Abstract: Herbs are largely unexplored source of drug repository. Medicinal plants have great potential for providing novel drug leads with novel mechanism of action. The present study describes the antibacterial activity, phytochemical profile and toxicological evaluation of Urtica dioica. U. dioica leaves were subjected to solvent extraction by hexane, ethyl acetate, chloroform, methanol and water. All the extracts were tested for antibacterial activity against five clinical isolates of Gram positive and Gram negative bacteria by disc diffusion method. Hexane extract showed good antibacterial activity against all the five bacterial strains, hence it was further purified using silica column chromatography and Minimum Inhibitory Concentration (MIC) of the semi purified fraction-B (FB) of hexane extract was determined by serial tube dilution method. FB showed MIC value of 31.25, 250, 7.81, 31.25 and 125 μg mL-1 against Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae and Shigella flexneri, respectively. FB was subjected to GC-MS analysis in search of potent antibacterial compound(s). Neophytadiene (26.97%) butyl tetradecyl ester (9.53%), Dibutyl phthalate (7.45%), Bis (2-ethyl hexyl) maleate (8.80%) and 1,2-benzenedicarboxylic acid (9.89%) were the major constituents which may be responsible for the antibacterial activity of FB of U. dioica. Sub-acute oral toxicity of FB was carried out in wistar rats at doses of 100, 200, 400 and 800 mg kg-1 Body Weight (b.wt.) to assess index of safety. FB showed MIC value as low as 7.81 μg mL-1 against Salmonella typhi, exhibiting that a promising antibacterial agent is present in this fraction. Hematological and biochemical parameters showed that the FB was safe at tested concentrations. Hence further purification of FB is required to obtained potential antibacterial compound.
INTRODUCTION
Microbial infections are the major cause of morbidity and mortality particularly in immune-compromised patients (Black et al., 1982). Although a numbers of antibiotics are available for treatment and management of microbial infections, development of resistance against present day antibiotics is a major health concern (Al-Bari et al., 2007). Therefore, there is a perpetual and urgent need to discover new antimicrobial compounds with high safety index (Erturk et al., 2006). Historically, medicinal plants have been a source of inspiration for novel drug compounds. Plant derived medicines have made large contributions to human health and well being. Phytochemicals may become the base for the development of medicines by providing a pharmacophore which could be used for the development of new drug with novel mechanism of action. Many laboratories across the globe are involved in systematic screening of plant species for discovering new bioactive compounds but still a very meager portion of this tremendous potential drug-repertoire has been scientifically screened (Menghani et al., 2011). Hence, there is a need for scientific evidence based validation of bioactive phytochemicals (Davies, 1994; Adeniyi and Ayepola, 2008; Karim et al., 2011). Isolation, purification and characterization of lead antimicrobial phytochemicals is an exciting area of search, especially developing drugs against multi drug resistance microbes (Rahman et al., 2000). A number of medicinal plants have been screened for antimicrobial activity in recent times (Premanath et al., 2011; Oboh, 2010) and efforts have been done to identify their active constituents (Tijjani et al., 2009). Safety of herbal extracts and their purified constituents is another major concern during natural product based drug discovery research (Hassan et al., 2011). The plant extracts possessing bioactivity are essentially evaluated for toxicity (Chavda et al., 2010). The extracts are usually tested for short or long term toxicity in animal models (Abdelgadir et al., 2010; Diallo et al., 2010). Non toxic extracts possessing good bioactivity may provide potential antimicrobial leads.
Urtica dioica or stinging nettle or common nettle, is a herbaceous perennial flowering plant, native to Europe, Asia, Northern Africa and North America and is the best-known member of the nettle genus Urtica (Emmelin and Feldberg, 1949). Urtica dioica is used in Indian traditional medicine for genitourinary ailments, kidney disorders, allergies, diabetes, internal bleeding and anemia, GI tract ailments, musculoskeletal aches, osteoarthritis and alopecia (Matsingou et al., 2001). However, only a few of these pharmacological activities have been experimentally proved (Lourdes et al., 2008). Recently anti-proliferative (Durak et al., 2004), anti hyperglycemic (Bnouham et al., 2003), antioxidant (Abdullin et al., 2002), antiarthritic (Riehemann et al., 1999), antidandruff, galactogogue and haemostatic (Hadizadeh et al., 2009) activity of U. dioica are also reported. The main phytoconstituents of U. dioica include lectins, plastocyanins, glycoproteins, carotenoids, fatty acids, sterols, flavonoids, polysaccharides, terpenes and lignans (Sajfrtova et al., 2005).
