Evaluation of the stimulatory and inhibitory effects of Malva sylvestris leaf extract on some beneficial and pathogenic bacteria from the colon Journal of Istanbul Veterınary Scıences

The aim of the present study was to evaluate the stimulatory and inhibitory effects of Malva sylvestris leaf extract on some selected beneficial and pathogenic bacteria from the colon to form a presupposition on its efficacy on intestinal health. The sensitivity of colon bacterial strains to M. sylvestris leaf extract was tested by a broth dilution method in the anaerobic cabinet. Malva sylvestris leaf extract stimulated the growth of Bifidobacterium bifidum from beneficial species starting from 0.06 mg/mL dose (P<0.05). The same stimulatory effect was observed for other beneficial species Bifidobacterium infantis and Lactobacillus acidophilus from 0.125 mg/mL dose (P<0.05) and that effect was more obvious for B. infantis. On the other hand, the extract did not have any effect on Lactobacillus casei up to 4 mg/mL dose. Malva sylvestris leaf extract also had a potential inhibitory activity against pathogenic Escherichia coli , Clostridium perfringens , and Staphylococcus aureus from 0.25, 2, and 4 mg/mL concentrations respectively (P<0.05). The dose of 8 mg/mL of the extract (MIC; minimal inhibitory concentration) completely inhibited Fusobacterium nucleatum (P<0.05), other enteropathogen, which is associated with colorectal cancer. It was concluded that M. sylvestris leaf extract at 0.06-8 mg/mL dose could have favorable effects on colon bacteria since the extract selectively promoted the most of the beneficial species’ growth at this dose range while it had a potential inhibitory or inhibitory effect on pathogenic ones. Investigating the effects of M. sylvestris leaf extract on other colon bacteria and testing the in vivo effectiveness will contribute to a better understanding of its efficacy on colon microbiota and intestinal health.

Colon constitutes a complex community of bacteria, over 99% of which are anaerobic bacteria (Macfarlane and Macfarlane, 2003). Commensal or resident bacteria of the colon are considered as "beneficial" since they have remarkably crucial influences for the physiology, immunity, and susceptibility to diseases of the host (Kau et al., 2011). Consequently, disturbance of the colon microbiota can cause to expansion of pathogenic species and subsequent abnormal physiological states (Ahn et al., 1998). Recently, many studies have been focused on potential prebiotic and antibiotic effects of medicinal plants and plant metabolites on beneficial and pathogenic bacterial species from the colon microbiota (Phoem and Voravuthikunchai, 2012;Thapa et al., 2012;Goker and Demirtas, 2020).
Malva sylvestris, commonly known as mallow, is a plant native to Europe, Asia, and North Africa with dark green leaves and red to blue-purple flowers (Elsagh et al., 2015). This plant has a long history of Introduction use in Mediterranean and European traditional medicine to treat inflammations, dermal infected wounds, bronchitis, and particularly digestive problems such as peptic ulcers, gastritis, enteritis, and colitis due to its high mucilage content and its colon cleansing properties (Gasparetto et al., 2012;Hamedi et al., 2015;Al-Rubaye et al., 2017). Aqueous extracts of aerial parts of M. sylvestris and also isolated polysaccharide of the plant were effective in preventing the inflammatory lesions of ulcerative colitis in the rats (Hamedi et al., 2015). The leaves have shown high effectiveness against swine constipation and in the treatment of mastitis in bovines when applied in enemas or compresses (Uncini-Manganelli et al., 2001). Phytochemical analyses of the M. sylvestris leaves have also relieved the presence of a wide range of phytochemical groups with antimicrobial properties such as flavonoids, alkaloids, phenolic acids, quinones, tannins, saponins, steroids, terpenoides, carotenoids, and unsaturated fatty acids (Barros et al., 2010;Dowek et al., 2020). There are reports about antibacterial activity of the extracts of M. sylvestris leaf on plant (Razavi et al., 2010), oral (Vahabi et al., 2019), and wound pathogens (Zare et al., 2012) and on some clinical isolates (Azadpour et al., 2016). However the literature is scarce regarding the effects of M. sylvestris leaf extract on colon bacteria especially on beneficial ones. Therefore, the aim of the present study was to evaluate the stimulatory and inhibitory effects of M. sylvestris leaf extract on some selected beneficial and pathogenic bacteria from the colon.

