Abstract
Withania adpressa (Coss.) Batt. (W. adpressa) is a wild medicinal plant in the family Solanaceae, which is used as an alternative medicine. The present study aims to investigate the chemical composition, antioxidant, and antibacterial potentials of polyphenol-rich fraction from the leaves of W. adpressa. Polyphenol-rich fraction was characterized by use of high-performance liquid chromatography (HPLC). Antioxidant potency was determined by use of 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and total antioxidant capacity (TAC) assays. Antibacterial activity was assessed against gram-positive and gram-negative bacteria by use of disc diffusion and microdilution assays. Chromatographic analysis by HPLC showed four compounds in the polyphenol-rich fraction including 1-O-Caffeoyl glucose, Luteolin-7-diglucuronide, Apigenin-O-pentoside, and Quercetin-3-O-glucuronide. Polyphenol-rich fraction exhibited important antioxidant activity as assessed by DPPH and FRAP assays, with IC50 and EC50 values of 14.27 ± 0.43 and 39.48 ± 0.81 µg/mL, respectively, while butylated hydroxytoluene (BHT) and Quercetin exhibited IC50 values of 28.92 ± 0.24 and 22.48 ± 0.54 µg/mL. Results of TAC showed that the polyphenol-rich fraction scored 781.74 ± 2.83 µg AAE/mg. Polyphenol-rich fraction showed an important antibacterial effect vs gram-positive and gram-negative strains recording inhibition zone diameters varying from 19.5 to 29.2 mm, while antibiotics were almost ineffective toward all strains except for E. coli. It can be concluded that W. adpressa polyphenol-rich fraction possesses promising phenols with strong antioxidant and antibacterial properties, which may help fight pathogenic bacteria and free radicals.
1 Introduction
Natural compounds generated from plants are a major source of medicines used in traditional pharmacopeia [1]. Even though modern pharmaceuticals have replaced natural preparations traditionally used to treat diseases, scientists and health organizations continue to value the use of alternative medicine in disease treatment due to its promising therapeutic effects [2]. The empirical usage of plants for medication has significantly contributed to the discovery of many interesting secondary plant metabolites with promising pharmacological properties [3]. For decenniums, medicinal and aromatic plants have played a significant role in medication development and pharmacological research as plants produce a wide range of compounds that are employed as treatments, crude materials for drug production, and models for chemically manufactured molecules [4]. Several plants possess a natural reservoir of pharmacologically active compounds, which is used for medication and drug conception history recorded [5].
Antioxidant agents play a crucial role in protecting the human body from numerous diseases including aging, cancer, malaria, neurological disease, and arteriosclerosis, as well as other pathological events [6]. The antioxidant properties of compounds derived from plants are gaining particular interest, which could be important in terms of their nutritional impact along with their role in health protection [7]. Eco-friendly antioxidants appear to be favored by food sector users for avoiding oxidative degradation induced by free radicals. Because of their carcinogenic risk, the use of synthetic antioxidants such as tertbutyl hydroquinone, propyl gallate, and butylated hydroxyanisole is no longer recommended [8]. Impacts of oxidative stress on human health have become a major concern and can involve in degenerative diseases if no antioxidants are used to contain free radicals [9].
Human pathogenic microorganisms have been claimed to be a major cause of disease and death around the world. Even though pharmaceutical industries permanently developed new antibiotics, drug resistance has grown and has become a global health issue [10]. The large spread of multidrug-resistant bacteria (MDR bacteria) is diminishing the efficacy of currently available antibiotics, resulting in significant treatment failure [11]. Scientists throughout the world are paying more attention to AMR since it has become a great burden affecting entire healthcare systems. According to the World Health Organization, AMR was classified as the top tenth global public health concern against humanity in 2019 [12]. Antibacterial phytochemicals are increasingly being used in food and medicine for both prevention and treatment of emerging infections [13].