In present study, in vitro bioassay guided purification of antibacterial compound(s) from leaves of U. dioica was carried out. The active sub fraction was subjected to in vivo toxicological evaluation to assess its safety index and GC-MS analysis to identify potential antibacterial lead compound(s).
MATERIALS AND METHODS
Collection and extraction of plant material: U. dioica leaves were collected from the Anantnag district of Kashmir valley, India in the month of October, 2009 and authenticated by the Centre of Biodiversity and Taxonomy (CBT), Department of Botany, University of Kashmir and a voucher specimen (28100 KASH) was deposited. The leaves were air dried, chopped in small pieces and grinded to obtain coarse powder. Powder (1 kg) was extracted successively with hexane, chloroform, ethyl acetate and methanol in soxhlet apparatus for 24 h. The extracts were filtered over Whatman No. 1 paper and the organic solvent extracts were concentrated under vacuum using rotary evaporator (Singh et al., 2002). The residue left after methanol extraction was soaked in distilled water with continuous stirring for 24 h. Filtered water extract was centrifuged at 5000 rpm for 10 min at room temperature to remove any suspended material and lyophilized. All the extracts were stored at 4°C until used for the antibacterial assay.
Antibacterial susceptibility test: All five solvent extracts were investigated for the antibacterial activity by disc diffusion method against clinical isolate of Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Klebsiella pneumonia and Shigella flexneri (Bauer et al., 1996) obtained from Govt. Medical College, Jhansi, UP, India. Sterile whatman filter paper discs with 6 mm diameter were impregnated with 2000 μg of each extract (dissolved in 8 μL DMSO) as per standard procedures (Chandrasekaran and Venkatesalu, 2004). Gentamycin (30 μg disc-1) was used as a positive control and a paper disc impregnated with DMSO (8 μL) was used as negative control. The bacterial cultures were grown in nutrient broth liquid medium at 35°C and kept under refrigeration (4°C) until use. The nutrient agar plates were prepared for disc diffusion assay and each bacterial strain at a concentration of 1.5x106 cells mL-1 (adjusted to the 0.5 McFarland turbidity standards) was used (NCCLS, 2005). All experiments were performed in triplicate and results were recorded by measuring the zones of growth inhibition around the discs. The test samples with inhibition zones equal to or greater than 7 mm, were considered active.
Bioassay guided isolation of active compound(s): A 60 cm long glass column with a diameter of 10 cm was filled with 1.5 kg silica gel, mess size 60-120. Hexane extract (HE, 100 g) was dissolved in a minimal amount of mobile phase and mixed with 200 g silica gel. The column was eluted with gradient mobile phase of hexane-ethyl acetate, 20 fractions of 50 mL each were collected, analyzed by silica gel TLC (Merck) and pooled to give 5 sub fractions FA, FB, FC, FD and FE. The subfractions were tested for antibacterial activity by disc diffusion assay.
Minimum Inhibitory Concentration (MIC): Serial tube dilution technique was used to determine MIC (Rahman et al., 2000). FB was dissolved in DMSO to prepare a stock solution. Ten serial dilutions of stock, ranging from concentration of 1000 μg to 0.97 μg mL-1 were prepared in the test tubes. The tubes were inoculated with 100 μL of bacterial strain inoculums at a concentration of 106 cell mL-1. Standard antibiotic Gentamycin was included in the assay for comparison. Nutrient broth with the inoculums only was used as growth control. All experiments were carried out in triplicates. The tubes were incubated aerobically at 37°C for 12-18 h and 50 μL of 0.2 mg mL 2-(4-iodophenyl)3-(4-nitrophenyl)-5 phenyltetrazolium chloride (INT) solution was added. The tubes were observed for color change (Anders et al., 2002). The concentration of FB at which a decrease in red color was apparent compared to the next dilution was taken as MIC value. Bacterial growth was indicated by the red color of INT reduced to formazan by bacteria.
