Evaluation of Antibacterial and Acute Oral Toxicity of Impatiens Tinctoria A. Rich Root

Background: Infections due to a variety of bacterial etiologic agents become common and are taking the big share of morbidity and mortality. On the other way, development of antibacterial drug resistance has been commonly reported from all over the world. As a solution of stated problems scientic studies have to be conducted on the traditional medicinal plants to develop new, effective and safe antimicrobial drugs since plants are important sources. Traditionally, the study plant (I. tinctoria A. Rich) used to treat fungal infections like ring worms that cause tinea pedis and it has also different medicinal values .These were some of the provoking information to undertake the evaluation of antibacterial activities with its oral acute toxicity study. Objectives: To evaluate the antibacterial activities and acute oral toxicity of aqueous, ethanol and ethyl acetate root extracts of Impatiens tinctoria A. Rich. Methods: The roots of Impatiens tinctoria A. Rich were extracted using solvents of water, ethanol and ethyl acetate. Agar well diffusion for preliminary antibacterial screening and agar dilution methods for determination of minimal inhibitory concentration were used. The minimum bactericidal concentration of the extracts was determined. by taking inoculums from all concentrations of the plant extract plates exhibiting invisible growth (from inhibition zone of minimal inhibitory concentration plates) and subcultures onto appropriate media plate. Finally, the plant extracts were subjected to oral acute toxicity study according to the organization of economic co-operation and development test Guidelines 420. Result: Gram positive bacteria were more susceptible to the extracts compared to gram negative bacteria especially against S. aureus and S. epidermis which are commonly found in the skin even though the traditional application is to control fungal infections and to toughen the skin. Ethyl acetate extract was more potent than ethanol and aqueous extracts. The LD50 was above 9600 mg/kg. Conclusion: This study provides scientic basis as the root of I.tinctoria A.Rich had a promising antibacterial activity in extract dependent manner in which ethyl acetate extract showed better potency. Therefore, the antibacterial potential and practically non toxicness of the study plant could take the attention of scientic for the development of new, effective and safe antimicrobial drugs by further studying the plant in different directions.


Introduction
Infectious diseases are the world leading cause of premature deaths, killing almost 13.4 million people per year. The World Health Organization (WHO) forecasts 13 million deaths attributed to this cause in 2050 (1,2). Infections due to a variety of bacterial etiologic agents become common and are taking the big share of the burden (1). Severe infections, including sepsis, meningitis, and pneumonia, are estimated to cause about a third of the 2.6 million neonatal deaths globally in which most of them are in less a uent regions of our planet (1). In Ethiopia, the top ve leading causes of premature mortality in 2015 were lower respiratory infections, tuberculosis, diarrheal disease, ischemic heart disease, and Human Immunode ciency Virus/Acquired Immune De ciency Syndrome that indicates the dominant fatality of the infectious diseases (3).
There is also an alarming increase in the incidence of new and reemerging infectious diseases of which some of them don't have drugs that act against them (4,5). For instance, over the past 40 years a minimum of 50 emerging infectious agents have been identi ed across the globe; approximately 10% of them are bacterial agents (5). Additionally, drug resistance has been commonly reported from all over the world (6). For example, the development of resistance to methicillin has decreased the usefulness of this antibiotic in treating serious staphylococcal infections within the community and hospitalized patients (6). Presently, approximately 60,000 people in Europe and United States die each year due to serious infections caused by antimicrobial resistant bacteria (7). The problem is also high in Ethiopia as indicated by few studies (8)(9)(10)(11).
In spite of such problems, medicinal plants have been used since ancient time to treat various diseases.
They are bases for most of traditional healing practices in which around 4.3 billion people of the world's population use herbal medicines for some aspect of primary healthcare (12). Surveys carried out in developed countries like Germany and Canada tend to show that no less than 70% of their population have used herbal remedies at least once that reaches 80% when we come to the emerging world (13).
Traditional remedies are the most important and sometimes the only source of therapeutics for nearly 80% of the Ethiopian population and 95% of the preparations are of plant origin (14).
As a solution of stated problems scienti c studies have to be conducted on the traditional medicinal plants to develop new, effective and safe antimicrobial drugs. Locally, Ethiopian women chop or mash the inside of the roots of I. tinctoria A. Rich in to a paste to dye the palms and nails of the hands and feet as a beauty treatment, to control fungal infections and to toughen the skin (15,16). The root decoction is also drunk against abdominal pains and as a purgative. The stem is chewed to treat mouth and throat diseases (17). In view of this, this study initiated to scienti cally justify the antimicrobial potential of this medicinal plant root extracts against selected bacteria. The acute oral toxicity evaluation was necessary to identify the range and concentration of dose that could be used and the possible clinical signs elicited by this medicinal plant.

