Phenolic Content and Antioxidant and Antimicrobial Activities of Malva sylvestris L., Malva oxyloba Boiss., Malva parviflora L., and Malva aegyptia L. Leaves Extract

Background. The plants brought by Arabs were of real therapeutic values. Ibn Al-Baitar, an Islamic scholar (pharmacist, botanist, and physician), in his encyclopedia wrote the detailed characterization of more than one thousand herbs describing their medicinal value, methods of preparation, and their route of administration. Objectives. The current investigation points towards the quantitative characterization of the phenolic contents among the four edible Malva plants species (Malva sylvestris L., Malva oxyloba Boiss., Malva parviflora L., and Malva aegyptia L.) and also towards assessing their antibacterial activity against one Gram-positive isolate (Staphylococcus aureus) and four Gram-negative strains Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, and Proteus vulgaris. It also aimed to evaluate the free radical scavenging activity of hexane, methanolic, aqueous, and acetone extracts of four Malva species. Methods. By utilizing the Folin–Ciocalteu procedure and gallic acid as a reference molecule, the phenolic contents were estimated. In addition, the broth microdilution method was used to evaluate four plants’ 16 extracts, and the DPPH (2,2-diphenyl-1-picrylhydrazyl) method was utilized to assess the abovementioned extracts against oxidative stress. Results. The results showed that the methanolic extract of M. oxyloba has the highest contents of phenols (191.54 ± 2.84 mg of GAE/g) and has the best antioxidant capacity with an IC50 value of 1.94 ± 1.84 µg/ml, which is very close to Trolox. Regarding the ferrous ion chelating activity of the extract, the methanolic extract of M. sylvestris exhibits appreciable activity with IC50 values of 52.7 ± 1.8 µg/ml. In addition, the plant extract and acetone extract of M. sylvestris showed a strong antibacterial activity with an MIC value of 0.0078 mg/ml. Conclusion. The methanolic extract of M. oxyloba has a pharmacological potential as a valuable natural product that can be utilized as a main ingredient in the design and development of new therapeutic formulations. It exerts multiple inhibitory properties against oxidative stress and bacterial growth. As such, it is emerging as a promising therapeutic agent for the treatment of various neurodegenerative diseases and many types of human infectious diseases.