Materials and Methods
Malva sylvestris leaf extract: The extract was provided by Kale Naturel Herbal Products Company, Ltd., Balikesir, Turkey. As specified by the manufacturer, M. sylvestris leaves were air dried, ground into 0 to 200 µm large particles and screened. Powdered plant leaves used for extraction in 80% ethanol (1/10, w/v) at 30°C for 4-5 h and then filtered. The extract was concentrated to 1/5 of its volume with a rotary vacuum evaporator at 35°C for 8 h. The drying process of the extract was performed using a laboratory-scale spray dryer. Afterwards, dry extracts liquefied in the mixer at an adequate ratio. Microorganisms and growth conditions: The assay was carried out using the following bacterial species:  (Hobson, 1969) for C. perfringens and F. nucleatum. Medium 2 was prepared anaerobically according to Hobson (1969) with only slight modification. Trypticase peptone was used instead of casitone in medium 2 (Table 1). Ruminal fluid which was used as a component of the medium 2 brought from the slaughterhouse, mixed, and filtered through three layers of cheesecloth to partition into liquid and solid (digesta) fractions. The liquid fraction was centrifuged at 15000 rpm, and the clear supernatant was used as a component of the medium (Table 1). All strains were grown at 37 o C for 24 h under an atmosphere of 80% N 2 , 10% CO 2 , and 10% H 2 in an anaerobic cabinet (Whitley DG250, Don Whitley, West Yorkshire, UK).
already containing 200 µl of two-fold serially diluted M. sylvestris leaf extract in the bacterial strain specific growth media. Final concentrations of extract were kept at the ranges of 0.015-8 mg/mL. Each strain was tested in triplicate wells. Plates were incubated for 24 h at 37°C in the anaerobic cabinet. Bacterial growth was detected with a microplate reader at 600 nm (Epoch, BioTek, USA). The minimal inhibitory concentration (MIC) was the lowest concentration of the extract that allowed no visible growth. A significantly lower OD600 value compared to control dose (0 mg/mL) was accepted as potential inhibitory activity (Ko et al., 2018) while significantly higher value was accepted as stimulatory effect (Das et al., 2015).

Statistical analyses:
Statistical analysis was carried out by the use of one-way ANOVA followed by Dunnett's test. Each well of a 96-well plate was an experimental unit. A value of P<0.05 was taken to indicate a significant difference.

Results
Effects of M. sylvestris leaf extract on colon bacteria are presented in Figure 1 and Figure 2. Malva sylvestris leaf extract did not have inhibitory effect on