Herbal material collection and preparation
The whole plant material of Impatiens tinctoria A. Rich was collected from Butajira, Southern Nations and Nationalities Region, Ethiopia and identi ed by a botanist. The collected root part washed with clean water, cut, and dried at room temperature, by using milling machine, the plant cutlets was milled to powder. The powder was weighted using electronic weighting balance and packed in polyethylene bags to avoid entrance of air and any other contaminant and stored in closed container with proper labeling for further extraction processes.

Extraction
The extraction was by mixing the root powder and extraction solvents (distle water,ethanol and ethyl acetate) with a proportion of 1 gram of powder and 20 ml solvents. After thoughly mixed macerated in rotary-shaker at 100 rpm for 24 hours and ltered through Whatman lter paper followed by concentration of it under reduced pressure (vacuum) by rotary evaporator at 40 °C to obtain the extract.
These concentrated extracts were kept in water bath set at 40 º C to avoid the remaining organic solvent and water (18,19).

Inoculums preparation
All strains were refreshed for the actual test with in Petri dishes containing nutrient agar, except S. pyogenes (ATCC 19615) and S. agalactiae (ATCC 12386) which were grown on 5% sheep blood nutrient agar, by incubation for 18-24hours at 37 °C. The grown bacteria few inoculums were harvested using 5 ml of nutrient broth, its absorbance was adjusted at 625 nm and diluted to attain viable cell count of 10 7 CFU/ml using spectrophotometer (20, 21).

Screening antimicrobial activity of the extract
Agar well diffusion method was used to evaluate the antimicrobial activity of the extract .The agar plate surface was inoculated by spreading a volume of the microbial inoculums, taken from adjusted suspensions, over the entire agar surface. Then, a hole with a diameter of 8 mm was punched aseptically with a sterile cork borer or a tip, and a volume (100 µl) of the antimicrobial agent or extract solution at desired concentration (100 mg/ml, 200 mg/ml and 400 mg/ml) was introduced into the well. Then, the plates were incubated under suitable conditions (at 37 °C for 18-24hours).The antimicrobial agent diffuses in the agar medium and inhibits the growth of the microbial strain tested. The presence of inhibition zones were measured by ruler and considered as indication for antimicrobial activity (22).

Minimum Inhibitory Concentration
All tested extracts were manipulated to determine their minimum inhibitory concentration (MIC) using agar dilution method by preparing different concentrations of extracts (from 64 mg/ml to 0.0625 mg/ml) through two-fold serial dilution. Then, incorporation of the prepared concentrations of the extracts into an agar medium (molten agar medium) followed by the inoculation of de ned microbial inoculums on to the agar plate surface. The incubation time is similar to the screening method. The MIC was considered as the lowest concentration which inhibits the growth of the respective bacteria under suitable incubation conditions was expressed in mg/ml (23).

Determination of Minimum Bactericidal Concentration
Streaks were taken from all concentrations of the plant extract plates exhibiting invisible growth (from inhibition zone of MIC plates) and subcultures onto appropriate media plate. The plates were incubated under suitable conditions depending upon the test microorganism (at 37 °C for 18-24hours).Then, examined for bacterial growth in corresponding to extract concentrations. Minimum Bactericidal Concentration (MBC) was taken as the concentration of plant extract that did not exhibiting any bacterial growth on the freshly inoculated agar plates. All assays were performed in triplicate (24).

