4.1. 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 significant 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 efficacy 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 significance 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 significant 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(significantly 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 efficacy of ethyl acetate extract supported by other previous studies on plant extracts (28–30). 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 (31–34). 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 confirms 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 (biofilm forming) bacteria and the most important causes of community associated and hospital acquired skin infections (36–38).
4.2. 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 scientific validation on the toxicity and adverse effects of medicinal plants. Therefore, scientific 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 confidence 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 first 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 LD50 is considered be > 9600 mg/kg. According to Hodge and sterner toxicity classification the root extract of I. tinctoria A. Rich is classified at least as practically non toxic herbal medicine as LD50 between 5000 to 15000 mg/kg is practically non toxic according to this classification (40).
The body weight of each mouse was carefully weighed at first day, 7th day and on the day of sacrifice. The body weights of tested animals of both control and treated groups were increased progressively throughout the study period though it was not statistically significant 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 confidence to state roots of I. tinctoria A. Rich did not interfere with the normal metabolism of animals.