The Withania genus has traditionally been used to control diseases including conjunctivitis, inflammation, anxiety, neurological diseases, bronchitis, ulcers, liver, and Parkinson’s disease [9]. Scientific reports mentioned that the Withania genus possessed biological properties such as cytotoxic, immunomodulatory, analgesic, healing, and anticholinesterase [14,15,16,17,18,19,20,21]. W. adpressa (Solanaceae) grows in North Africa and Mediterranean areas, known for its pharmacological properties including immunomodulatory anti-inflammatory effects [22,23].
To the best of our knowledge, very few pharmacological reports on W. adpressa have been documented, and therefore, the current research was undertaken to study the chemical composition, antioxidant and antibacterial properties of phenol compounds from the leaves W. adpressa.
2 Materials and methods
2.1 Plant material
In March 2021, a high growth period for flowers, W. adpressa was collected from the Maghrebian Sahara. Next, the plant specimen was deposited at the University Herbarium under the reference A2/WdBF21 after being identified by a botanist (A2/WdBF2). After that, the leaves were carefully cleaned and dried for 1 week in the dark and a well-ventilated place before being subjected to polyphenol extraction.
2.2 Extraction of total polyphenols
The dried leaves of W. adpressa were cut into small pieces by use of an electric apparatus. Briefly, extraction of total polyphenols was undertaken by mixing 50 g of plant powder with 150 mL of methanol (50°C) for 3 h prior to filtration under reduced pressure (40°C). Afterward, the obtained extract was dissolved in 250 mL of distilled water flowed by three successive extractions by use of 200 mL hexane, chloroform, as well as ethyl acetate before concentration under reduced pressure by use of a rotary evaporator (40°C) [24].
2.3 Determination of phenolic compound content
The content of polyphenols was estimated by use of previously described method [25]. Briefly, 1 mL of total polyphenols was combined with 5 mL of the reagent Folin–Ciocalteu (1/10) and 4 mL of sodium bicarbonate solution (0.7 M). Later, the mixture was vigorously shaken and incubated at ambient temperature for 120 min. The absorbance was carefully measured and the concentration of polyphenols was calculated by use of the regression equation of the gallic acid calibration curve (0–1 mg/mL) and the obtained findings were expressed as milligram equivalents of gallic acid per gram sample (mg EGA/g).
2.4 Chemical composition of phenols
The polyphenolic extract was analyzed by use of the Agilent 1100 High-Performance Liquid Chromatography (HPLC/DAD) with a 150 mm × 4.6 mm I.D, dp = 5 m, and Zorbax Eclipse XDB C8 column. Briefly, 10 µL of sample was injected for analysis. The flow rate was determined to be 1 mL/min and the column temperature was 25°C; acidified water (0.1% acetic acid) and acetonitrile made up the mobile phase. Identification of phenols was done by performing comparison of retention times and UV-DAD spectra with those of standards under the same conditions [26].
2.5 Antioxidant activity
2.5.1 DPPH free radical scavenging activity
The antioxidant capacity of polyphenol-rich fraction from the leaves of W. adpressa was determined by use of DPPH assay [27]. Briefly, different concentrations of polyphenol-fraction (concentration ranging from 0.1 to 1 mg/mL) were mixed with 1000 µL of DPPH (0.004%). Following incubation for 20 min in the dark, the absorbance was carefully measured at 517 nm. Methanol was used as a negative control, while Quercetin and BHT were used as positive controls. Results were given as the inhibitory concentration of phenols required to reduce 50% DPPH radicals (IC-50) as follows:
The inhibition percentage of DPPH was calculated as follows:
where T 0 is the absorbance of standard reference and T r is the absorbance of sample test.
