Antibacterial properties of traditional Sudanese medicinal materials against selected enteric bacterial strains

Folklore medicine in Sudan used medicinal materials to treat inestinal infections caused by bacterial infections or contamination of food. Methanolic and aqueous extracts of different parts of Acacia nilotica (L.) Delile gum, Haplophyllum tuberculatum Juss.aerial parts, Hydnora abyssinica A. Br.fruits, Nigella sativa L seeds, Rhynchosia minima (L.) DC. roots,and Usnea molliuscula lichen were tested for antibacterial properties at a concentration of 100 mg/mL against 20 intestinal isolates including Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Salmonella typhi, Salmonella para typhi B,Staphylococcus aureus, and standard bacterial strains (Bacillus subtilis (NCTC 8236), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 35657), Salmonella typhi (ATCC 1319106) and Staphylococcus aureus (ATCC 25923), using the Agar Diffusion Method. Standard antibiotics were used as standards drug for antibacterial effect. The highest enhancing properties were observed in methanol extracts and the lowest in aqueuos extract. U. molliuscula methanolic extract was the most active among all tested plant extracts, while, the aqueous extract of H. tuberculatum had a promising level of efficacy among the aqueous extracts tested. Most responsive Gram-negative clinical isolates bacteria were S. para typhi B and P. aeruginosa. Most susceptible standard bacteria were B. subtilis (NCTC 8236). Obtained results from investigated plants confirm their antibacterial potential and usefulness in the treatment of intestinal infections.


INTRODUCTION
The increasing incidence of antibiotic resistance has been increasing throughout the world in the last few decades (Abdallah, 2011).This has led to higher mortality in humans. Consequently, one of the most intensive researched fields is the search for material with high antibacterial potency, with folklore medicine being a prime area.In this regard Sudan has a long history of traditional use of plants to treat primry health issues.However, very little research is directed towards understanding their potential for curing gastrointestinal E-mail: sanatayyab@uhb.edu.sa.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License tract infections,caused by various bacteria. Spread and prevelence of microbial resistance is geting more frequent worldwide (WHO, 2001). Search for new antimicrobial substances is the major weapon to combat the microbial resistance through developing new antibacterial materials to substitute with inefficient ones.
Combination of native cultures and different traditions are factors formed by Sudanese traditonal medicine.The extremely large diversity of plants in the area, different cultures due to the variation of climatic zones, and the distinctive geography created Sudanese herbal medicine. 11% of the population has access to prescribed health care.Therefore, research on the best pharmacological influence and possible undesirable side effects or toxicity is essential to enhance potency and protection of Sudanese folklore medicine .
Ethnobotanical survey in the Blue Nile State, Southeastern Sudan of medicinal materials used by folklore healers was carried out. Fifty three plant species within 31 families and 47 genera were detected as being used to treat one or more diseases. The most commonly mentioned manifestations were gastro intestinal tract disorders, diseases/infestations, stiffness, and respiratory tract disorders (Musa et al., 2011).
Traditional medicine in Sudan still has the most rational source of therapy of several ailments and bacterial infections.Traditional medicine is distinguished by a special fusion of Islamic, Arabic, and African tradition. Sudanese folk plants have been reported to be characterized with a wide range of folk medicinal uses including different bacterial infections and digestive system disorders (Karar and Kuhnert, 2017). Different extracts of Usnea revealed a variable effect of antibacterial properties against S. aureus, E. coli, V. cholerae, S. dysenteriae and S. flexneris.The methanol extracts were the most active against tested bacteria; all tested organisms showed no antibacterial susceptibility against aqueous extracts of tested lichens (Sinha and Biswas, 2011). U.molliuscula extract showed high antimicrobial potential against all tested Gram (+ve) bacteria inclusive penicillin-resistant S aureus and methicillin-resistant S.aureus (Weckesser et al., 2007). Studies revealed the effects of N.sativa seed extracts have dose dependent antibacterial activities on the tested organisms (Hosseinzadeh et al., 2007;Chaudhry and Tariq, 2008;Yoruk et al., 2010;Mishra, 2011;Pichette et al., 2011;Haloci et al., 2012;Monika et al., 2013).
