EJBR2021v11i3art356 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2021; 11(3): 356-366 DOI: http://dx.doi.org/10.5281/zenodo.5129324 Phytochemical characterization, antioxidant and antibacterial activity of Salvia officinalis (L.) extracts from the Tiaret region Rachida Bouteldja 1,2,*, Radhouane Doucene 3, Hebib Aggad 1, Fatima Zohra Abdi 4, Hamza Belkhodja 4, Mustapha Abdali 1, Khaled Zidane 3, Siham Abaid 5 1 Laboratory of Hygiene and Animal Pathology, Institute of Veterinary Sciences, University of Tiaret, Algeria 2 Faculty of Natural and Life Sciences, Ibn Khaldoun University of Tiaret, Algeria 3 Laboratory Reproduction of Farm Animals, Institute of Veterinary Sciences, University of Tiaret, Algeria 4 Laboratory of Bioconversion, Microbiology Engineering and Health Safety, University of Mustapha Stambouli, Mascara, Algeria 5 Laboratory of Eco-development of Spaces, Department of Environmental Sciences, Faculty of Natural and Life Sciences, University of Djilali Liabes, Sidi Bel Abbes, Algeria * Corresponding author: E-mail: bouteldjarachida.92@gmail.com Received: 29 April 2021; Revised submission: 25 June 2021; Accepted: 23 July 2021 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2021. Licensee Joanna Bródka, Poland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: This work aims the valorization of a medicinal plant known by its traditional use, Salvia officinalis L. (Lamiaceae), by phytochemical characterization and evaluation of the antioxidant and antibacterial activity of their extracts. The antioxidant activity was assessed by the DPPH method and the antibacterial potential was determined by the diffusion method. The quantitative determination revealed that the ethanolic extract has a content of 8.04% for polyphenolic content and 17.4 % for flavonoids. The DPPH radical scavenging activity of S. officinalis showed that the ethanolic extract of S. officinalis presented the higher antiradical effect manifested with IC50 of 0.106±0.001 mg/ml. In addition, the antibacterial activity showed the strong capacity of S. officinalis methanolic extract to inhibit B. subtilis, M. luteus, E. coli and S. aureus with a diameter inhibition zone of 27.06±1.49; 15.43±2.23; 11.6±0.52 and 11.5±2.17 mm respectively. While the activity of the ethanolic extract was 26.62±2.97 mm against B. subtilis, 16.51±2.36 mm against M. luteus, 13.62±0.55 mm for S. aureus, P. aeruginosa (12.30±1.59 mm). The macrodilution method (MIC) showed a range of 625 to >5000 µ g/ml. The study of the antioxidant and antibacterial activity of extracts of S. officinalis suggested that this plant represented a natural source of bioactive molecules with very important biological activities. Keywords: Salvia officinalis L.; Polyphenols; Flavonoids; Antioxidant; Antibacterial. Abbreviations: MIC: Minimal Inhibitory Concentration, DPPH: 2,2-diphenyl-1-picrylhydrazyl (C18H12N5O6), IC50: Median inhibitory concentration, S. officinalis: Salvia officinalis, AK: Antibiotic Amikacin. Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 357 European Journal of Biological Research 2021; 11(3): 356-366 1. INTRODUCTION Medicinal plants make a big part of modern medicine by their richness in bioactive compounds of different parts of the plant. These molecules of natural origin participate in various fields such as cosmetic, food technology and pharmaceutical [1, 2]. Bioactive molecules are extra-nutritional constituents participating in physiological or cellular activities in humans and animals by providing biological activities; antioxidant, anti-inflammatory, anti-carcinogenic protect against metabolic disorders [3, 4]. The treatment of complex diseases requires the development of synthetic drugs. These products can cure various pathologies and cause adverse effects on human health with a more or less severe intensity [5]. To determine this growing threat, research is focusing on medicinal plants as the main resource for the therapeutic bioactive compound in addition to their use in health care by approximately 80% of the world's population [6]. In fact, they are sources of biologically active compounds; phenolic compounds with antioxidant properties and antimicrobial potentials [7]. Oxidative stress is an imbalance