Discussion
Many species of bacteria have adapted to grow in the colonic lumen with concentrations up to 10 11 or 10 12 cells/g of luminal contents (Guarner and Malagelada, 2003). Of these bacterial groups, the bifidobacteria is one of the most important genera in the human and animal intestinal tract with its role in the fermentation of the complex carbohydrates (Crociani et al., 1994), producing vitamins, enhancing immunity, and inhibiting invasion of potential pathogens (Shen et al., 2011). Malva sylvestris leaf extract promoted the growth of Bifidobacterium species in a dose-dependent manner in the present study. The density of B. infantis nearly doubled in the high-dose groups. Same stimulatory effect was observed for L. acidophilus which is the other beneficial bacteria of the colon, which provides health-promoting effects (Shen et al., 2011). Malva sylvestris is rich in polysaccharides such as mucilages, which are particularly responsible for the therapeutic effects of the plant in the gastrointestinal disorders (Gasparetto et al., 2011). It is reported that the mucilage content of M. sylvestris leaves is 17.2% which is 2.3 times higher than in roots and flowers (Karawya et al., 1971). The mucilages consist mainly of glucuronic acid, galacturonic acid, galactose, rhamnose, glucose, sucrose, fructose, and trehalose, but uronic acid, fucose, mannose, arabinose, xylose, raffinose, and 2″-O-a-(4-O-methyl-a -d-glucuronosyl)-xylotriose have also been found (Gasparetto et al., 2011). All these sugars are harvested from mucin throughout the gastrointestinal tract by saccharolytic members of the colon microbiota such as bacteroides, bifidobacteria, and lactobacilli genera (Shen et al., 2011;Bäumler and Sperandio, 2016). These sugars are crucial for the metabolism of colon bacteria as vital carbon sources. Crociani et al. (1994) showed that 96% of B. bifidum strains are the only consumers of porcine gastric mucin among 290 bifidobacteria strains tested, and B. infantis was the only species that fermented Dglucuronic acid. Consequently, sugars in the mucilage content of the M. sylvestris leaf extract could be responsible from the stimulatory effects on the growth of beneficial bacteria except L. casei in the present study.
Escherichia coli, C. perfringens, and S. aureus can often cause foodborne infections and they are associated with gastroenteritis (Ørskov and Ørskov, 1992;Rajkovic, 2014). Malva sylvestris leaf extract exhibited a potential inhibitory activity against E. coli, C. perfringens, and S. aureus from 0.25, 2, and 4 mg/ mL concentrations respectively, however it did not inhibit these bacteria completely. Dowek et al. (2020) reported that methanolic extract of M. sylvestris leaves showed potential antimicrobial activities against S. aureus, a clinical isolate, as that recorded in this study and the zone of inhibition for S. aureus was almost 47.2% of the zone for positive control antibiotics. The rate of antibacterial effect was 30.6% relative to negative control for the highest dose in our study. Extract of M. sylvestris leaves also did not have an inhibitory activity on S. aureus (ATCC 25923) in another study (Azadpour et al., 2016). The extract prepared from the root, leaves, and flowers of M. sylvestris at 7.5 mg/mL dose exhibited antibacterial effect of 50% of positive control against a clinical isolate of S. aureus while the maximum antibacterial activity was recorded at 15 mg/mL that was almost twice the highest dose (8 mg/mL) used in this study (Walter et al., 2011). The findings about weak antibacterial activity of M. sylvestris leaf extract on E. coli in the present study are also in accordance with the results of the studies in which E. coli strainswithout intestinal isolate-was little affected by the methanolic extracts of M. sylvestris leaves (Dowek et al., 2020) and resistant to the methanolic extract of M. sylvestris aerial parts (Dulger and Gonuz, 2004). On the other hand, there is no report about the effects of M. sylvestris extract on C. perfringens. However, 1 mg/mL of the extract from the leaves of M. parviflora, Egyptian mallow, had a growth inhibition percentage of less than 1% relative to negative control on the same strain of C. perfringens used in this study (Omar et al., 2006). The inhibition percentage of the extract on C. perfringens was 4.8% relative to negative control in the present study (without statistically significant difference). However, the inhibition percentage of the extract increased to 67.1% at the highest dose of 8 mg/mL.
The other enteropathogen, F. nucleatum, is obviously associated with colorectal cancer and promotes the development of colorectal neoplasms (Shang and Liu, 2018). The growth of F. nucleatum was inhibited completely by 8 mg/mL of M. sylvestris leaf extract in the present study. The ethanolic extract of M. sylvestris leaves was also reported to inhibit the same strain of F. nucleatum at 1 mg/mL concentration in a previous study (Benso et al., 2015). Malva sylvestris leaves were purchased from a local farmer in the northeast Brazil in that study. Phytochemical studies revealed that leaves of M. sylvestris are rich in flavonoids and phenolic acids that dominated by luteolin and chlorogenic acid (Terninko et al., 2016) whose antimicrobial effects were reported (Lou et al., 2011;Qian et al., 2020). However, composition of the extracts can vary according to collection location, climatic conditions, soil characteristics, possible differences in the plant genotypes, harvest time, handling types, storage conditions, and extraction method (Fidan et al., 2019). Therefore, the difference between inhibitory concentrations in this and above study (Benso et. al., 2015) might be due to one or more of these factors.

Conclusion
As a conclusion, M. sylvestris leaf extract at 0.06-8 mg/mL dose could have favorable effects on colon bacteria since the extract selectively promoted the most of the beneficial species' growth at this dose range while it had a potential inhibitory or inhibitory effect on pathogenic ones. Investigating the effects of M. sylvestris leaf extract on other colon bacteria and testing the in vivo effectiveness will contribute to a better understanding of its efficacy on colon microbiota and intestinal health.