produced by the different extracts are measured in millimeters³⁴. All tests were conducted in triplicate.

Determination of minimal inhibitory concentrations (MIC):

Dilution techniques are more suited to determine MIC values to estimate the concentration of the tested antimicrobial agent in the agar medium, This method is used to determine the minimal inhibitory concentration (MIC) for extracts that have a ≥ 10mm inhibition zone³⁵.

Statistical analysis:

The data in vitro is displayed as a mean standard deviation (SD), The statistical analysis was carried out using the GraphPad Prism software (version 5.0). The data were analyzed using one-way analysis of variance (ANOVA) and the Tukey test for multiple comparisons.

RESULTS AND DISCUSSION:

Extraction yield:

The results achieved (Table 1) show that the extraction percentages vary among the plant extracts according to the solvent applied

Table 1. Extract yields, total phenolic and flavonoid content of H.albus extracts

Extract	Yield (%)	Polyphenols (mg GAEq/gE)	Flavonoids (mg QEq/gE)
CrE	13,34	90,44 ±0,687	22,62 ±0,22
ChE	1,989	45,19 ± 4,22	25,82 ± 0,018
EAE	1,46	186,55 ± 0,47	25,87 ±1,12
AqE	7,02	73,93 ±1,383	10,30 ±0,12

Among the various fractions of HA extracts, CrE represents the highest yield (13.34%), followed by AqE (7.02%), ChE (1.989%) and the lowest yield is that of EAE (1.46%).

Extraction is the initial stage in separating the desired natural compounds from plant matter. The choice of extraction method is of significant importance in the process of isolating, identifying and using phenolic compounds³⁶. However, there is no standard extraction method, though. Solvent extraction is the most used approach among the others for obtaining phenolic compounds. Of these methods, organic solvent extraction is the most widely used because of its ease of use, efficiency and broad applicability³⁷.

Our results show that the more polar solvents (methanol and water) have better extraction yields than the less polar solvents, and that a large proportion of the crude extract constituents retained in the aqueous fraction. Low-molecular-weight organic compounds with functional groups³⁸. This suggests that components such as proteins, carbohydrates, water-soluble vitamins and salts were extracted with the aqueous phase in an aqueous organic solvent mixture, contributing to the overall extraction yield³⁹.It has been documented that using an aqueous-alcoholic mixture (methanol/water) at room temperature as the first maceration step extracts polar molecules as well as those with a medium or low degree of polarity⁴⁰. Methanol increases cell membrane permeability, making it easier to extract of a wide range of substances⁴¹.

Total polyphenols, and flavonoids contents:

The results indicate that all plant extracts are rich in polyphenols. EAE showed the highest polyphenol content, reaching 186.55 mg GAEq/gE, the ChE had the lowest polyphenol amount, measuring 45.19 mg GAEq/gE. In terms of flavonoids, EAE and ChE extracts showed the highest quantities, reaching 25 mg QEq/gE, on the other side, the AqE showed a relatively lesser flavonoid content.

The crude extract of HA prepared in this study was distinguished by a higher polyphenol content than that reported by⁴², where they obtained a value of 22.15 ± 0.026 mg GAEq/g. However, the flavonoid content (16.44± 0.17 QEq/g) of the species studied appears to be slightly lower compared to this finding. Additional results were previously documented in a study by⁴³ on the same species obtained in another country (Libya), who estimated the polyphenol content at 48.54 ± 7.82 mg GAEq/g, which was lower than the value we obtained in our study. As for flavonoids, their value (27.39 ± 0.87 mg QEq/g) exceeded that obtained from our samples. While another study on the same plant from Batna⁴⁴ (eastern Algeria) gave 111.1 ± 1.82 μg GAE/mgE and 24.31 ± 0.62 μg EQ/mgE respectively. This polyphenol content seems slightly higher than ours, while we obtained the same value for flavonoids. Our results suggest that H. albus is a promising source of phenolic compounds.

The differences in overall polyphenol content can be attributed to differences in the solubility of biomolecules extracted from plant material, and to the specificity of the solvents used⁴⁵. The solubility of polyphenols mainly depends on factors such as the presence of hydroxyl OH groups, their molecular dimensions and length of hydrocarbon chains⁴⁶. This difference in phenolic, and flavonoid content values could be due to external elements such as environmental and geographical variations, the location in which the plant was collected, climate such as sunlight, humidity and rainfall influence soil quality and therefore plant maturity⁴⁷, extraction method, extraction time, solvent used and temperature may also affect the content of secondary metabolites⁴⁸, while internal factors (genetic diversity) influence the content of secondary metabolites.