between the production of reactive oxygen species or free radicals and their removal by antioxidants. This phenomenon results from multiple external factors such as exposure to X-rays, ozone, smoking, air pollutants, nutritional deficiencies and industrial chemicals. It stimulates molecular attacks on biological membranes and tissues and causes or leads to several neurodegenerative diseases such as Parkinson's and Alzheimer's [8, 9]. Nowadays, with the appearance of side effects of synthetic drugs as well as the increase of resistance to conventional antibiotics, research is focusing on medicinal plants as the main resource for therapeutic agents to overcome this growing threat. Salvia officinalis L. (Sage) is a very popular plant in the traditional pharmacopeia of the Tiaret region (Algeria) for the treatment of various pathologies. Sage is certainly the queen of aromatic herbs and one of the oldest cultivated plants. It is an evergreen plant of the Lamiaceae family and belongs to the genus Salvia grouping more than 800 species. This group includes herbaceous plants (annuals and perennials) and semi- shrubs [10, 11]. The different parts of Salvia officinalis due to their richness in bioactive compounds such as flavonoids, alkaloids, tannins, glycosides can be essential elements for the inhibition of pathogenic bacteria and the reduction of different pathologies [11]. This research aims to evaluate the biological activity of the ethanolic (80%), methanolic (80%) and aqueous extracts of Salvia officinalis L. by the determination of polyphenolic and flavonoids contents followed by the study of the in vitro antioxidant and antibacterial activity against ATCC strains. 2. MATERIALS AND METHODS 2.1. Plant material and extraction The aerial parts (leaves and stems) of Salvia officinalis were collected in April 2017 in the region of Tiaret (Algeria) (35° 23′ 17″ North, 1° 19′ 22″ East). The plant was washed and dried in the open air and then grinded to obtain a powder. The identification of the species was carried out by the botanical laboratory of the National Superior School of Agronomy (Algiers). The extracts were prepared by maceration with a ratio of 5 g of powder mixed with 50 ml of the solvent (methanol 80%, ethanol 80% form BIOCHEM Chemopharma and distilled water) under continuous stirring with a shaker. After 24 hours, the mixture was filtered through filter paper and the filtrate was evaporated and dried at 40°C. The resulting residue was stored at a temperature of -20°C [12]. Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 358 European Journal of Biological Research 2021; 11(3): 356-366 2.2. Quantitative analysis 2.2.1. Total Phenolic Content The determination of total phenolic content was carried out by the Folin-Ciocalteu method [13]. A volume of 0.2 ml of different dilutions (1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 mg/ml) of ethanolic, methanolic, and aqueous extracts of S. officinalis was mixed with 1 ml of the freshly prepared Folin-Ciocalteu reagent (BIOCHEM Chemopharma) at 10% and 0.8 ml of sodium carbonate (Na2 CO3) at 7.5%. The mixture is incubated for 30 minutes at room temperature and then read by a spectrophotometer (Biochrom Libra S6) against a blank at a wavelength of 765 nm. Results were expressed in mg equivalent of gallic acid/g dry plant [14]. The tests were repeated in triplicate (Means ± SEM with n=3). 2.2.2. Total Flavonoid Content The determination of flavonoids is done by the aluminum chloride method [15]. A volume of 0.5 ml of each dilution was mixed with the various extracts and added to 1.5 ml of methanol (95%), 100 µ l of AlCl3 prepared at 10% (w/v) plus 100 µ l of sodium acetate (1 M) and 2.8 ml of distilled water. The mixture was stirred and incubated in the dark at room temperature for 30 min. The blank was made by replacing the extract with 95% methanol and the absorbance was determined by spectrophotometer (Biochrom Libra S6) at 415 nm. The results were expressed in mg quercetin equivalent/g dry plant [16]. The tests were repeated in triplicate (Means ± SEM with n=3). 