Introduction
e history of phytotherapy started with our ancestors, who had the knowledge of the herbs and knew that the consumption of certain kinds of herb resulted in the soothing effect of several types of diseases. Recently, herbal remedies have become a controversial issue all over the world as large numbers of herbals are utilized in large quantities in cosmetics, as food preservatives and as nutritional supplements and as natural ingredient in large number of drugs in many of the pharmaceutical industries. In fact, people around the world are more moving towards or prefer the herbal medicines due to the belief that these phytotherapeutic products are natural and safer than synthetic medicines [1]. In recent years, the term oxidative stress has been gaining interest among many research scientists, and this has led to the development of numerous techniques in the prevention of diseases caused by the action of the harmful free radicals that cause oxidative stress. In addition, they studied the possible roles of antioxidants in protecting the body from many dangerous diseases caused due to oxidative stress. Common free radical such as hydroxyl (OH_), superoxide (O 2-), and nitric monoxide (NO − ) are considered as causative agents for many chronic diseases such as cancer, diabetes, cardiovascular diseases, rheumatoid arthritis, and other oxidative diseases. At the same time, there are many antioxidant compounds, mostly probably derived from plants, such as vitamin E, vitamin C, creatinine, and other phenolic compounds have the ability to protect the body against the diseases caused by free radicals [2].
It has been observed that the bacterial strains are being increasingly resistant to the antibiotics that are available in the market due to their overuse. e available reports suggest that the overuse of the antiseptic in house and hospitals also contributed to the increase in the resistance of bacteria. However, if the patients with bacterial infection, that is, resistance to antibiotics, did not receive effective treatment may lead to long-term infections that may result in high mortality as was observed in the patients with pneumococcal infections and typhoid fever [3].
Malva (Malvaceae family) is a very common plant growing in wildly in the Middle Eastern countries and commonly known as mallow and has been used by Greek and Romans as an emollient and laxative; also, it has been used to treat some diseases associated with alimentary canal and respiratory disorders. In Asian countries, it is used to treat injuries and inflammations. People use Malva species in different ways; many use it in their food; they eat it raw mixed in a salad or cooked as a vegetable. Other people use its flowers for medicinal purpose to treat injuries and different skin disorders [4]. e intensive use of Malva species among Arabian population urges us to study and identify the medical and chemical characteristics of Malva sylvestris L., Malva oxyloba Boiss., Malva parviflora L., and Malva aegyptia L.
In a study established by Tabaraki et al., they identified the bioactive molecule from Malva sylvestris using GC-MS technique and found that the major compound in the methanolic extract was 2-methoxy-4-vinylphenol [7]. Moreover, M. sylvestris anthocyanins contents reduced plasma total triglycerides and cholesterol and also protected rats from ethanolinduced gastritis due to the high contents of mucilage [8][9][10].
Hot poultice of Malva parviflora used in traditional medicine to treat wounds, swelling, sores, bruises, and broken limbs. It contains tannin, flavonoid, polyphenol, saponin, resin, and alkaloid. Several investigations showed that M. parviflora has antiulcerogenic, analgesic, antidiabetic, anti-inflammatory, antibacterial, and neuroprotective activities [11]. roughout the literature, no previous studies were carried out to investigate the constituents and the pharmacological activities of M. oxyloba and M. aegyptia. is prompted us to search for new source of safer and inexpensive antioxidants from four edible species of M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia plants. e extracts were prepared in aqueous solution, hexane, acetone, and methanol solvent; these extracts were tested for their antioxidant activity using DPPH method, and to estimate their total phenol contents using the Folin-Ciocalteu assay, the relationship between antioxidant activity and total phenol contents was correlated; their antimicrobial properties were also studied.

Extraction.
e exhaustive maceration was used to extract M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia plants materials by soaking 150 g of powdered samples sequentially into 500 ml of distilled water, n-hexane, ethyl acetate, and methanol in an enclosed glass bottle with occasional shaking at room temperature for 72 hrs. ereafter, the macerated materials were then filtered through a Whatman No. 1 filter paper and then evaporated under reduced pressure in a rotary evaporator set at 35°C in a water bath until the solvents completely evaporated, while the aqueous extracts were freeze-dried. e final dried extracts were stored at 4°C in a refrigerator.

Total Phenol Content.
e total phenol contents of M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia plants four extracts were estimated calorimetrically utilizing the Folin-Ciocalteu procedure and gallic acid as a reference molecule [12][13][14]. Briefly, from each plant fraction, 1 mg/ml aqueous solution was prepared for this analysis, while the reaction mixtures were prepared by taking 0.5 ml of the aqueous solution from each plant fraction, 2.5 ml of 10% Folin-Ciocalteu's testing agent dissolved in water, and 2.5 ml of 7.5% NaHCO 3 in aqueous solution. ereafter, the produced mixtures were kept in a thermostat at 45°C for 45 min. e absorbance was estimated utilizing a spectrophotometer at a wavelength of 765 nm. All of the studied samples were tested in triplicate for each plant fraction, and the mean values of absorbance obtained were reported. e same method was repeated for the standard molecule gallic acid, and then the required calibration curve was constructed. Based on the measured absorbance, the concentration of gallic acid equivalent was determined in mg of GAEq/g for each working plant sample.
2.6. Free Radical Scavenging DPPH Method. According to the method described by Jothy et al. [15] with some modifications, the antioxidant activity was determined using DPPH scavenging assay. A stock solution of Trolox and 16 plant extracts in methanol was prepared with a concentration of 1 mg/mL; from the stock solution by serial dilution method, various concentrations were prepared. en, 0.002% DPPH was prepared. A concentration ratio (1:1:1) of the above prepared solution, DPPH, and methanol were arranged, respectively. ese solutions were incubated in a dark place for 30 min, while methanol was utilized to zero the UV-Vis-spectrophotometer. en, the absorbance readings were measured at 517 nm. Antioxidant activity is given as percent DPPH scavenging that is calculated as where M is the absorbance of the control sample and S is the absorbance of the tested extracts. e results are documented as an IC 50 value which is the inhibitory concentration required to decrease 50% of the activity.