2.5.2 Ferric reducing antioxidant power (FRAP) assay
Ferric reducing antioxidant potency of the polyphenol-rich fraction was conducted as described in earlier work [28]. Briefly, 0.1 mL of various concentrations of the sample was combined with 501 µL of phosphate buffer solution with 0.20 M, a pH of 6.6, and 501 µL of potassium ferricyanide (10 mg/mL). After an incubation period of 20 min at 50°C in a water bath, 501 µL of a 10% acetic trichloride solution, 501 µL of distilled water, and 100 µL of FeCl3-6H2O (1 mg/mL) were poured into the medium. Consequently, the absorbance was carefully measured at 700 nm vs a blank containing all reagents. Results were given as effective concentration to have 50% antioxidant effect (EC50). BHT served as a positive control.
2.5.3 Total antioxidant capacity (TAC)
TAC of polyphenol-rich fraction test was carried out as described earlier [29]. In summary, 1 mL of a solution composed of ammonium molybdate (4 mM), sulfuric acid (0.6 M), and sodium phosphate (28 mM) was mixed with 100 µL of polyphenol-rich fraction solubilized in methanol. Thereafter, the reaction solution was incubated at 95°C for 90 min and the absorbance was read at 695 nm. Ascorbic acid and methanol were, respectively, used as negative and positive controls. The ascorbic acid was used to make a calibration curve, which was used to calculate the overall antioxidant capacity. TAC was expressed as microgram of ascorbic acid equivalent per milligram sample (µg EAA/mg).
2.6 Antibacterial activity
2.6.1 Bacterial strains
Antibacterial effects of polyphenols were investigated against five bacterial strains including two gram-positive and three gram-negative: Klebsiella pneumoniae (G−), Escherichia coli (G−), Acinetobacter baumannii (G−), Staphylococcus aureus (G+), and Streptococcus pneumoniae (G+).
2.6.2 Bacterial suspension
To summarize, bacterial suspension was prepared by culturing fresh colonies aged 24 h in Mueller-Hinton medium before being aseptically suspended in 0.9% of sodium chloride. The optical density of bacterial suspension was 1–2 × 108 CFU/mL as recommended concentration for experiment [30].
2.6.3 Disc diffusion method
The bacterial strains’ sensitivity to polyphenols was tested by the disc diffusion method as described in earlier work with some modifications [31]. Briefly, the Petri plates (9 cm) containing Mueller-Hinton-Agar medium were inoculated with 1 mL of fresh bacterial cultures before being dried for 10 min. Afterward, 0.6 cm sterile discs were impregnated with 5 μL sample and positive controls before being deposed on the Petri plates’ surface. Next, the plates were incubated at 36°C for 24 h prior to reading inhibition zone diameters in mm.
2.6.4 Minimum inhibitory concentration (MIC)
The microdilution dilution assay was used to evaluate the polyphenol-rich fraction MICs as reported in earlier work [32]. Briefly, polyphenols were diluted in 0.2% agar, while positive control was diluted in Mueller-Hinton Bouillon (MHB) medium. Next, the MHB medium (50 μL) was placed into microplate wells. Afterward, 100 μL of each agent was deposited in the first followed by ½ dilution except for the last well, which served as positive growth control. Next, the inoculation was carried out by depositing 50 μL of the bacterial suspension in all wells except the first well (serves as negative growth control). The bacteria culture was incubated at 37°C for 24 h prior to reading results by use of 1% 2,3,5-triphenyl tetrazolium chloride wherein wells containing bacterial growth became pink due to the activity of the dehydrogenases, while the wells without bacterial growth remained uncolored after 2 h of incubation.
2.7 Statistical analysis
Findings presented here were expressed in mean values with standard deviations of triplicate tests. Shapiro–Wilks test was used to test for normality of distributions, while the t-test was used to check for homogeneity of variances. Analysis of variance was performed, with Tukey’s HSD test as a post hoc test for multiple comparisons. A significant difference was considered at P < 0.05.