2.3. Antioxidant activity The antioxidant activity was performed by the DPPH (2,2-diphenyl-1-picrylhydrazyl, C18H12N5O6) radical scavenging assay. The protocol followed was the one described by Braca et al. with some modifications. An equal volume of the different dilutions (0.009 to 5 mg/ml) of the methanolic, ethanolic and aqueous extracts of S. officinalis was mixed with a volume of 0.004 % (w/v) methanolic solution of DPPH (Sigma Aldrich). Incubation was carried out for 30 min at room temperature. The absorbance was read against a control prepared for each concentration at 517 nm after 30 minutes of incubation in the dark and at room temperature with a spectrophotometer (Biochrom Libra S6) [17]. The tests were repeated in triplicate (Means ± SEM with n=3). The percentage inhibition was calculated by the following equation: % Inhibition = [(Ac -Ae)/Ac] × 100. With: Ac: Absorbance of the control solution which contains an equal volume of methanolic solution of DPPH and methanol. Ae: Absorbance of extract. 2.4. Antibacterial activity The antibacterial potential of S. officinalis extracts was evaluated against seven referenced bacterial strains: Micrococcus luteus ATCC 14452, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027 and Enterococcus faecalis ATCC 29212, from the laboratory of Animal Hygiene and Pathology of the Veterinary Institute of Ibn Khaldoun University "Tiaret". 2.4.1. Agar diffusion method The agar diffusion method was used to evaluate the antibacterial activity of the extracts of S. officinalis [18, 19]. Petri dishes are filled with Mueller-Hinton medium (BIOCHEM Chemopharma) in a liquid state and allowed to solidify, then the bacterial suspension is adjusted to 0.5 McFarland and seeded Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 359 European Journal of Biological Research 2021; 11(3): 356-366 with a sterile swab. Using sterile forceps, impregnate a Whatman paper disc (6 mm diameter) in 10 µ l of each extract (ethanolic, methanolic and aqueous at a concentration of 20 mg/ml of the plant), and another for the solvent used as a negative control and another one an antibiotic (Amikacin 30 µg) represents the positive control. The disk is placed with slight pressure on the seeded agar. After diffusion of the extract for 2 hours at 4°C, the plates are maintained at 37°C for 24 hours. Antimicrobial activity is determined using a caliper by measuring the diameter of the zone of inhibition (mm) [18]. The tests were repeated in triplicate (Means ± SEM with n=3). 2.4.2. Macrodilution method The minimum inhibitory concentration was performed by the macrodilution method. For this purpose, a series of 9 dilutions were prepared in Mueller-Hinton broth (BIOCHEM Chemopharma) (78 µ g/ml to 10000 µ g/ml) of the three extracts (methanolic, ethanolic and aqueous) of S. officinalis. The bacterial suspensions used were standardized to 0.5 McFarland (108 CFU) and then diluted 1:100 (106 CFU). Finally, 1 ml of the suspension was added to a tube containing 1 ml of the different dilutions prepared previously. A tube containing an antibiotic (Amikacin 30 µ g) considered as a positive control and a tube with 1 ml of Mueller- Hinton broth considered as a negative control. The tubes are incubated at 37°C for 24 hours, then 100 µ l of the tubes without bacterial growth are spread by a rake in plates containing solidified Mueller-Hinton agar and incubated at 37°C for 24 hours. After incubation, the MIC is the concentration of the plates with no growth [19, 20]. The tests were repeated in triplicate (Means ± SEM with n=3). 2.5. Statistical analysis The statistical analysis of the data was performed using the Statistica software (version 8.0.725.0) via Anova with a single factor followed by the LSD test. 3. RESULTS AND DISCUSSION 3.1. Quantitative analysis 3.1.1. Total Phenolic Content The results of polyphenol content are presented in the Table 1. The polyphenol content of S. officinalis extracts revealed a highly significant (p ≤ 0.001) methanolic against aqueous extract with a ratio of 5.72 vs. 4.51%, respectively. In addition, we have noted a highly significant increase (p ≤ 0.001) in ethanolic extract of 8.04% with the methanolic extract. Our results show the richness of the ethanolic extract of S. officinalis in polyphenols compared to the methanolic and aqueous extract. Pop et al. showed that the ethanolic extract of S. officinalis has a high