Ferrous Ion-Chelating Activity.
e samples were estimated for the ferrous ion-chelating activity as described by Chua et al. [16] Briefly, 750 µL of the sample in test material (2-500 µg/mL) or standard substance (1-50 µg/ml) were incubated with 50 µL ferrous chloride solution (FeCl 2 ) (2 mM) for 5 min. e reaction was initiated by the addition of 50 µL ferrozine solution (5 mM) into the mixture and allowed to stand for 10 minutes in dark. e absorbance of the reaction mixture was measured at 562 nm. All experiments were performed at least in triplicate. e chelating effect was calculated as a percentage using the following equation. EDTA was used as a positive control in this assay.
where Acontrol is the absorbance of the control reaction mixture without the test compounds, and Asample is the absorbance of the test compounds. IC 50 values, which represented the concentration of the extract that caused 50% of Fe 2+ ion chelation, were calculated from the plot of chelating percentage against concentration.

Antimicrobial Test.
For determination, the antimicrobial activity for four plants' 16 extracts, a micro broth dilution method was employed. Some colonies from the used bacterial pathogens were planted in agar broth for 24 hrs. A little amount of the bacteria was added inside a 5 ml of Mueller-Hinton tube and was mixed. en, the turbidity was compared with McFarland standard tube 0.5 (1.5 × 10 8 cfu/ ml). en, 2 ml of the bacterial suspension was diluted with 4 ml broth. e four Malva species extracts were prepared by dissolving 50 mg of each in 1 ml DMSO (100%) with a 50 mg/ ml concentration. en, 100 μl from Mueller-Hinton was placed in a sterile 96-well microplate. By using a micropipette, a volume of 100 μl of plant extracts was added into the first well and mixed. Serial dilution was done by transferring 100 μl from well to next well up to well 11 from which 100 μl was discharged after mixing process; well 12 had only broth without plants extracts, so bacterial growth will be noticed. However, a serial dilution was established under sterile conditions. After that, a 10 μl of bacterial suspension was employed into all wells but not in well 11. While well 12 must be turbid, if not we should know that something wrong happened with our media or bacteria. en, the used microplate was incubated for 24 h. Each plant extract was examined in duplicate. MIC is defined as the minimum concentration of the antibacterial agents that resulted in nonvisible turbidity compared with a control well.

Statistical Analysis.
e extraction yields, total phenols, and antioxidant results of the four studied plant species were expressed as means ± SD standard deviation. Statistics were compared utilizing unpaired t-tests.

Phenolic Content.
For the evaluation of total phenol content, the absorption values of several concentrations of the gallic acid standard are listed in Table 2. From the calibration curve of gallic acid (Figure 1), the following equation was used to estimate total phenol content in M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia plants aqueous, hexane, methanol, and acetone extracts [14].
where y is the absorbance at 765 nm and x is the total phenol content of the four Malva plants species various extracts.
Obviously, Table 3 indicates that acetone extract of M. sylvestris, the methanolic extract of M. oxyloba, and hexane extracts of M. aegyptia and M. parviflora plants have the highest total phenols among the other screened solvents.