3 Results and discussion
3.1 Analysis of chemical composition
The extraction yield of polyphenols from the leaves of W. adpressa was determined to be 13.81 ± 0.76 mg%. Polyphenol content evaluated by gallic acid calibration (Y = 0.005X + 0.028; R 2: 0.996) was determined to be 15.38 ± 0.72 mg EGA/g. This result is comparable to polyphenol content in Withania frutescens L. which was determined to be 10.015 ± 0.063 mg EGA/g [33]. Polyphenol contents in leaves and roots of Withania frutescens L. indigenous to Morocco were found to be 12.04 ± 0.61 and 53.33 ± 1.20 mg EGA/g, respectively [34].
The variation phenol content in plants can be influenced by different environmental factors, such as temperature, relative humidity, climatic conditions, and soil and/or factors related to post-harvest treatments (harvesting, drying, and extraction) [35]. Analysis of the polyphenol-rich fraction profile by HPLC/DAD identified four polyphenolic compounds namely 1-O-Caffeoyl glucose, Luteolin-7-diglucuronide, Apigenin-O-pentoside, and Quercetin-3-O-glucuronide (Table 1; Figures 1 and 2). These results are consistent with those reported by El Moussaoui and coauthors [36], who showed that phenols namely Luteolin and Apigenin are present in the genus Withania.
Chemical profile of polyphenol-rich fraction from the leaves of Withania adpressa Coss. ex Batt
| Tr | Identification compound | Standard used | Ref. |
|---|---|---|---|
| 1.069 | 1-O-Caffeoyl glucose | Caffeic acid | 20 |
| 1.274 | Luteolin-7-diglucuronide | Luteolin | 22 |
| 1.595 | Apigenin-O-pentoside | Apigenin | 21 |
| 1.645 | Quercetin-3-O-glucuronide | Quercetin | 33 |
Chromatogram profile of polyphenol-rich fraction from the leaves of Withania adpressa Coss. ex Batt.
Chemical structure of compounds in the polyphenol-rich fraction from the leaves of Withania adpressa Coss. ex Batt.
3.2 Antioxidant activity
Polyphenol-rich fraction was able to reduce free radicals in a dose-dependent manner. The IC50, as determined by the DPPH assay was 14.27 ± 0.43 µg/mL, was lower than that observed for positive controls such as BHT and Quercetin that exhibited IC50 values of 28.92 ± 0.24 and 22.48 ± 0.54 µg/mL, respectively. Similarly, the findings of the FRAP assay also revealed significant antioxidant potency polyphenol-rich fraction with an EC50 value of 39.48 ± 0.81 µg/mL, which was slightly greater than that recorded for standards (Figure 3). The findings of the TAC test demonstrated powerful antioxidant effect of polyphenol-rich fraction with an EC50 value of 781.74 ± 2.83 µg EAA/mg which was also greater than EC50 values of standards such as Quercetin and BHT (Figure 3).
Antioxidant activities of polyphenol-rich fraction from the leaves of Withania adpressa Coss. ex Batt.
The antioxidant activity seen in the polyphenol-rich fraction might be attributed mostly to the phenols revealed by HPLC such as 1-O-Caffeoyl glucose, Luteolin-7-diglucuronide, Apigenin-O-pentoside, and Quercetin-3-O-glucuronide. These chemicals might act individually or in synergy resulting in scavenging free radicals [37]. The chemical composition of the polyphenol-rich fraction from W. adpressa was rich in 1-O-Caffeoyl glucose and Luteolin-7-diglucuronide known for their antioxidant power, particularly 1-O-Caffeoyl glucose had strong antioxidant activity with DPPH radical scavenging rate up to 90.4%. [38]. Luteolin-7-diglucuronide showed better antioxidant activity against DPPH radicals, superoxideradical-anion in cell-free (xanthine oxidase), and cellular (polymorphonuclear neutrophils) [39]. Apigenin-O-pentoside from Petroselinum crispum was responsible for its excellent antioxidant activity [40]. By use of DPPH and FRAP assays, antioxidant activity of quercetin derivatives including quercetin-3-O-glucuronide was investigated by Lesjak and coauthors who showed that quercetin-3-O-glucuronide displayed notable antioxidant activity, even higher than butylated hydroxytoluene [37].