GC-MS analysis: GC-MS analysis of FB was carried out by using Perkin Elmer-Clarus 500 GC-MS with PE-5 (equivalent to DB-5) capillary column (length-30 m) and helium as carrier gas (flow rate-3.3 mL min-1). Samples were analyzed with the column held initially at 100°C for 1 min after injection, then increased to 170°C with 10°C/min programme rate and up to 215°C with 5°C/min programme rate. Final temperature was increased to 270°C with 10°C/min heating ramp for 10.5 min. The injections were performed in split mode (30:1) at 250°C. Detector and injector temperatures were 260 and 250°C, respectively. Pressure was established as 50 psi. Run time was 35 min. Temperature and nominal initial flow for Flame Ionization Detector (FID) were set as 250°C and 3.1 mL min-1, correspondingly. The constituent compounds were determined by comparing their retention times and mass weights with those of authentic samples obtained by GC as well as the mass spectra from the Wiley and Nist database searches by GC-MS.
Toxicity studies: The FB was subjected to in vivo sub-acute toxicity studies. Thirty male wistar rats weighing about 150-200 g, obtained from Central Drug Research Institute (CDRI), Lucknow were used in the experiment. Animals were kept for acclimatization at ambient temperature of 25°C and 45-55% relative humidity, 12 h each of dark and light cycles and were fed pelleted diet and water ad-libitum. Animal experimental protocols were in accordance with the recommendations of the institutional animal ethical committee. Animals were divided into five groups of six rats in each. First group served as a control group and the remaining four groups (A, B, C and D) received the doses of 100, 200, 400 and 800 mg kg-1 body weight of FB, respectively, for 14 days by gavaging (Mosaddik and Haque, 1999). The behavior and body weight of the animals was observed daily. Animals were sacrificed on 15th day and blood was collected from the jugular vein with the help of sterilized hypodermic syringe. Half of the blood was transferred into Ethylene Diamine Tetra Acetic acid (EDTA) vials for hematological studies. Serum for various biochemical investigations was isolated from other half. Hematological and biochemical parameters were determined using standard procedures and reagents supplied by diagnostic kit (Siemens Medical Solution Diagnostics Ltd, India).
Statistical analysis: All experiments were performed in triplicate. Statistical analysis was carried out using GRAPHPAD Prism 5. Results were expressed as Mean±SD. The statistical analysis involving two groups was performed by means of paired t-test and Analysis of Variance (ANOVA) followed by Tukey’s test. p<0.01 was considered significant and p<0.001 was considered highly significant.
RESULTS AND DISCUSSION
Herbal drugs are in great demand currently due to their varied pharmacological properties and source of novel bioactive compounds. Natural plant product based antibiotic drug discovery attained paramount importance as newly discovered drugs are likely to be effective against multi drug resistant microbes. There is only one report of antimicrobial activity from Urtica dioica plant (Uzun et al., 2002) and no antimicrobial the compounds are isolated so far, hence this study was planned to isolate antibacterial molecule(s) from this plant.
Antibacterial susceptibility test: The HE exhibited good antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Klebsiella pneumoniae and Shigella flexneri. The antibacterial activity produced by HE was comparable to that of the standard antibiotic Gentamycin (Table 1). The crude HE showed good antibacterial activity against all the tested bacteria as compared to other extract. The results suggest that antibacterial principle lies in non polar solvent extract as compared to polar extracts. There are a number of reports of hexane extracts of plant possessing antibacterial activity (Elzaawely et al., 2005). These results indicate that the extracting solvent has a definite effect on bioactive principles. Usually, the extract having large inhibition zone diameter with low minimum inhibitory concentration can be recognized as more potent drug than that of small inhibition zone diameter and high minimum inhibitory concentration (Semwal et al., 2009).
Bioassay guided isolation of active compound(s): HE was fractionated on silica column and 20 fractions of 50 mL each were collected. The fractions were analyzed by silica TLC and similar fractions were pooled to give 5 sub fractions FA, FB, FC, FD and FE. All subfractions were tested for antibacterial activity by disc diffusion assay. FB showed potent antibacterial activity against gram positive and negative bacteria as compared to other fractions in disc diffusion assay (Table 2).