Antioxidant Activity.
e antioxidant activities of hexane, methanol, acetone, and aqueous M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia plants extracts were evaluated using DPPH free radical method. In addition, in the current study, Trolox, the powerful antioxidant compound, was used as a reference positive control. e DPPH inhibition effects for each studied Malva plant species are calculated in Tables 4-7 Table 8, which showed that among M. sylvestris extracts, the acetone extract has the highest IC 50 value of 4.07 ± 1.04 µg/ml, among M. oxyloba extracts, the methanolic extract was the best one with IC 50 value of 1.94 ± 1.84 µg/ml, and among M. aegyptia and M. parviflora extracts, the hexane extracts were the best with IC 50 values of 6.9 ± 1.42 µg/ml and 22.9 ± 1.47 µg/ml, respectively. e ferrous ion chelating activity of the different extracts is mentioned in Table 9. It is interesting to mention that the aqueous extract showed better activity than the other extracts; the IC 50 values were ranged from 52.7 ± 1.8 to 95.5 ± 3.4 µg/ml. e hexane extract of different plants was inactive; this might be due to the absence of polar chemical compound which is able to form chelate with the ferrous ion. e acetone extract of the plant exhibited weak activity. e IC 50 values ranged from 315.5 ± 7.0 to 425.5 ± 10.2 µg/ml. e ferrous ion chelating activity of M. sylvestris was better than the other extracts.  Table 10.