3.3 Antibacterial activity
Polyphenol-rich fraction showed important activity vs gram-negative and gram-positive strains wherein the inhibition diameter varied from 19.5 to 29.2 mm. The largest diameter was 29.2 and 28 mm against E. coli and K. pneumoniae, respectively. On the other hand, we found that all of the bacterial strains were resistant to commercially available antibiotics used as positive controls except for E. coli and kanamycin which were moderately sensitive to these antibiotics (Table 2).
Inhibition results of W. adpressa polyphenol-rich fraction against bacteria
| Diameter of the inhibition zone (mm) | |||||
|---|---|---|---|---|---|
| Bacterial strains | PTW | Antibiotics | |||
| OX | S | K | ZOX | ||
| E. coli | 29.20 ± 1.14a | 12.50 ± 1.17a | 16.00 ± 1.40a | 17.50 ± 0.70a | 13.00 ± 1.40a |
| K. pneumoniae | 28.00 ± 1.44b | 0 | 0 | 16.50 ± 0.70b | 0 |
| A. baumannii | 19.50 ± 0.91b | 0 | 0 | 0 | 0 |
| S. pneumoniae | 19.50 ± 2.10c | 0 | 0 | 0 | 0 |
| S. aureus | 24.50 ± 2.50c | 0 | 0 | 0 | 0 |
Kanamycin (K), oxacillin (OX), streptomycin (S), ceftizoxime (ZOX).
The row values with the same letters differ significantly (means SD, n = 3, one-way ANOVA; Tukey’s test, P = 0.05); Shapiro–Wilks test (P more than 0.1); t-test (P less than 0.01).
Our results showed that MIC values were less (more potent) against all bacterial strains, where the MIC value ranged from 0.027 to 0.067 mg/mL, reflecting the strength of the inhibitory effect of polyphenols. Furthermore, the same results were obtained with the antibiotics used as positive controls (Table 3).
MICs results of W. adpressa polyphenol-rich fraction against bacteria
| MIC (mg/mL) | |||||
|---|---|---|---|---|---|
| Bacterial strains | PTW | Antibiotics | |||
| OX | S | K | ZOX | ||
| E. coli | 0.045 ± 0.003a | 0.031 ± 0.003a | 0.041 ± 0.001b | 0.052 ± 0.003a | 0.064 ± 0.009a |
| K. pneumoniae | 0.031 ± 0.004a | 0.023 ± 0.006ns | 0.041 ± 0.005a | 0.026 ± 0.007a | 0.023 ± 0.001ns |
| A. baumannii | 0.034 ± 0.001a | 0.014 ± 0.007a | 0.039 ± 0.004b | 0.024 ± 0.007a | 0.016 ± 0.004a |
| S. pneumoniae | 0.076 ± 0.001a | 0.022 ± 0.003a | 0.045 ± 0.003a | 0.032 ± 0.005a | 0.027 ± 0.003b |
| S. aureus | 0.027 ± 0.005a | 0.012 ± 0.004a | 0.036 ± 0.002c | 0.029 ± 0.001b | 0.017 ± 0.001a |
Kanamycin (K), ceftizoxime (ZOX), oxacillin (OX), streptomycin (S).
The row values with the same letters differ significantly (means SD, n = 3, one-way ANOVA; Tukey’s test, P = 0.05); Shapiro–Wilks test (P more than 0.1); t-test (P less than 0.01).
ns = non significant.
