Biomedical Research

Abstract

Alkanna is a traditional medicinal plant; Bedouin of Saudi Arabia use the roots of the plant for the treatment of many skin diseases. Water and ethanol extracts of Alkanna roots were evaluated for their antibacterial activity against four pathogenic bacteria responsible for skin infections i.e. Pseudomonas areuginosa ATCC 27853, Bacillus subtilis (clinical strain), Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25928. The agar well diffusion method was employed for the antibacterial screening of crude extracts. Ethanol extract inhibited the growth of all bacteria, as depicted by the zone of inhibition of bacteria around the well filled with extract. The maximum zone of inhibition was observed against B. subtilis (28.0 mm) followed by S. aureus (24.7 mm), P. areuginosa (18.7 mm) and E. coli (15.3 mm). The water extract showed minor antibacterial activity against B. subtilis and S. aureus however, it was found ineffective against other two pathogens. The streak method was used to determine the Minimum Inhibitory Concentration (MIC). The MIC value of ethanol extract against B. subtilis and S. aureus was 2 μl/ml, whereas, against E. coli and P. areuginosa it was 4 μl/ml. The qualitative analysis of the ethanol extract was conducted by the GC-MS. The major compounds detected in the ethanolic extract of Alkanna were akanin, acetyl-alkanin, ethane, 1, 1-dichloro-, 1H-enzotriazole, 4-nitro, 2- chloroethyl (methylsulfonyl) methanesulfonate and 2, 5-cyclohexadien-1-one, 4-diazo-. Besides that several other napthoqinones and esters were detected in the extract. Results of present study manifest that extract of Alkanna has the potential to inhibit the growth of pathogens responsible for skin infections.

Keywords

GC-MS analysis, Alkanna tinctoria, Antimicrobial activity, Pathogenic bacteria, Skin infection.

Introduction

Natural products are a major source of discovery and development of new medicines due to the chemical diversity exhibited by plants. Hundreds of plant metabolites are reported to have many pharmacological activities [1]. Major population of the world still depends upon the herbal treatment of common ailments. With the increase in the dependency on the herbal medicines, scientists are paying more attention on exploring the medicinal plants for isolating bioactive compounds and developing new medicines. Members of genus Alkanna are known for medicinal and other properties from the ancient time. As evident, Hippocrates documented the use of alkanet root for the skin ulcers treatment. Similarly, Theophrastus and Dioscorides described the properties of Alkanna [2]. Traditionally, alkanet, are used for treating ulcers, inflammation and wounds. It is also used in dying cloth, colouring fats, cosmetics and food [2,3]. Naphthoquinone pigments, alkannin and its derivatives have been detected in the extracts of alkanet [4,5]. Several of these compounds are recorded for cytotoxic, antimicrobial, anti-leishmanial and antiinflammatory activities [6-9]. The ointments, helixderm and histoplastin red contain alkannin as the active ingredient [5].

Bedouin of Saudi Arabia still use Alkanna roots for treating burns, skin wounds and other skin diseases [10]. Streptococcus and Staphylococcus are the common pathogens of skin infection. After burn injury diverse microorganisms such as gram positive and gram negative bacteria and yeast colonizes the burn wounds. Presently S. aureus and P. areuginosa are the most prevalent source of burn wound infection and are of great concern due to the development of resistant strains [11]. B. subtilis a ubiquitous bacterium is found on skin and also in epithelial wounds [12]. To find out the role of Alkanna in treating skin infections, the antibacterial activity of crude extract of A. tinctoria roots and the chemical composition of crude extract of Alkanna by GC-MS analysis was conducted.

Materials and Methods

Microorganisms

Four human pathogenic bacteria responsible for skin infection viz. Pseudomonas areuginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25928, Bacillus subtilis (clinical strain) were procured from the King Khalid Hospital, King Saud University, Riyadh, Saudi Arabia. Stock cultures of bacterial strains were maintained on the slants of nutrient agar, all stock cultures were stored at 4°C.

Collection of Alkanna roots and preparation of crude extracts

The roots of Alkanna were purchased from the local market of Riyadh, Saudi Arabia. For the preparation of water extract, dried powder of the sample (20 g) with 100 ml of distilled water was kept on water bath for 4 h at 45°C. After that solution was filtered by Whatman filter paper no.1 and the filtrate so obtained was used for the study. The ethanolic extract was prepared with help of Soxhlet extractor. Dried powder of the sample (20 g) and 96% ethanol (100 ml) were placed in the extraction tube and the apparatus was run for 24 h to get the extract. The liquid extract was evaporated to dryness in the oven at 50°C. The dried residue was scraped and dissolved in 96% ethanol to make the final volume of 2 ml. The so obtained crude extracts were used for further study.