H. tuberculatum showed antimicrobial evaluation against a variety of strains, revealed moderate effect against B. subtilis, S. choleraesuis and E. coli, gentamycin sulphate; it has 75% potency as antibacterial agent on S.aureus and E.coli. It is the most potent inhibitor against plant pathogenic bacteria and fungi (Al-Burtamani et al., 2005;Sabry et al., 2016;Abdelgaleil et al., 2020). Available literature shows that no earlier study has been performed on the antibacterial characters of A.nilotica gum until 2013.Study of methanolic extract of A. nilotica revealed moderate antibacterial effect (14-18 mm) against E. coli (ATCC 25922);aqueous extracts of A. nilotica had no effects of antibacterial characters against all tested bacteria (Mahjoub, 2013). Studies of antimicrobial evaluation of different extracts of A.nilotica revealed high (˃18 mm) to modrate (14-18 mm) activity for different extracts which consider a good source of natural antibiotic for the therapy of different transmittable diseases (Sravani et al.,2014;Banjar et al., 2017;Al Alawi et al., 2018;Shehu et al., 2018;Ali et al., 2020). Ethanolic extracts of 8 species of R.minima were investigated for their antibacterial activity and phytochemical screening against clinical isolates (B.subtilis,E.coli, P.aeruginosa and S.aureus); they showed equal or nearly equal antibacterial characters against all tested bacteria (El-Kamali and El-Amir, 2010).The essential oils of R.minima exhibited antioxidant and antimicrobial activities against E. coli, S aureus and C. albicans, but not active against C. perfringens and K. pneomoniae (Gundidza et al., 2009). Methanolic extract of H.abyssinica showed moderate antimicrobial potential. Analysis there are tanins and phenols in the plant root extracts (Saadabi and Ayoub, 2009).
A research was done to determine the antibacterial potential of six medicinal materials U. molliuscula lichen, N .sativa seeds, H. tuberculatum aerial parts, A. nilotica gum, R. minima roots and H.abyssinica fruits. They were obtained from Omdurman market on the origin of antimicrobial potentials with documented details for antibacterial assay against bacterial strains associated with intestinal infections. These investigations determine antibacterial potential efficacy of selected medicinal plants.This selection was guided in the first place by ethnobotanical claim in traditional medicine suggestive of their antibacterial activity and secondly by defficiency or insufficiency of information in published works on antibacterial and antioxidants potency of their extracts. This will provide baseline data for developing new antibacterial compounds in the treatment of intestinal infections, based on their folklore use.

MATERIALS AND METHODS
Plants' taxonomy and authentication were established by Prof.Hatil H.Elkamali via differentiation with herbs specimens of Botany Department, Faculty of Science and Technology, Omdurman Islamic University during the spring season of 2012. The plants wew dried in the shade for three weeks.The air-dried plants' parts were crushed and turned into coarse powder. It was further reduced to powder using a mechanical grinder.
Two hundred grams of all air-dried plants were soaked for 24 h with 50% methanol (MeOH) in a round bottomed flask. Liquor (crude extract) was filtered with whatman grade1qualitative filter papers and then rotor-evaporated.The filtrate was dried at room temperature.Then the dried extracts were sterilized and kept in airtight containers at room temperature until used for further tests. At the time of testing for antibacterial activities extracts were prepared at a concentration of 100 mg/mL in methanol. One hundred g of dried plant material was crushed coarsely into powder, which was further reduced to powder using a mechanical grinder.Then it was dissolved with purified water (1L), and left for 24 h at room temperature.With whatman grade 1 qualitative filter papers the mother liquor was filtered.Thus, the aqueous extract (10%) was obtained.