Antioxidant Activities of H. albus Extracts:

Total Antioxidant Capacity (TAC) by Phosphomolybdenum Method:

Results indicated that, the four tested extracts (Table 2) exhibited antioxidant potential in a concentration-dependent manner with varying efficiencies, which could be ascribed to the prevalence of polyphenols. The EAE is deemed to be excellent reducer with a lower EC₅₀close to that of synthetic standard (p < 0.001). Lower antioxidant potential was registered for AqE, while the CrE and ChE exhibited a moderate antioxidant activity.

It is also widely accepted that the presence of reductants acting as electron donors might explain the reducing capacity of the samples investigated in this study. Polyphenols are known to be effective electron donors in this regard⁴⁹. Singh et al. (2018)⁵⁰ had reported that total antioxidant capacity of the extracts relies to the intrinsic amount of polyphenols and flavonoids in the plants³⁸. The significant trapping action of EAE extract bodes well for the utilization of H. albus as a natural antioxidant in oxidative stress.

DPPH radical scavenging ability:

The DPPH test is commonly employed for the evaluation of antioxidant activity because it is a simple, fast and inexpensive method⁵¹.

The obtained results (Table 2) revealed that the extracts have remarkable scavenging activity against DPPH. EAE extract displayed the highest anti-free radical potency to disrupt DPPH radicals with a lower IC₅₀ value of 0.021±0.0002 mg/ml, followed by AqE and CrE. These fractions proved significantly higher activity (P<0.001) than the antioxidant standard, BHT (0.0±0.002 mg/ml). While ChE displayed moderate activity (0.126±0.002 mg/ml). These results were superior to those obtained by⁴², an almost similar result was quoted in a study of⁵² on the aerial part of the plant from Saudi. Furthermore, study of⁴³ found that H. albus possesses low antioxidant activity despite its richness in polyphenols. An earlier study found that the scavenging potency of plants can be responsible to their phenolic content⁵³. Flavonoids are highly involved because of their remarkable antioxidant properties as hydrogen-donating substituents⁵⁴.

Hydroxyl radical scavenging

From the results listed in Table 2, the OH^*free radical scavenging illustrate that all extracts are able to scavenge this reactive species in a dose-dependent manner. The CrE was effective in quenching hydroxyl radical formation and expressed by an IC₅₀ value of 0.255±0.003 mg/mL, followed by ChE (0.331±0.009 mg/mL) and EAE (0.484±0.004 mg/mL) (p ≤0.001), whereas the AqE extract was characterized by the lowest Scavenging capacity (0.57±0.006 mg/mL). It's worth mentioning that BHT proved to be the strongest inhibitor than the extracts tested (0.101±0.013 mg/mL) (p ≤0.001). There was no correlation between the polyphenol content of the extracts and the ability to neutralise hydroxyl radicals. This may be due to the nature of the components contained in each extract.

Hydroxyl radicals are the highly active and reactive of oxygen's free radicals, with a short lifespan which react where they are produced and cause serious damage to neighbouring molecules. Specifically, direct interactions with DNA, result in its breakdown degradation⁵⁵, thereby playing an important role in cancer formation⁵⁶. So displacement of hydroxyl radicals is essential to protect cells from damage.

Previous studies have shown the significance of polyphenolic compounds as antioxidants because of their hydroxyl groups, which provide plant products the ability to scavenge free radicals. Because of their antioxidant properties, flavonoids have a strong capacity to neutralize reactive oxygen species and harmful free radicals. The primary active groups in flavonoids that are able to scavenge OH* are their phenolic hydroxyls. This OH-scavenging capacity of flavonoids is in line with previous studies on their inhibitory action, probably due to their ability to provide active hydrogen via hydroxyl substitution^57,58

β-Carotene/linoleic acid bleaching assay:

All extracts exhibited significant protective capabilities for β-Carotene in comparison to the standard antioxidant BHT (98.16 ± 0.74%). Notably, the greatest level of antioxidant potential was achieved with ChE, this was followed by the two fractions EAE and CrE, which demonstrated a similar linoleic acid inhibitory effect, while AqE seems to have less inhibitory activity. These values are still lower than those of BHT used as a positive control. This impact is attributable to inhibition of linoleic acid oxidation and/or displacement of the peroxide radicals resulting from this oxidation⁵⁹. The transfer of a hydrogen atom from phenolic compounds to free radicals is most likely the reason of the antioxidant action of plant extracts, which prevents β-Carotene from whitening. The chain-like propagation of lipid oxidation in biological systems can be stopped by these fractions and act as antioxidants at early and later stages of lipid peroxidation⁶⁰, the flavonoids present in the plant studied also effectively inhibit oxidation. Their appropriate structure and lipophilicity may also contribute to their ability to penetrate deep into the patterned lipid bilayer, thus enhancing their efficacy in inhibiting lipid oxidation⁶¹, or the inhibitory activity may return to the presence of other compounds than phenols, like sulfur compounds⁶². The study of⁴⁴ indicates that the methanolic extract exhibits significant antioxidant potential, boasting an activity level of 76.0%, a value almost similar to the recorded in our study. Similarly, the authors note that the chloroformic fraction of the leaves demonstrates comparatively lower activity, with an antioxidant activity of 43.24±0.92% versus the 67.27±0.854% observed in our chloroformic fraction.