| Table 1: | Antibacterial activity of different solvent extract of leaves of Urtica dioica |
| H: Hexane extract, C: Chloroform extract, M: Methanol extract, E: Ethyl-acetate extract, A: Aqueous extract, Ct: Control (Dimethyl Sulphoxide, DMSO), Sd: Standard (Gentamycin). The results were represented as inactive (-, inhibition zone diameter ≤7 mm), moderate activity (+, inhibition zone diameter ≤10 mm), good activity (++, inhibition zone diameter ≤15 mm) and very good activity (+++, inhibition zone diameter>15 mm) | |
| Table 2: | Antibacterial activity of different column eluted fractions of Hexane Extract (HE) of Urtica dioica |
| FA: Combined fractions 1-4, FB: Combined fractions 5-7, FC: Combined fractions 8-10, FD: Combined fractions 11-14C, FE: Combined fractions 15-19, Sd: Standard; Gentamycin and Ct: Control; DMSO. The results were represented as inactive (-, inhibition zone diameter ≤7 mm), moderate activity (+, inhibition zone diameter ≤10 mm), good activity (++, inhibition zone diameter ≤15 mm) and very good activity (+++, inhibition zone diameter >15 mm) | |
| Table 3: | The Minimum Inhibitory Concentration (MIC) of the fraction FB of Hexane Extract (HE) |
Minimum Inhibitory Concentration (MIC): The MIC values of FB were 250, 7.81, 31.25, 125 and 31.25 μg mL-1 against Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumonia, Shigella flexneri and Staphylococcus aureus, respectively (Table 3). The MIC as low as 7.81 μg mL-1 of a semi purified fraction against gram negative bacteria is suggestive of good antibacterial potential of the compounds of FB. Hence FB may yield potential molecules in the treatment of infections caused by pathogenic bacteria which have developed resistance against the known antibiotics (Singleton, 1999).
GC-MS analysis: GC-MS analysis of FB (20 compounds) is shown in Fig. 1. The GC-MS Retention Time (RT), molecular formula, molecular weight and percentage peak of the individual compounds were presented in the (Table 4). FB showed five major compounds i.e., 2,6,10-Trimethyl,14-ethylene-14-pentadecne (Neophytadiene) (26.97%) butyl tetradecyl ester (Phthalic acid) (9.53%), dibutyl ester (Dibutyl phthalate) (7.45%), Bis(2-ethyl hexyl) maleate (8.80%) and 1,2-benzenedicarboxylic acid (9.89%) which may be responsible for antibacterial activity. Antimicrobial activity of plant essential oil containing neophytadine (Palic et al., 2002) and other aromatic carboxylic acids and ester is reported (Roy et al., 2010). Neophytadiene is also reported to possess antibacterial activity as well as helping in treatment of headache, rheumatism and some skin disease (Suresh et al., 2010). Fatty acids are also known to have antibacterial and antifungal activities (Russel, 1991). Fatty acid esters namely phthalic acid, dibutyl ester, bis(2-ethyl hexyl) maleate and 1,2-benzenedicarboxylic acid were reported to possess anti inflammatory (Li et al., 2004) and antibacterial activity (Modupe et al., 2010). Essential oils rich in terpenes have been shown to possess good antibacterial activity (Taylor et al., 1996). The appreciable presence of the terpenes (neophytadiene 26.97% and olean-1-ene 1.97%) in the FB of HE, could explain its antibacterial activity against the tested bacterial strains. Since, Urtica dioica is reported to possess a wide range of medicinal value, these compounds may be analyzed for other biological activities reported from this plant.
Toxicity studies: Increase in popularity and scarcity of scientific studies on safety of herbal remedies have raised concern regarding toxicity and adverse effect (Saad et al., 2006). Body weight is one of the sensitive indicators of toxicity if it is monitored frequently and correctly during the study (Wilson et al., 2001). Average body weights of all rats before and after treatment with FB are presented in Fig. 2. After 14 days, control group gained an average weight of 2.46% and the experimental groups A, B, C and D gained an average weight of 3.76, 2.71, 2.81 and 3.11%, respectively.
| Fig. 1: | Chromatogram of FB of hexane extract Urtica dioica |
| Table 4: | GC-MS analytical report for fraction FB of Hexane Extract (HE) |
| Fig. 2: | Body weight changes before and after the treatment of Fraction B of Hexane extract of Urtica dioica. Each bar represents Mean+SEM of 6 rats |
No significant treatment related variation was observed in body weight and behavior of wistar rats during 14 days exposure to FB of HE indicating safety of the semi purified plant extract.