Oral acute toxicity
Acute toxicity study was performed according to the organization of economic co-operation and development (OECD) test Guidelines 420 (Acute Oral toxicity-Fixed dose procedure) with slight modi cation. Healthy young adult, nulliparous and non pregnant female albino mice was used. The testing animals were randomly selected from 8-12 weeks old mice, marked to permit individual identi cation, and kept in their cages for 5 days prior to dosing to allow for acclimatization to the laboratory conditions. The animals were fasted 3-4hours (food withdrawn but not water) prior to dosing after which the animals weighed to determine the fasted body weight. Each animal, at the commencement of its dosing, was 25-33gm weight (25).
The starting dose was 300 mg/kg which was increased by bi-fold till 9600 mg/kg. Five animals were used for each dose. Treatment of animals at the next dose was delayed until assuring con dent of survival of the previously dosed animals. The extract was calculated according to the body weight and dissolved in a consideration of the administered volume not exceed 1 ml/100 g of mice body weight. Then, the solvent alone for control groups and dilueted extract for treated groups were administered with oral guabage. After administration each mouse was closely observed for the rst 30 minutes, hourly during the rst six hours, two hourly during the rst 24 hours, and daily for a total of 14 days. All observations like changes in breathing, alertness, restlessness, diarrhea, behavioral pattern, mortality and consumption of food and water were systematically recorded. Moreover, the change in body weight was measured at initial day and at 7 and 14 post treatment days (25).

Data analysis and interpretation
The extracted data was examined for its completeness and checked for consistency. Then, entered into excel spreadsheet, exported to Minitab 16 software and analyzed. The statistical differences of the antibacterial activity of crude extracts on each bacteria and the effect of each extracts on body weight of the albino mice were carried out by employing one way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. The experimental data was expressed as Mean ± Standard Deviation (SD). The result considered statistically signi cant at P < 0.05.

Antibacterial activity screening by well method
The antibacterial activity of aqueous, ethanol and ethyl acetate extracts of the roots of I.tinctoria A. Rich were screened against selected bacteria. A total of 13 bacteria by agar well diffusion assay were assessed at a concentration of 100, 200, 400 mg/ml for each extract in triplicates.

Antibacterial activity against gram positive bacteria
The average zone of inhibition formed by all tested concentrations of ethanol and ethyl acetate extracts against S. aureus was better compared to the aqueous extract, signi cantly different at P 0.05. Each extract has produced notable inhibition zone against MRSA at all concentrations. S. epidermidis produced the largest average zone of inhibition when compared to the other tested gram positive bacteria in which the inhibition zone against this bacterium ranges from 38.0 ± 1.0 mm by 100 mg/ml aqueous extract to 43.7 ± 1.5 mm by 400 mg/ml ethyl acetate extract. Comparisons of the mean growth inhibition zones for S. aureus at 100, 200 and 400 mg/ml concentrations of the three tested extracts showed no signi cant differences (P < 0.05). It indicates absence of concentration dependent inhibition difference which was also observed on MRSA and S. epidermidis.
All extracts showed less inhibition zone against the two Streptococci bacteria (S.pyogen and S.agalactiae) when compared with other tested gram positive bacteria with each corresponding concentration. Erythromycin (15 µg) showed better inhibition diameter against the two tested streptococci than the three extracts at all concentrations that were signi cantly different at p 0.05. In extract type dependent manner, better inhibition against S. pyogen and S. agalactiae were obtained by ethyl acetate extract followed with ethanol extract. The least inhibition diameter 17 ± 0.0 mm at 100 mg/ml aqueous extract against S.agalactiae was recorded when compared with all tested gram positive bacteria.
The three extracts showed better inhibition on 400 mg/ml than 100 mg/ml and 200 mg/ml concentrations to E. faecalis with a signi cant difference at P 0.05. The effect of vancomycin (30 µg) on this bacterium revealed less inhibition to all extracts and doses, with a signi cant difference at P 0.05, except the aqueous extract at 100 mg/ml concentration. Similarly, erythromycin (15 µg) that produced 21.3 ± 2.3 mm inhibition zone was greater than the inhibition of aqueous extract at 100 mg/ml concentration and lesser than or equal to other extracts at all concentrations. The potency of almost all extracts on this bacterium was better than S. pyogenes and S. agalactiae, but lesser than S. epidermidis, S. aureus and MRSA (Table 1).   The positive control, cipro oxacin (5 µg/ml), showed signi cantly higher inhibition of the growth of all gram-negative bacteria compared to all tested extracts at all concentration (P < 0.05). On the other hand, among the extract type, aqueous extract has showed signi cantly lower inhibition activity against the tested gram-negative bacteria (P < 0.05). Moreover, no statistically signi cant different inhibitions were observed against E. coli, S. typhimurium, P. aeroginosa, K. pneumoniae and P. mirabilus at a concentration of 100 mg/ml of aqueous extract compared to the treatment of negative control (distilled water and 5% tween 80) (P < 0.05). Table 2 showed the inhibition zone diameter and association of the total seven tested gram negative bacteria were used to assess the antibacterial activity of the extracts.   (Table 3).  Key notes: n = 5 ,"present" means at least 1 out of the 5 mice showed the symptom, "not seen/normal" means no mice showed the symptom .