Antimicrobial screening:

After a detailed review of the available literature, there are a few studies on the antimicrobial activity of fractional polyphenolic samples from Algerian Hyoscyamus albus. Wherefore, the current study was conducted to investigate the antimicrobial abilities of plant fractions using the agar diffusion well in Mueller Hinton agar. The results are represented in table 3. The findings have indicated that this plant fractions exhibit a dose-dependent significant inhibitory effects toward most tested bacteria strains, both on gram positive and negative pathogenic bacteria tested with different diameters of dependent concentration.

Table 3. Antimicrobial activity of H. albus’s extracts against bacteria and yeast. Gentamicin and ketoconazole served as positive control, H₂O and DMSO served as negative control

Bacteria	Main inhibition zone (mm)
Bacteria	CrE	ChE	EAE	AqE	Gent	keto
*Gram negative*
E. coli	11 ±0,57	10±0,57	11,5±0,57	-	28	-
P. aeruginosa	12± 1,15	11±0,57	17,5±0,00	8,5±0,57	27	-
S. typhimurium	-	10,5±0,57	-	-	16	-
P. mirabilis	10,5±0,57	19±0,57	-	-	23
K. pneumonia	12±0,57	11.5±1,15	14±0,00	-	25	-
A. baumannii	11,5±0,57	12.5±0,57	12±0,00	-	26
S. marcescens	-	-	10±0,00	-	nt	-
*GramPositive*
S. aureus	27± 1,15	32,5±0,57	24±0,57	10±1	34	-
B. cereus	23,5±1,15	15±1,15	18±1,15	18±1,15	26	-
B. subtilis	13± 1,15	23±0,57	15±0,57	13,5± 2,12	34	-
*Fungi*
C. albicans	-	-	11±0,57	-	-	30
H₂O	-	-	-	-	-	-
DMSO	-	-	-	-	-	-

keto: ketoconazole, Gent: gentamicin; Values represent the average of 03 measurements (n=3) ± SD; nt: not tested

The screening of antimicrobial potential (Table 3) revealed that the diameters were confined between 8,5 mm as a minimal inhibition zone to 32,5 mm for the studied strains, The highest average was reported for the pathogenic species S. aureus. The CrE induced inhibition zones on all strains except for two bacteria, S. marcescens and S. typhimurium, and the yeast C. albicans. It exerted the highest inhibition zone versus the Gram-positive bacteria S. aureus, and lowest activity against P. mirabilis. Indeed, The AqE, was completely inactive against all Gram-negative tested strains except on P. aeruginosa with a low activity of 8,5mm, this strain was previously resistant to crude extract whereas a good inhibitory effect of AqE toward the bacterial growth was obtained on the Gram positive strains: B. subtilis, B. cereus, and S. aureus.

The ChE showed a great antibacterial effect versus all the three Gram-positive strains and a moderate effect toward gram-negative, while S. marcescens was highly resistant. The yeast behaved in the same way as S. marcescens against this fraction. S. typhimurium was resistant to the plant fractions except to ChE with a minimum activity (10,5mm). Furthermore, only the EAE was active versus S. marcescens, which previously recorded resistance to the plant extracts. The EAE appears to have the broadest spectrum of activity by inhibiting the proliferation of eight bacterial strains (P. aeruginosa, E. coli, K. pneumonia, A. baumannii, S. marcescens, B. subtilis, B. cereus, S. aureus) and Candida yeast, but with a low diameter (11 mm).

All recorded antibacterial activities are less marked than the positive control used (gentamicin), except for the ChE on S. aureus, which was likely similar to the gentamicin used as a reference drug. A similar finding was found by⁴⁴ which showed a good effects against all the bacterial strains (P. aeruginosa (ATCC 27853), E. coli (ATCC 25922), S. aureus (ATCC 25923)) and clinical strains (P. mirabilis, S. aureus, P. aeruginosa and E. coli) except on the Candida albicans. As regards the higher activity of the ChE, since it is rich in flavonoid aglycones, it had a spectrum of activity as broad as they exert an inhibitory action on the growth of S. typhimurium, A. baumannii, S. aureus, and B. subtilis.