The antibacterial testing was investigated by well-agar diffusion technque (Cheesbrough, 1984). Two hundard and 50 millilitres of decontaminate nutrient agar (Oxoid) were used for antibacterial testing.Suspension of 106 cells was monitored in the inoculum size of each test bacteria.Two millilitres of the inoculum suspention obtained from 24 h cultures of bacteria were supplemented with 250 ml of nutrient agar (Oxoid) and then mixed; they were inoculated on soft agar (20 mL) and flowed on 10 cm diameter sterilized petri dishes and agar plates and allowed to solidify. After solidifying, a sterile cooled-flamed cork borer using four wells (10 mm in diameter) was bored in the agar and the agar discs were removed. One hundred microliters (100 µl) of each extract solution to each plant extract was added to each well with a pipette and the plate was held for 2 h at room temperature for diffusion of extract into agar. The plates were incubated at 37°C for 24 h. Result of tested plants were evaluated to test their antibacterial activities and expressed as the diameters of the inhibition zones which were calculated as the adjacent mm. Methanol serves as negative control. Axiom laboratories, New Delhi 1100055 Multidisc was used for antimicrobial susceptibility testing of tested clinical isolates (4 E. coli, 5 P. aeruginosa, 5 P. mirabilis, 3 S. aureus, 2 S. typhi, and 1 S. para typhi B ).
Co-Trimoxazole (BA) at concentration of 25 mcg showed promising effect (1Z=24 mm) against S.para typhi B no. (17), and moderate effect (14-18 mm) (1Z=16-14 mm) against E.coli no.(9), P.aeruginos no.(15) and S. aureus no.(14) ( Table 2). P. mirabilis no. (3) exhibits antibiotic-resistant (<14 mm) to BA and showed a high degree (˃18 mm) of sensitivity (1Z=18 mm) to the methanolic extracts of U. molliuscula. P. mirabilis has the ability to cause various human diseases; it is primarily corresponding with urinary system disorders and is a major health concern due to its complications and frequent recurrence. Usnic acid rich in phenols is accountable for the antioxidant activities of U.molliuscula methanolic extract; it possesses strong antioxidant activities against different antioxidant systems in vitro. It is considered as the source of natural antioxidants. It can be used simply as possible food complement, and in pharmaceutical implementations (Elkamali and Mahjoub, 2015). B.subtilis (NCTC 8236) showed promsing (˃18 mm) degree of sensitivity (1Z =24mm) to methanolic extracts of U.molliuscula, which exhibit antibiotic-resistant to Co-Trimoxazole and Ceftizoxim; same goes to Piperacillin/Tazobactam, Chloramphenicol and Ciprofloxacin. Our results support that the traditional therapeutic information for U.molliuscula, in near future, can surely change the traditional antimicrobial agents to which there is increased occurrence of drug interactions. The study recommends that U.molliuscula is promising for increasing phytomedicines with antibacterial potential. More studies on the selection and distinguishing of active concepts and assessing possible synergism among extract components for their antimicrobial potentiels depending on the main results obtained might be considered enough (Ali et al., 2012).