Discussion
Over thousands of years, the human orientations towards herbal products have been increasing significantly, for food purposes or for the medicinal use. Millions of people around the world believe in the efficacy and safety of the plants as being a natural resource in many areas of their lives. Many valuable medicines have been derived from plant source such as digoxin, vincristine, pilocarpine, atropine, and many others [17].
In recent years, some groups of natural compounds have received remarkable attention including polyphenols and simple phenols, which are an essential group of secondary metabolites with a wide diversity in structures and are biosynthesized in different parts of the plants for various functions such as protection, pigmentation, and pollination [18]. Various investigations have linked a high phenolic diet to the prevention of several cardiovascular, metabolic, infectious, and cancer diseases due to their pharmacological and biological activities [19].
Total phenolic content of all the plant extracts of all the four species of Malva were estimated using the Folin-Ciocalteu assay.  Table 2: Absorption values of several concentrations of the standard gallic acid [14].     [20]. Another study performed by           Journal of Chemistry 7 Tabaraki et al. found that the total phenolic content of M. sylvestris methanolic extract was 15.11 ± 0.28 mg GAE/g [7]. However, another study established by Beghdad et al. showed that the total phenolic content of the M. sylvestris leaves extract was 24.12 ± 0.72 mg GAE/g [21]. In the last two decades, the health benefits of antioxidant molecules obtained from various species of vegetables and plants have become the major focus of the researchers due to the fact that many of these dangerous diseases such as cancer, diabetes, atherosclerosis, and many other stressrelated diseases can be prevented by the appropriate use of these products [22].
Several reports suggest that, due to the side effects, adverse reactions, and possible carcinogenic effects of using chemically synthesized antioxidants, there is a universal agreement that the chemical antioxidants need to be replaced with natural ones. erefore, the present study aims to explore the antioxidant capacities of the M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia hexane, methanol, acetone, and aqueous extracts. Due to the side effect, adverse reaction, and the possible carcinogenic effect that come along the continuous use of chemically synthesized antioxidants, several reports suggest that there is a universal agreement on the replacement of the chemically synthesized antioxidant with the natural ones. erefore, the present study was aimed to explore the antioxidant capabilities of the Malva species with various extracts.
In the current study, the antioxidant capacities were estimated using the DPPH scavenging method and comparing the obtained results with a positive control compound Trolox that has IC 50 1.86 µg/ml. e plants, in general, showed high antioxidant activity ranging from 1.94 ± 1.84 to 72.44 ± 1.36 µg/ml. Particularly, the methanolic extract of M. oxyloba showed the highest antioxidant capacity with IC 50 value of 1.94 ± 1.84 µg/ml among the other methanolic extracts studied; this was followed by the acetone extract of M. sylvestris (4.07 ± 1.04 µg/ml), which had the highest antioxidant activity among the other acetone extracts studied. In addition, the hexane extract of M. aegyptia showed the highest antioxidant activity (6.9 ± 1.42 µg/ml) among the other hexane extracts, and finally, the aqueous extracts of M. aegyptia showed the highest antioxidant (7.11 ± 2.15 µg/ml) among the other aqueous extracts. e lowest antioxidant activity was observed in all the four extracts of M. parviflora. is study shows that methanolic extract of M. oxyloba has the IC 50 value almost similar to the value of Trolox (1.86 µg/ml), which suggests that this extract can be used as a natural alternative therapy for the prevention and treatment of various degenerative diseases and disorders.
Afolayan and his coworkers reported the IC 50 value of M. parviflora to be 94.3 ± 0.03 µg/ml [20]. In another study, Beghdad et al. showed that M. sylvestris leaves extract had an antioxidant IC 50 value of 16.691 ± 0.2 µg/ml [21]. is suggests that the results obtained in the present investigation have the highest IC 50 values when compared to other studies indicating that the plant species has the highest free radical scavenging activity which may be due to their total phenolic content, and in the present studies, the total phenolic content was the highest among all the reported literatures.
Bacterial resistance to many of the recently used modern antibiotics has becomes a common phenomenon. is has prompted many researchers to look for new chemical from natural origin or plant origin which is safer and effective in overcoming the resistance issue; to study the antimicrobial activity from plant origin, many experts went to study herbal antimicrobial effect trying to solve bacterial resistance problem and to develop new drugs [23].
In the present study, all the four extracts of the Malva species were used to explore the antimicrobial potentials against the five bacterial strains using the broth microdilution method.
In the present study, the antimicrobial activity of M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia species hexane, methanol, acetone, and aqueous extracts was conducted against five bacterial strains using the broth microdilution method. e results showed that M. sylvestris acetone extracts inhibited the bacterial growth with the highest MIC values of 0.125 mg/ml against S. aureus and 0.078 mg/ml against P. vulgaris among the other acetone extracts. In addition, the hexane extract of M. aegyptia  appreciable activity against E. coli with MIC value of 0.078 mg/ml. In a study conducted by Razavi et al. [10], they found that the methanolic extract of M. sylvestris inhibited the growth of Escherichia coli and Staphylococcus aureus with MIC values of 32 and 36 mg/ml, respectively [10]. At the same time, in the present study, the methanolic extract of M. sylvestris inhibited the growth of the Escherichia coli and Staphylococcus aureus with MIC values of 6.25 and 12.5 mg/ ml, respectively.
In fact, due to the high phenolic contents of M. sylvestris, M. oxyloba, M. parviflora, and M. aegyptia species, the hexane, methanolic, acetone, and aqueous extracts of these species showed strong antibacterial activities, and several prior researches generally confirm that the phenolic compounds affect strongly the microbial growth [24]. ese finding indicated that the acetone extract of M. sylvestris and the methanolic extract of M. oxyloba can be used as a suitable candidate in the manufacturing of natural phytoalexins and as a natural alternative to synthetic antibiotics. In line with previous studies, the current study found a strong positive correlation between antioxidant activities of plants extracts and total phenolic content, which implies that the studied plant's phenols are the major contributor to the antioxidant activity in these extracts. Naturally, occurring phenols have an antioxidant activity as these molecules have the ability to scavenge free radicals by a single-electron transfer mechanism which decreases the oxidative stress in life cells and tissues [7].

Conclusion
In view of the abovementioned results, the methanolic extract of M. oxyloba is a pharmacologically valuable natural product that can be utilized as the backbone for the design and development of new therapeutic formulations. It exerts multiple inhibitory properties against oxidative stress and bacterial growth. As such, it represented promising therapeutic agents for the treatment of various neurodegenerative diseases and many types of human infectious diseases.
It may be concluded from our studies that the methanolic extract of the M. oxyloba is of great pharmacological value, as this natural product can be utilized in the design and development of the new therapeutic formulations as the main ingredient. Due to its inhibitory properties against oxidative stress and bacterial growth, it may be one of the promising therapeutic agents to treat various neurodegenerative diseases and many of the infectious diseases in human.

Data Availability
e data used to support the findings of this study are available within the manuscript.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.