Interestingly, it’s noticed that the extracts have remarkable antibacterial action, which extends almost over all the strains of the collection. It was very evident that the effectiveness observed in the AqE against B. subtilis, despite its lack of polyphenols and flavonoids, was certainly due to the presence of one or more plant-based component groups other than flavonoids and polyphenols, such as tannins, glycosides, saponins, etc., that are involved in antibacterial function either alone or in combinations. The efficacy observed for EAE could be due to certain flavonoids belonging to the class of flavonols causing changes in the cytoplasmic membrane, leading to their cleavage, or isoflavones inhibit topoisomerase, thus blocking the synthesis of DNA^15,63,64. The cytotoxic effect of the extract has been documented, which could play a role in the effect against bacteria⁶⁵. These findings are best illustrated by⁶⁶. They explained the antimicrobial activity of aerial parts of H. albus by the presence of all classes of secondary metabolites, encompassing saponosides, quinones, anthraquinones, flavonoids, alkaloids, coumarins, and tannins. Also, this could be partly attributed to a higher alkaloid content, as reported by the same authors⁶⁶ who emphasized the richness of this plant in alkaloids; these results were similar to those reported by ^67,68.

It should be emphasized that these extracts possess a greater effect on positive bacteria than on negative ones; the reason for this higher sensitivity could be related to the differences in their cell wall compositions. Indeed, the presence of the lipopolysaccharide layer in the wall of Gram-negative bacteria makes the cell impervious and prevents the entry of foreign molecules⁶⁹. However, the cell wall of Gram positive bacteria has only peptidoglycan⁷⁰. Which is an ineffective permeability barrier; it facilitates the passage of molecules inside the cell and explains the sensitivity of these bacteria. For this purpose, the resistance of S. typhimurium and S. marcescens bacteria is not surprising; it is related to the nature of their external membrane.

It is clear that the antibacterial activity of the reference antibiotics was remarkably higher than that of our extracts; because the uses of pure components like antibiotics often offers a stronger antimicrobial action than a complex component combination, such as plant extract^71,72. Finally, these are extracts that contain a large amount of diverse compounds. Therefore, they are likely to contain compounds that, when purified, have activity comparable to that of a chemical agent.

The minimum inhibitory concentration (MIC):

The fractions that exhibited a good activity were retested on the bacteria itself to calculate the minimum inhibitory concentrations (MIC).

Table 4 summarizes the quantitative antibacterial activity of plant extracts as MICs (mg/mL). The four extracts have outstanding antibacterial properties. The CrE, ChE, and EAE have a remarkable antibacterial effect, with the lowest MIC values against Gram positive bacteria. The minimum value was found by ChE against Bacillus subtilis with 0.03±0.00 mg/mL. This reflects that the extract is the most active given their MICs were below 500 μg/ml. Taking into account that extracts with a MIC of fewer than 500 μg/ml are considered active⁷³. This effect was slightly higher than that of ⁴³, but close to that of⁴⁴.

CONCLUSION:

From the previous, In addition to their antioxidant capacity, the extracts showed moderate to robust antibacterial efficacy against Gram-positive and Gram-negative bacteria that were employed in the experiment. The crude extract also displayed anti-oxidant properties; these activities might be attributed to the specific actions of particular phytochemical compounds such as polyphenols. These outcomes bolster the traditional applications of this plant. Nevertheless, further studies will be needed to isolate and characterize the compounds involved for these activities and the identification of their mechanisms of action. The use of Hyoscyamus albus L. as an antibiotic alternative was recommended due to the increasing antibiotic resistance to microorganisms linked with infectious disorders, besides the unpleasant side effects of antibiotics. However, further investigations are needed to establish the practical value of the therapeutic application.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Dr. Sari Djamel, Department of Science of Nature and Life, M’sila University, Algeria, for plant identification.

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Received on 09.05.2024 Modified on 05.08.2024

Research J. Pharm. and Tech 2024; 17(11):5515-5524.

DOI: 10.52711/0974-360X.2024.00843

Extract/ standard	IC₅₀ (mg/mL)	EC₅₀ (mg/mL)	IC₅₀ (mg/mL)	Inhibition (%)
Extract/ standard	DPPH radical scavenging	Total Antioxidant Capacity	Hydroxyl radical scavenging	β-Carotene bleaching assay
CrE	0,05±0,0007^***	0,126±0,01^***	0.255±0.003^**	66,38±0,01^*
ChE	0,126±0,002^***	0,133±0,006^***	0.331±0.009^***	67,27±0,32^*
EAE	0,021±0,0002^***	0,050±0,001^ns	0.484±0.004^***	66,92±0,84^*
AqE	0,063±0,0003^*	0,19±0,013^***	0.57±0.006^***	63,79±0,38^**
BHT	0,087±0,001	0,031±0,003	0.101±0.013	81,10± 0,11