Antibiotics
Ampicillin/ sulbactam (20 mcg) sensitivity (1Z = 20-18 mm) to the methanolic extracts of U. molliuscula. The appearance and rapid development of antibiotic resistance by infectious bacterial isolates are critical threats to the international public health; it leads to a significant threat to public health worldwide due to the bounded therapy options and unconcerned discovery of new types of antibiotics (Trojan et al., 2016).Incorporating sensitivity data within study may be worth it in fulfilling treatment strategies for diseases.P.mirabilis no. (16) showed antibiotic-resistant to synthetic antibiotics and showed good (14-18 mm) degree of sensitivity (1Z=14 mm) to the methanolic extracts of U. molliuscula. S. para typhi B no. (17) showed promising degree of sensitivity (1Z=20 mm) to the methanolic extracts of U.molliuscula same extent as to OF. Plant chemical constituents as alkaloids, flavonoids, tannins,and phenolic compounds work as protection mechanisms contrary to predation by many microbs,insects and herbivores. Flavonoids have antibacterial potential through their capability to complex with extracellular and disolved proteins and to complex with bacterial outer cell walls (Vijayasanthi et al., 2012). E.coli no.(6) exhibited antibiotic-resistant to OF and showed a good (14-18 mm) degree of sensitivity (1Z=16 mm) to the methanolic extracts of H.tuberculatum same extent as to Piperacillin/Tazobactam. P.aeruginosa no. (7) showed fairly high effect (1Z = 18 mm) against H.tuberculatum methanolic extracts in the same extent as to OF.Phytochemical constituents of H tuberculatum revealed polyphenolic compound as resveratrol,myricetin and quercetin flavonol kaempferol and rutinand rosmarinic acid (Abdelkhalek et al.,2012). Evidence indicated that bioactive substances (antioxidants) possess extranutritional charateristics and advanced role in fooddisease association.Radical scavenging activity under physiological conditions needs additional research to show and to determine whether there is any link between their radical scavenging properties and their antimicrobial potential (Ramadan et al., 2003).

DISCUSSION
U. molliuscula methanolic extracts revealed high (˃18 mm) antibacterial effect (1Z = 20 -18 mm) against P.aeruginosa, S.aureus, and P.mirabilis. There is need for more research on the isolation and recognition of active ingradients and to assess the probable synergism between extract constituents for their antibacterial activities based on the results obtained (Ali et al., 2012). P. aeruginosa, S. aureus, and P. mirabilis showed antibiotic-resistant against U.molliuscula extracts. There is need to discover and develop new antimicrobial medications for the treatment of diarrhoea and other bacterial infections due to P.mirabilis and E. coli through support scientific base and inclusion of traditional practices in present system of medicines.
Only one of all S. aureus clinical isolates was more Mahjoub 561 sensitive and effective against methanolic extract of U.molliuscula than Gram (-ve) antibiotics (Tables 2 and  3). Lichen metabolite usnic acid exhibits antimicrobial activity against plant and man infectious microorganism, including antimicrobial efficacy against antibiotic-resistant bacterial strains (Ingólfsdóttr, 2002). Aqueous extract of U. molliuscula lichen did not exhibit antibacterial properties against all tested bacteria.The obtained results may account for the reason people in Sudan refuse to use aqueous extract countiniously as a treatment of gastrointestinal tract. It is speculated that it may possess anti-inflammatory activities, where it is used in Sudan as a bitter stomachic, for cough and also by women to relive menstrual pain (Kheir, 1966). Nigella sativa methanolic extract showed moderate (1Z=14-18 mm) potency (1Z=16 mm) against S.para typhi B (Table 1).More and more beneficial effects of N.sativa should be explored in order to maximize its utility in effective treatment and cure for various diseases.A single strain of S. typhi B clinical isolates was resistant to most of the plant extracts analyzed.This bacterial species is of importance in gastrointestinal tract infections and deserves a wider investigation, including a large number of strains.Aqueous extract did not exhibit antibacterial potentials against all tested bacteria.Thymoquinon possesses anti-inflammatory activities,protects the cell membrane integrity through inhibition of lipid peroxidationm and alters levels of leukotrienes and prostaglandins favoring cytoprotection of the gastric mucosal cells (Kanter et al., 2005).Scavenging activity of the free radical ions was increased due to effectiveness of human serum albumin (HSA) isoforms ('N' form at pH 7.4 and 'B' form at pH 9.0) in the presence of Thymoquinone (TQ), the main constituent in N. sativa (Ishtikhar et al., 2015).