Body Weight
A weekly body weight has weighed on initial day, 7th and 14th days of the six groups as displayed on Table 5. At these days all treated groups didn't show any statistically signi cant difference changes in the body weight compared with control groups (p < 0.05). Key notes: Values are expressed as Mean ± SD (n = 5) one-way ANOVA followed by Tukey's multiple comparison tests (P 0.05), "Means" that do not share a superscript letter are signi cantly different (only column wise).

Antibacterial activity
The inhibition zone of the most susceptible bacteria (S. aureus, S. epidermis and MRSA) in the well method assay did not show any signi cant difference (p < 0.05) at the tested concentration of 100 mg/ml, 200 mg/ml and 400 mg/ml for most tested extracts. Therefore, these bacteria have resulted with similar susceptibility at both the lowest and highest tested extract concentration in this method.
These observations could possibly be explained as the effect of these concentrations might be the maximal e cacy portion on the dose-response curve where as the steepest portion might be below 100 mg/ml concentration with the assumption of the dose-response curve is sigmoidal curve. From this the minimum effective dose might be ≤ 100 mg/ml. The remaining tested gram positive bacteria S. pyogenes, S. agalactiae and E. faecalis also did not show any signi cance difference inhibition at 200 mg/ml and 400 mg/ml concentration for ethanol and ethyl acetate extracts which was a similar scenario with most of gram negative bacteria.
The antibacterial activity of the extracts against MRSA resulted with a highest inhibition zone, lowest MIC and MBC value of 35.7 ± 1.2 mm, 4 mg/ml and 8 mg/ml, respectively. All extracts at all concentrations (100 mg/ml, 200 mg/ml and 400 mg/ml) showed a better antibacterial activity than vancomycin (30 µg) on the well method with a statistically signi cant difference at (p < 0.05). This might be due to the ability of the extracts to inhibit penicillin-binding proteins of the bacteria that are involved in the synthesis of peptidoglycan which is impossible by the antibiotic methicillin. Therefore, it could be a good alternative as a natural product, as we are now in a situation where, in some cases, the glycopeptides antibiotic vancomycin, is the only option for antimicrobial therapy even its non susceptibility in S. aureus is on the increase (6,26).
The well method zone of inhibition was in line with the MBC and MIC value concentration for most of the tested microorganisms except S. pyogenes and S. agalactiae that might suggest the consistency of the testing methods. The inconsistency of the two organisms might be due to the usage of 5% sheep blood muller-hinton agar. The sheep blood might in some extent decrease the looseness of the media that lead a weak diffusion of extracts than the pure muller-hinton agar that used for other bacteria. On the other way, these two bacteria might be susceptible for large molecules or hydrophobic molecules of the extracts constituents which did not diffuse easily as other studies support it (27). These might be the reasons that the two organism record better MIC and MBC value than those bacteria that had longer inhibition zone than them. For instance S. agalactiae and E. faecalis on 400 mg/ml ethyl acetate extract showed inhibition zone of 24.0 ± 1.0 mm and 29.7 ± 1.5 mm,respectively(signi cantly different at p < 0.05). This value was inversed as S. agalactiae records 2 mg/ml and 8 mg/ml where as E. faecalis records 8 mg/ml and 16 mg/ml of MIC and MBC value, respectively.
As observed from the inhibition zone, MIC and MBC value of the extracts the study plant also showed antibacterial activity against gram negative bacteria in extraction solvent dependent manner. Of the extracts ethyl acetate extract showed better antibacterial activity against all gram negative bacteria. For example, K.peumoniae and P.aeroginosa had > 64 mg/ml of both MIC and MBC on water and ethanol extracts where as ethyl acetate extract had 16 mg/ml MIC and 32 mg/ml MBC which was a great difference in between. This notable better e cacy of ethyl acetate extract supported by other previous studies on plant extracts (28)(29)(30). Thus, of the extracts ethyl acetate extracts might has a better penetration ability of the outer membrane of gram negative bacteria and disturbing cellular function, metabolism, and loss of cellular constituents, leading their inhibition and death of the bacteria.
It has been found that the gram positive bacteria were more susceptible to the extracts compared to gram negative bacteria. Many other studies on different medicinal plants also revealed as gram positive bacteria tend to be more sensitive to the antimicrobial properties of plant extracts than gram negative bacteria (31)(32)(33)(34). These could be due to gram negative bacteria have an outer membrane that is composed of high density lipopolysaccharides that serves as a barrier to many environmental exposures including antibiotics (35).
In addition, this study con rms as the roots of I. tinctoria A. Rich had also a promising antibacterial activity especially against S. aureus and S. epidermis which are commonly found in the skin even though the traditional application is to control fungal infections and to toughen the skin (15,16). Hence, locally dying of skin, applying on cloths and different materials might prevent infection transmission of Staphylococci (S. aureus, MRSA and S. epidermidis), the most abundant skin-colonizing (bio lm forming) bacteria and the most important causes of community associated and hospital acquired skin infections (36-38).