Determination of antibacterial activity by agar well diffusion method

Antibacterial assay: The agar well diffusion method was employed for the antibacterial screening of crude extracts [13]. Fresh culture of bacteria to be tested were inoculated in 10 ml aliquots of nutrients broth and incubated at 37°C for 24 h. After required incubation the cultured broth was adjusted to a turbidity of 0.5 Mac Farland standards (10⁸ CFU/ml). From the quantified broth, 10 μl bacterial culture was evenly spread over the entire surface of the nutrient agar plate. On the agar plate 4 wells/plate were made with the help sterilised cork borer and each well was filled with 50 μl crude extract, ethanol solvent was used as the negative control. Gentamycin (30 μg) was used for determination of susceptibility of tested pathogen to antibiotic. The plates were incubated at 37°C for 24 h. After 24 h, the antibacterial activity was determined from the size of the diameter (mm) of clear zone of surrounding the well.

Determination of minimum inhibitory concentration (MIC): The streak method was used to determine the Minimum Inhibitory Concentration (MIC) as described by Hancock [14]. Crude extracts were dissolved in nutrient broth and serially diluted in eppendorf tubes. 10 μl of overnight actively growing culture of the bacteria (1 × 10⁶ CFU/ml) was transferred to the different eppendorf tubes. The tubes were incubated for 24 h at 37°C. The following morning, streaking was done from all samples on nutrient agar plates, agar plates were incubated overnight at 37°C. Next day plates were observed for the bacterial growth. MIC value was judged by the lowest concentration of the extract concentration that inhibited growth.

GC-MS analysis of crude extracts

To determine the chemical composition of crude extract, the gas chromatography coupled with mass spectrometer (GC-MS) analysis was conducted. The analysis was performed with Perkin-Elmer (Clarus 500, USA) gas chromatography coupled with (Clarus 500, USA) mass spectrometer (MS) equipped with RTx-5 column (30 × 0.32 nm). The oven temperature was set 50 to 150°C at 3°C/min, it was held isothermal for 10 min and raised to 250°C at the rate of 10°C/min. The carrier gas was Helium (1 ml/min). In this analysis neither internal nor external chemical standards were used. Interpretation of the resultant mass spectra was done by using NIST database and by studying the fragmentation pattern of such compound resulted from mass spectrometry analysis.

Statistical analysis

Experiments were performed in triplicate. The Standard Deviation of the mean (SD) of experimental results were calculated using SPSS statistics software.

Results

The water and ethanol extracts of A. tinctoria were evaluated for their antibacterial activity against four pathogenic bacteria responsible for skin infections (Table 1). Results depict that the ethanol extract had excellent potential in inhibiting the growth of all four pathogens tested, maximum zone of inhibition was observed against B. subtilis (28.0 mm) followed by S. aureus (24.7 mm), P. areuginosa (18.7 mm) and E. coli (15.3 mm). The water extract showed minor antibacterial activity against B. subtilis (8.3 mm) and S. aureus (8.3 mm) whereas, it was found ineffective against other two pathogens. In general, the pathogen, B. subtilis was observed to be most sensitive pathogen whereas, least was E. coli. The antibiotic, gentamycin (30 μg) was found much more effective than any of extract evaluated. Results of determination of Minimum Inhibition Concentration (MIC) of ethanol extract of A. tinctoria roots reported in Table 1 showed that the MIC values were depended on the pathogenic strain tested and the dilution used. The pathogens, B. subtilis and S. aureus were inhibited at a concentration of 0.2%. The MIC value of ethanol extract for E. coli and P. areuginosa was 0.4%.

Table 1. In vitro antibacterial activity and minimum inhibitory concentration (MIC) values of water and ethanol crude extract of A. tinctoria roots.

The ethanol extract selected for the chemical analysis as it showed excellent antibacterial activity against all the tested pathogens. The qualitative analysis of the ethanol extract was conducted by the GC-MS (Table 2). The major compounds detected in the ethanolic extract of Alkanna were akanin, acetyl-alkanin, ethane, 1, 1-dichloro-, 1H-benzotriazole, 4- nitro, 2-chloroethyl (methylsulfonyl) methanesulfonate and 2, 5-cyclohexadien-1-one, 4-diazo-(napthoquinone). Besides that several other napthoqinones and esters were detected in the extract.