All plant aqueous extracts except H. tuberculatum do not possess significant antibacterial efficacy against tested bacteria.Phytochemical investigation revealed the presence of Terpenes and β-phellandrene,limonene, βocimene, β-caryphyllene,myrcene, and α-phellandrene, the most rich oil components (Al-Burtamani et al., 2005).It is speculated that they may possess anti-inflammatory characteristics. The potential therapeutic assessment of the medicinal plants has been the subject of continual research for their anti-inflammatory componants, including the terpenes which have pharmacological actions (Souza et al., 2014). H. tuberculatum aqueous extract showed moderate (1Z=14-18 mm) potency (1Z=18-16 mm) against clinical isolates E. coli and P. aeruginosa. Antibacterial activity of the extract showed promising result against P. aeruginosa and E. coli clinical isolates, the most common one and problematic among opportunistic pathogens.Resistance to antimicrobial drugs may be a problem. The use of an appropriate combination therapy is important. This plant plays vital role in man health, possesses different pharmacological properties and bioactive materials; therefore, it might participate in various drug productions. Its cultivation is very important.
U. molliuscula, H.tuberculatum, and N.sativa extracts show positive microbial potency. These plants may be considered as important sources for new antimicrobial drugs. Therefore they will contribute to the devolopment of new methods to treat infectous diseases and intestinal disorders caused by some pathogenic strains.U molliuscula in folk medicine is used widely for the treatment of different diseases,but scientifically few of them have been invetigated .Thus more scientific research should be harmonized to investigate unutilized activities. Thymoquinone is a phytochemical compound found in the plant N.sativa; it was found to be effective against Gram -positive bacteria (Kokoska et al., 2008). Depending on doses the oil showed antibacterial activity against all tested bacteria (Salman et al., 2008). Thymoquinone and Thymohydroquinone may be used for the treatment of infections alone or in combination with some antibiotics, especially in case of highly susceptible Gram (+ve) bacteria S.aureus (Halawani, 2009).More and more beneficial effects of N. sativa should be explored in order to maximize its utility for effective treatment and curing of various diseases (Naz, 2011).
Methanolic extract of A. nilotica, R. minima, H. abyssinica and aqueous extracts of C. phelypaea did not exhibit antibacterial properties against all tested bacteria except A. nilotica against E. coli (ATCC 25922) (Tables 1).It is speculated that it may possess anti-inflammatory activities. The antioxidant activity of H. abyssinica aqueous extract and A. nilotica extracts reported by Mahjoub (2013) revealed that the solvent extracts exhibited strong to modrate antioxidant activity as compared to other plants .Aqueous and methanolic extracts of R. minima showed the presence of alkaloids, flavonoids, tannins, trepenoids, glycoside and steroid were absent (Mali and Mahale, 2008). In recent years, the trend towards natural products that are considered as antioxidant, antimicrobial, anti-inflammatory and similar agents is rapidly increasing in the prevention and treatment of diseases. Accordingly, various speculations on natural products could arise and lead to information on pollution (Sevindik et al., 2017).

Conclusion
The results demonstrate that the extremely active plant was U. molliuscula. Methanolic extracts of U. molliuscula of all extracts possessed significant antibacterial efficacy. All the plants' aqueous extracts did not clearly show antibacterial activities against all tested bacteria except H. tuberculatum. U. molliuscula showed high antibacterial activity against P. aeruginosa, S. aureus, P.mirabilis and S.para typhi, B clinical isolates; also B. subtilis (NCTC 8236) showed high sensitivity (1Z = 4mm) to methanolic extracts of U.molliuscula . All plants' aqueous extracts were less effective against the bacterial growth of all tested Bacteria except H. tuberculatum; they showed high effect against P.aeruginosa and moderate effect against E.coli clinical isolates.Aqueous extract of U.molliuscula, N. sativa, A. nilotica. R. minima, and H. abyssinica were found to be ineffective against all tested bacteria. Studied medicinal materials of U. molliuscula were satisfactory on the basis of their antibacterial properties. An adequate toxicological testing must be carried out to confirm the capability of using these plants to fight against infectious dieases.

CONFLICT OF INTERESTS
The author has not declared any conflict of interests.