Acute toxicity
The evaluation of the toxic characteristics is usually a preliminary step in screening medicinal plants for pharmacological activity. But, there is a lack of scienti c validation on the toxicity and adverse effects of medicinal plants. Therefore, scienti c knowledge towards acute oral toxicity study is much needed since it helps to identify the dose that could be used subsequently and to reveal the possible clinical signs elicited by these medicinal plants under investigation. In addition, in order to increase the con dence on medicinal plants or preparations safety to human being the data of toxicity studies should be obtained (39).
The oral acute toxicity study of the tested plant extracts was carried out on albino mice at a single dose of 300, 600,1200,2400,4800 and 9600 mg/kg body weight and was continuously monitored for rst 4 hours, followed for a period of 14 days daily for any toxic effect after the treatment period. Major changes in behavior and mortality were not observed in all groups. However, drowsiness and erection of fur were observed in each mouse of treated groups of 4800 and 9600 mg/kg body weight. These signs were disappeared after the 4th hours almost among all of the mice that showed the symptom. The extract seems to be safe at a dose level of 9600 mg/kg, and the LD 50 is considered be > 9600 mg/kg.
According to Hodge and sterner toxicity classi cation the root extract of I. tinctoria A. Rich is classi ed at least as practically non toxic herbal medicine as LD 50 between 5000 to 15000 mg/kg is practically non toxic according to this classi cation (40).
The body weight of each mouse was carefully weighed at rst day, 7th day and on the day of sacri ce.
The body weights of tested animals of both control and treated groups were increased progressively throughout the study period though it was not statistically signi cant changes (p < 0.05). The body weight changes serve as a sensitive indication of general health status of animals (41). Therefore, the normal increment in body weight and the zero death report could give con dence to state roots of I. tinctoria A. Rich did not interfere with the normal metabolism of animals.

Conclusion
This study provides scienti c basis as the root of I.tinctoria A.Rich had a promising antibacterial activity in extract dependent manner in which ethyl acetate extract showed better potency. Gram positive bacteria especially S.aureus and S.epidermidis were more susceptible to the extracts compared to gram negative bacteria. On the other way, the acute oral toxicity study of the aqueous extracts of the plant were appeared to be safe up to the maximum tested dose that classify I.tinctoria A.Rich at least with in practically non toxic category. Therefore, the antibacterial potential and practically non toxicness of the study plant can take the attention of scienti c communities for the development of new, effective and safe antimicrobial drugs by further studying the plant in different directions.

Declarations
Ethics approval and consent to participate