The hematopoietic system is very sensitive to toxic compounds (Mukinda and Syce, 2007) and it ranks with liver and kidneys in pre clinical and clinical safety evaluation (Bloom, 1993). Analysis of blood parameter is relevant in risk evaluation as the changes in hematological system have a higher predictive value for human toxicity, when the data translated from animal studies (Rhiouani et al., 2008). The hematological profiles of the experimental and control group wistar rats were determined at 15th day after treatment with FB in this study. No significant variation in the values of Red Blood Cell Count (RBC), White Blood Cell count (WBC) (Fig. 3), neutrophils, monocytes, basophils and eosinophils (Fig. 4). Significant decrease in level of lymphocytes was observed (Fig. 4) in group D. Hemoglobin percentage (Hb), Mean Corpuscular Hemoglobin Concentration (MCHC), bilirubin and total protein showed no significant variation in any of the treatment group as compared to control except significant decrease in Packed Cell Volume (PCV) in group A.
| Fig. 3: | Effect of fraction B of Hexane extract of Urtica dioica on Red Blood Corpuscles (RBC) and White Blood Cells (WBC) count in wistar rats. Each bar represents Mean+SEM of 6 rats |
| Fig. 4: | Effect of fraction B of Hexane extract of Urtica dioica on Differential Leucocytes Counts (DLC) in wistar rats. Each bar represents Mean+SEM of 6 rats. *Significantly different from control group at p<0.05 |
Significant increase in Mean Corpuscular Volume (MCV) in group C and D, while significant increase in Mean Corpuscular Hemoglobin (MCH) was observed in group B, C and D (Table 5).
Detoxification and clearance of toxic substances from the body are primarily a function of liver and kidneys and they are often the first to be affected by toxic herbs (Elvin-Lewis, 2001). Damage to these organs often results in elevation in clinical chemistry parameters. Biochemical parameters from serum were determined after treatment with FB and compared with control group. No variation in liver function markers like Aspartate amino Transferase (AST), Alanine amino Transferase (ALT) (Fig. 5). Alkaline Phosphates (ALP) was significantly decreased in treated animals in group B, C and D as compared to control (Fig. 6), however this decrease was with in the normal reference range of ALP values. Cholesterol (CHO), High Density Lipoprotein-cholesterol (HDL-C), Triglycerides (TG), Low Density Lipoprotein-cholesterol (LDL) and very low density lipoprotein-cholesterol (VLDL-C) (Fig. 7) were found under the normal reference values in treated animals. CHO, HDL-C, LDL-C and VLDL-C, TG can give useful information on the lipid metabolism as well as predisposition of the heart to atherosclerosis and its associated coronary heart diseases (Toyin et al., 2008). High blood cholesterol concentrations are an important risk factor for cardiovascular disease (Abolaji et al., 2007). Therefore, the reduced levels of serum CHO at 100, 200, 400 and 800 mg kg-1 body weight of the FB may be clinically beneficial and indicates hypolipidemic activity.
| Table 5: | Effect of 14 days oral administration of FB of hexane extract (HE) on hematological and biochemical parameters |
| *p<0.05, **p<0.01 | |
| Fig. 5: | Effect of fraction B of Hexane extract of Urtica dioica on Aspartate amino Transferase (AST) and Alanine amino Transferase (ALT) level. Each bar represents Mean+SEM of 6 rats |
| Fig. 6: | Effect of fraction B of Hexane extract of Urtica dioica on Alkaline Phosphatase (ALP) level in wistar rats. Each bar represents Mean+SEM of 6 rats. *Significantly different from control group at p<0.05 |
| Fig. 7: | Effect of fraction B of Hexane extract of Urtica dioica on Cholesterol (CHO), High Density Lipoproteins (HDL), Triglycerides (TG), Very Low Density Lipoproteins (VLDL) and Low Density Lipoproteins (LDL) level in wistar rats. Each bar represents Mean+SEM of 6 rats |
Cholesterol lowering activity of U. dioica is reported in hypercholesterolemic rats (Nassiri-Asl et al., 2009). The TG, HDL, LDL and VLDL cholesterol did not change significantly (p>0.05) indicating that the mechanism involving lipid alteration may not have been disturbed by the administration of the FB at these doses.
CONCLUSIONS
Hexane extract of Urtica dioica showed good antimicrobial activity against all the tested pathogenic clinical strains of bacteria. The extract showed better activity against gram negative bacteria as compared to gram positive. Owing development of multidrug resistant strains treating gram negative bacterial infection is more difficult, it will be interesting to purify the active subfractions for potential lead antibacterial compounds. GC-MS analysis of the subfraction FB indicated that terpenes, fatty acid esters and phenols are the active antimicrobial principles present in FB. FB was non toxic in wistar rats up to the tested concentration of 800 mg kg-1 bw indicating that it is safe for in vivo consumption.
ACKNOWLEDGMENTS
The authors are thankful to Bundelkhand University, Jhansi, India, for providing research facilities for undertaking this work.