level of polyphenols compared to methanolic extract [33]. Uygun et al. presented that the ethanolic extract of S. amplexicaulis was richer than methanolic and the aqueous extract [34]. Other comparative studies between the aqueous and the ethanolic extract at 50% and 96% showed that the 50% was the richer on phenolic content followed by the concentration 96% and finally the aqueous extract [21]. According to the results of the determination of polyphenols from S. officinalis a specific rate for each extract is presented. We can explain that the diversification of polyphenol levels can be translated into several parameters, including climate change, which has been a very important factor in their richness of phenolic compounds and similarly in their biological activity [35]. Or to the experimental conditions; temperature, time and method of extraction, the nature of the solvent and their percentage [36, 37]. Naczk and Shahidi, explained that phenolic compounds can guide the formation of insoluble complexes by their association with Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 360 European Journal of Biological Research 2021; 11(3): 356-366 plant components, as in the case of proteins and carbohydrates. Their solubility is based on their chemical nature in the plant, from simple to very highly polymerized, which can affect by the polarity of the solvents used [38]. 3.1.2. Total Flavonoids Content Flavonoid content was always related to polyphenols and richness in bioactive compounds. The flavonoid content in S. officinalis presented in Table 1. It showed a highly significant rate (p ≤ 0.001) of ethanolic extract versus methanolic and aqueous extract (17.4±0.2 vs 14.33±0.98 and 14.46±0.3%). Table 1. Polyphenols and flavonoids content of methanolic, ethanolic and aqueous extracts of S. officinalis. Plant extracts Polyphenols content % Flavonoids content % Me Ex 5.72*** 14.33±0.98 Et Ex 8.04### 17.4±0.2●●● Aq Ex 4.51 14.46±0.3 *** Highly significant difference of methanolic vs. aqueous extract. ###Highly significant difference of ethanolic vs. methanolic extract. ●●● Highly significant difference in ethanolic vs methanolic and aqueous extract. This study reveals the high content of flavonoids in the ethanolic extract of S. officinalis versus the methanolic and aqueous extracts. These results were corroborated with those obtained by Alimpić Ana et al. where the ethanolic extract of S. amplexicaulis has a high flavonoid content than methanolic extract [22]. According to Duletić-Laušević et al. The ethanolic extract at 50% of S. officinalis of different varieties presented a high level of flavonoids compared to extract at 96% and aqueous [21]. The evolution of flavonoid levels was influenced by several genetic and environmental parameters, including climate, area, temperature, fertility, parasites and diseases; similarly, extraction efficiency depended on the diffusion rate and solubility of the solvent [23, 39]. This was demonstrated by Do et al., where the ethanolic extract presented a ratio 4 times higher than the aqueous extract according to polarity [40]. 3.2. Antioxidant activity The results obtained DPPH radical scavenging assay as a function of the evolution of antioxidant activity are presented in Table 2, showed that the ethanolic extract of S. officinalis presented a highly significant antioxidant capacity p ≤ 0,001 versus the methanolic and aqueous extracts with an IC50 of 0.106±0.001; 2.82±0.05 and 1.74±0.005 mg/ml respectively compared to IC50 of ascorbic acid (0.13 mg/ml). Table 2. IC50 of the methanolic, ethanolic, aqueous extracts of S. officinalis and ascorbic acid. Methanolic extract Ethanolic extract Aqueous extract Ascorbic acid IC50 (mg/ml) 2.82±0.05 0.106±0.001*** 1.74±0.005 0.13 *** Highly significant difference in ethanolic vs methanolic and aqueous extract. Antioxidants are chemical or biological agents capable of neutralizing the potentially damaging action of free radicals. To respond to free radical attacks, some types of antioxidant defenses have been developed. Their function is to maintain the balance of the cells [24]. Rasmy et al. revealed that the ethanolic extract (80%) of S. officinalis has better free radical scavenging potential than the aqueous