Table 2. The GC-MS analysis of ethanol extracts of A. tinctoria roots.

Discussion

The results of antibacterial activity of water and ethanol extracts of A. tinctoria showed that the ethanol extract had a potential to inhibit the growth of all bacterial pathogen tested. Whereas, the water extract showed almost negligible activity against these pathogens. The reasons for lesser antibacterial activity by water extract could be due to a lower concentration of antibacterial compounds in the extract or may be water was not a suitable solvent for the extraction of active ingredients of the Alkanna roots [15]. Cowan [16] documented that most of the active antimicrobial compounds of plants are obtained through the extraction with ethanol or methanol solvent. Moreover, the alkanet roots contain dyestuffs which are soluble in alcohol, ether and oils but insoluble in water. May be the bioactive ingredients are the part of non-water soluble constituents as it is also evident from the present results that ethanol extract gave much better results than water extract. In general, the pathogen, B. subtilis was found to be most sensitive pathogen whereas, least was E. coli. It is interesting to note that the ethanol extract was also effective against the gram-negative bacteria (E. coli and P. areuginosa) which are generally resistant to antibiotic. Khan et al. [17] reported that leaves extract of A. tinctoria prepared in ethanol, chloroform hexane and water had showed potential activity against S. aureus in addition these extracts were also effective to A. baumannii, E. coli, P. aeruginosa. Sengul et al. [18] reported that the methanolic extract of A. tinctoria had antimicrobial activity against 9 out of 32 microorganisms tested.

Results of determination of Minimum Inhibition Concentration (MIC) of ethanol extract of A. tinctoria showed that the pathogens tested in the current study with various concentration of ethanol extract exhibited different level of inhibition, which means that the crude extract alone was responsible for the inhibition of pathogenic species. This is the first report of determination of MIC value of ethanolic extract of Alkanna roots. In a study, the MIC of aqueous Alkanna leaves extract against A. baumannii and S. aureus was recorded to be 12.5 mg/ml whereas, against E. coli and P. aeruginosa it was 25 mg/ml [17]. The flavonoid sarothrin isolated from A. orientalis resulted in inhibiting the growth of Mycobacterium smegmatis (MIC 75 μM) and S. aureus (MIC>800 μM) [19]. Ogurtan et al. [20] reported the second-degree burn in rabbit model was healed by the root topical preparation in olive oil. Traditionally the Alkanna roots extract mixed with olive oil are used to treat burn wounds. Two bacterial species S. aureus and P. areuginosa are the most prevalent infective agents of the burn wounds. Results of present study manifest that extract of Alkanna has the potential to inhibit the growth of these pathogens. Hence the present study proves the role of Alkanna in treating burn wounds and skin infections.

After evaluating the antibacterial activity of water and ethanol extract of A. tinctoria, the ethanol extract of Alkanna was selected for the chemical analysis as the extract showed excellent antibacterial activity. The qualitative analysis of the ethanol extract was conducted by the GC-MS. The major compounds detected in the ethanolic extract of Alkanna were akanin, acetyl-alkanin, ethane, 1, 1-dichloro-, 1Hbenzotriazole, 4-nitro, 2-chloroethyl (methylsulfonyl) methanesulfonate and 2, 5-cyclohexadien-1-one, 4-diazo- (napthoquinone). Besides that several other napthoqinones and esters were detected in the extract. The current GC-MS analysis of ethanol extract clearly showed the presence of important bioactive chemicals which were reported earlier by various workers, such as, alkanin, shikonin and naphthoquinone and their derivatives [21]. Through chromatographic purification Tung et al. [4] isolated two major naphthoquinones, alkannin and angelylalkannin from the roots of A. tinctoria. Assimopoulou et al. [15] reported that dichloromethane extract of A. tinctoria roots contains mainly alkannin esters. In a review Ivancheva et al. [1] illustrated the presence of naphthoquinone, alkannin or shikonin, Alkannan and alkane’s esters in the roots of A. tinctoria. Some of these compounds are characterised as bioactive compounds [8,9]. Papageorgiou et al. [21] concluded that napthoquinone pigment with naphthezarin system is requisite for the activity. The present GC-MS analysis of ethanol extract reveals the presence of many napthoquinones, chemicals noticeable for their antibacterial activity.

Conclusion

The present study proves the role of Alkanna roots in treating skin infections. The ethanolic crude extract of A. tinctoria roots, can be a source new antibacterial compound which are yet to be explored.

Acknowledgements

The author extended their appreciation to Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RG-1435-086.

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