The results presented here showed that polyphenol-rich fraction from W. adpressa had strong antibacterial effects vs all tested bacteria, even at low concentrations. These results are comparable to those reported elsewhere [34], wherein it was reported that W. frutescens crude extracts from the roots possessed antibacterial activity opposed to gram-positive and gram-negative bacteria with inhibition zone diameters ranging from 8 to 15 mm, and MIC of 2.8 mg/mL vs A. baumannii [34]. Al-Ani and coauthors reported that Withania somnifera ethanol extracts exhibit antibacterial effects against gram-negative and gram-positive bacteria such as S. aureus, E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis, and S. paratyphi [41]. Previous research reported that alkaloids present in both aqueous and methanolic extracts of W. somnifera leaf and root, as possible phytoconstituents for their potent antibacterial activity against E. coli, Staphylococcus aureus, and Salmonella typhimurium [42]. Polyphenol-rich fraction investigated in this work is composed of four phenols (Table 1), which may work in synergy or individually resulting in bacteria death. Extracts from Luffa cylindrical rich in 1-O-Caffeoyl glucose exhibited antibacterial activity against B. cereus, B. megaterium, B. subtilis, S. aureus, S. lutea, E. coli, S. typhi, P. aeruginosa, S. paratyphi, S. dysenteriae, V. mimicus, and V. parahaemolyticus [43].
The antibacterial effects of polyphenol-rich fraction might be attributed to the luteolin-7-diglucuronide since it was reported that this compound exhibited potent antibacterial activity against S. aureus [44]. Ethanol extract from Petroselinum crispum, which was found to be rich in Apigenin-O-pentoside, exhibited good antibacterial activity against S. typhi, S. aureus, and Klebsiella pneumonia, thus confirming its antimicrobial potency against a wide range of microorganisms [45]. Extracts from Persicaria perfoliata containing quercetin-3-o-glucuronide was found to be also active against Escherichia coli, Propionibacterium acnes, and Staphylococcus aureus [46]. Gram-positive Bacillus cereus and Micrococcus luteus were shown to be the most sensitive bacteria to caffeic acid in a separate study, while E. coli and Staphylococcus aureus were found to be the most resistant bacteria [47]. The MIC for drug-resistant bacteria was 4 mg/mL, and the inhibition diameter varied from 10.52 to 19.12 mm with apigenin as previous work reported [48]. Polyphenols’ antibacterial action is affected by hydroxylation and structural features that render bacteria susceptible [49].
The sensitivity of bacterial strains to the polyphenols could be explained by their potential action in the cytoplasmic membrane, resulting in structural disorganization of polysaccharides, fatty acids, and phospholipids, which modify cytoplasmic membrane permeability resulting in bacteria death [31,43]. El bouzidi and coauthors [18] reported that antimicrobial activity may be due to withanolide identified in the genus Withania.
4 Conclusion
The outcome of this work stated that the polyphenol-rich fraction from the leaves of W. adpressa exhibited excellent antioxidant and antibacterial potencies against clinically drug-resistant pathogenic bacteria. The chemical characterization of the W. adpressa polyphenol-rich fraction revealed the presence of four compounds, 1-O-Caffeoyl glucose, Luteolin-7-diglucuronide, Apigenin-O-pentoside, and Quercetin-3-O-glucuronide, which are responsible compounds for the recorded activities. In brief, the results presented here can serve as a valuable natural source for future research concentrating on determining the polyphenol-rich fraction of single purified chemicals. Assessment of the potential undesirable effects on nontarget organisms will value polyphenol-rich fraction prior to further use.
Acknowledgment
The author extends his appreciation to Researchers Supporting Project number (RSP-2022R437), King Saud University, Riyadh, Saudi Arabia.
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Funding information: This study was funded by Researchers Supporting Project number (RSP-2022R437), King Saud University, Riyadh, Saudi Arabia.
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Author contributions: This work was completely performed by AMS.
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Conflict of interest: The author declares that there is no conflict of interest.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: All data reported here are available from the author upon request.
Abbreviations
- MH
-
Mueller-Hinton medium
- EAA/mg
-
ascorbic acid equivalent per milligram of sample
- EGA/g
-
equivalents of gallic acid per gram of sample
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© 2022 Ahmad Mohammed Salamatullah, published by De Gruyter
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