extract [41]. Duletić- Laušević et al. proved the strong capacity of radical inhibition by the ethanolic extract of S. officinalis Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 361 European Journal of Biological Research 2021; 11(3): 356-366 followed by the dichloromethane, acetate and chloroform [23]. Mekhaldi et al. showed the high capacity of methanolic extract of S. officinalis compared to the essential oil [42]. According to the values obtained, a difference in inhibition capacity depending on the solvent used was observed. The ethanolic extract provided a high free radical scavenging capacity. The latter can be translated by the best extraction power of antioxidants and their richness in bioactive compounds. The latter represent a diverse range of molecules that are not necessary for cell life but play a major role in the interaction between cells and the environment [25]. Zhou and Yu, reported significant effects between the solvents applied in the extraction of antioxidants and the power of inhibition of free radicals of DPPH [43]. Tosun et al. showed a positive correlation between phenolic compounds and antioxidant activity. In addition, the extraction quality was linked to several parameters, including the polarity of the solvents applied in the extraction, the separation technique where organic solvents were more efficient than water because of their capacity to extract polyphenols [44]. The latter were known for their major participation in antioxidant activity [45, 46]. Several works proved that the ethanolic extract 80% was more effective compared to methanolic (80%) and aqueous because of its polarity. Their polarity provided a strong extraction capability of phenolic compounds and flavonoids [47-49]. 3.3. Antibacterial activity 3.3.1. Agar diffusion method Antibacterial activity against Gram-negative and Gram-positive bacteria of ethanolic, methanolic and aqueous extract of S. officinalis using the disc diffusion method by determination of zone of inhibition (Table 3). Table 3. Results of antibacterial activity of S. officinalis extracts using the agar diffusion method by determination of inhibition zones (mm). Inhibition Zone (mm) Microorganisms Me Ex Et Ex Aq Ex AK S. aureus 11.5 ± 2.17 13.63 ± 0.55 0 19.83 ± 0.28 E. coli 11.6 ± 0.52 13.8 ± 1.53 0 23 ± 1 M. luteus 15.43 ± 2.23 16.51 ± 2.36 9.6 ± 5.54 20 ± 1 B. subtilis 27.06 ± 1.49 26.62 ± 2.97 2.55 ± 4.40 21 B. cereus 7.34 ± 0.64 10.66 ± 0.28 9.02 ± 0.95 18.66 ± 0.57 E. faecalis 9.14 ±1.82 10.83 ± 0.28 6.45 ± 5.60 9.21 ± 0.4 P. aeruginosa 10.34 ± 1.22 12.30 ±1.59 9.11 ± 0.83 8.78 ± 0.91 Me Ex: Methanolic extract, Et Ex: Ethanolic extract, Aq Ex: Aqueous extract, AK : Antibiotic amikacin (30 µ g). The results of antibacterial activity by agar diffusion method show the antibacterial effect of methanolic and ethanolic extracts of I against the tested strains with a zone of inhibition between 7.34-26.62 mm. On the other hand, the aqueous extract shows no effect against S. aureus and E. coli with low activity against most strains. Ghezelbash et al. showed that the ethanolic extract of S. officinalis stems were capable of inhibiting E. coli, S. aureus, B. cereus [12]. Abd-Elmageed and Hussein, demonstrated that ethanolic and aqueous extracts of S. officinalis have the potential to inhibit S. aureus, P. aeruginosa, E. coli, B. cereus, M. luteus and B. subtilis [50]. The ethanolic extract of S. officinalis can eliminate multi-resistant bacteria such as S. aureus and E. coli [51]. The activity of ethanolic and methanolic extracts inhibited the growth of P. aeruginosa, B. subtilis, E. coli and S. aureus [52]. Similarly, essential oils of S. officinalis reacted against B. Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 362 European Journal of Biological Research 2021; 11(3): 356-366 subtilis, B. cereus, S. aureus, M. luteus and E. coli [53]. In addition, other studies have shown that plant extracts belonging to the Lamiaceae family, such as sage, have biological activity against bacteria and yeasts. This inhibition can be explained by the richness of S. officinalis extracts in bioactive compounds such as phenolic compounds, alkaloids and terpenoids [11]. Several works have revealed that alkaloids, flavonoids, saponins, tannins, cardiac glycosides and terpenoids play a major role in the antibacterial activity through their ability to inhibit the growth of bacteria such as E. coli, P. aeruginosa and S. aureus [26-28, 54, 55]. Phenolic compounds cause disruptions in the cell wall leading to changes in permeability, leading to leakage of cell continuum or interfering with membrane proteins by a change in structure because of the strong correlation between toxicity and hydrophobicity of different phenolic compounds. In addition, bioactive compounds cause inhibitions of cell wall construction, microbial genetic material replication, biofilm formation, attachment motility and cell communication [29- 31]. 3.3.2. Minimal Inhibitory Concentration (MIC) Our results of the minimal inhibitory concentration reveal a diversity of the bacterial inhibition capacity according to the extract used and the tested germ. These results show a low antimicrobial efficacy of methanolic extract of S. officinalis for 71.42% of tested bacteria (S. aureus, E. coli, M. luteus, B. cereus and P. aeruginosa) with a MIC equal to 5000 µ g/ml, inactive (MIC >5000 µ g/ml) for B. subtilis and high sensitivity of MIC = 625 µ g/ml against E. faecalis. On the other hand, the ethanolic extract is inactive against M. luteus, B. subtilis, B. cereus (MIC >5000 µg/ml), S. aureus, P. aeruginosa, E. coli (MIC = 2500-5000 µ g/ml) and E. faecalis (MIC = 625 µ g/ml). The MIC of the aqueous extract is >5000 µ g/ml for E. coli, E. faecalis, S. aureus and 1250-2500 µ g/ml on B. subtilis and M. luteus, respectively and 625 µ g/ml against B. cereus and P. aeruginosa. Figure 1. (A): MIC by macrodilution method, (B): inoculation of the tubes with no turbidity. These results are backed by those obtained in the work of Daoud et al., which show that the ethanolic extract of S. officinalis provides a MIC >5000 µ g/ml against S. aureus [56]. Ali and Aboud, carried out a study the antibacterial activity of S. officinalis extracts on E. coli, E. faecalis and S. aureus and found a variable MIC of 2500-5000 µ g/ml for the methanolic extract and 2500-10000 µ g/ml for the aqueous extract [57]. A B Bouteldja et al. Phytochemistry, antioxidant and antibacterial activity of Salvia officinalis extracts 363 European Journal of Biological Research 2021; 11(3): 356-366 4. CONCLUSION According to the results obtained, it can be concluded that the ethanolic extract (80%) of Salvia officinalis L. can play a biological role as an antioxidant product by scavenging free radicals and antibacterial activity. The quantitative evaluation of polyphenols and flavonoids for the extracts of S. officinalis exposes that the ethanolic extract is the richest in polyphenols and flavonoids in comparison with the methanolic extract and the aqueous. On the other side, the results of antioxidant activity by the DPPH test and the antibacterial activity revealed that the ethanolic extract of S. officinalis provides a better free radical scavenging power and antibacterial activity compared to the other extracts. All the results obtained from this study indicate the possibility of using ethanolic extract of S. officinalis in the therapeutic field as an antioxidant and antibacterial. Authors' Contributions: RB, RD, HA designed and carried out the research. RB, RD, AM carried out the experiments. RB, RD, FZA, HB, KZ,SA analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. Conflict of Interest: The authors have no conflict of interest to declare. Acknowledgements: We would like to thank the Engineers working in the Laboratory of Animal Reproduction Farm and Laboratory of Animal Hygiene and Pathology of the Institute of Veterinary Sciences University Ibn khaldoun Tiaret. We are also grateful to the Directorate General of Scientific Research and Technological Development "DGRSDT". REFERENCES 1. Sharma S, Kulkarni Sh K, Chopra K. Curcumin, the active principle of turmeric (Curcuma longa), ameliorates diabetic nephropathy in rats. Clin Exp Pharmacol Physiol. 2006; 33: 940-945. 2. Wu S, Chappell J. Metabolic engineering of natural products in plants; tools of the trade and challenges for the future. Curr Opin Biotechnol. 2008; 19: 145-152. 3. 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