Abstract: Background and Objective: Many plant extract have been reported to have an antimicrobial and antioxidative activities, for instance, Salmonella typhimurium, recognized as the main causes of food contaminations and may induce various human infections. In addition, hydrogen peroxide (H2O2) induced reactive oxygen species and the absence of their scavenge systems in cells leads to oxidative stress. The present study is focused on an essay to determine the antimicrobial and antioxidant properties of aqueous extract of Urtica urens. Materials and Methods: The antibacterial activity was tested in albino rats as a model using S. typhimurium infection. Mice were initially infected by S. typhimurium and then treated with Urtica urens-extract. Oxidative stress was induced in male Wistar rats by a single intraperitoneal injection of 1 mM of H2O2. Results: The extract (3 mg kg–1 b.wt.) treated animals was found to have significant effects on mortality and the numbers of viable S. typhimurium recovered from feces. The extract was fed to albino rats, followed by H2O2. Biochemical evaluation of the treatment has been tested at different enzymatic levels, such as glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation (MDA). The antioxidant assay showed a significant decrease of the MDA level and increase in the GSH and SOD. Although, clinical signs and histological damage were rarely observed in the treated mice, the controls showed a signs of lethargy and histological damage in the liver, spleen and intestine. Conclusions: Urtica urens-extract has the potential to provide an effective treatment for salmonellosis and oxidative stress.
INTRODUCTION
Primary civilizations have used medicinal plants as the ultimate source of therapeutic aids1. In spite of the huge synthetic products into modern medicine, almost half of them are directly obtained from plants, many plants exhibit a unique complex combination of secondary metabolites which give an effective effect in therapy process for instance, caffeoylmalic acid, organic acid, chlorogenic acid, flavonoids, coumarins, steroids and scopoletin2. Moreover, aerial parts of plants are rich in inorganic minerals and vitamins etc. Which have significant antioxidant and antibacterial properties3,4. Many modern drugs which have contributed well in medical interventions were based or extracted from medicinal plants. Adequate drugs include the curare alkaloids, penicillin and other antibiotics, cortisone, reserpine, podophyllotoxin and other therapeutic agents5.
Urtica urens (UU), which belongs to the family of Urticaceae and commonly known as nettle apple has been used extensively as a traditional medicine in many countries6 for the treatment of anemia, rheumatism and arthritis, eczema, asthma, urinary gravel, stomach complaints, skin infections and as an anti-haemorrhagic7.
Furthermore, Urtica urens is reported by an antioxidant and antibacterial effects8,9. The constituents of UU include caffeoylmalic acid, flavonoids, chlorogenic acid, gallic acid and caffeic acid which are well known for their therapeutic properties2,10. The main medicinal uses of nettles historically were internally as a tonic and highly nutrient food11. However, till the date, no studies regarding the antimicrobial and antioxidant activity of Urtica urens have been conducted. Therefore, the objective of the present study was to determine the protective effect of feeding the aqueous extract of U. urens to albino rats of the Wistar strain against S. typhimurium and the toxic effects of H2O2 by biochemical and histopathological methods.
MATERIALS AND METHODS
Plant material: Urtica urens is a perennial plant with stinging hairs belonging to the family Urticaceae, under the division Spermatophyta, subdivision of Angiospermae, class Dicotyledonae, group Apetalae, order Urticales. Urtica urens were collected from the Bizerta region of Tunisia on April, 2014 (South Mediterrranean). The botanical identification of U. urens was carried out by Professor Ben Nasri-Ayachi, Sciences Faculty of Tunisia.
Preparation of crude plant extracts: For aqueous extraction, 10 g of air-dried powder was boiled on slow heat in distilled water for 30 min. The extract then filtered using Whatman filter paper No. 1 and centrifuged at 5000×g for 10 min as discussed by Folcara et al.12. The extract supernatant was collected each 30 min and concentrated to a final volume equal to one fourth of the original one. The final solution was used to perform antibacterial and antioxidant activities. Finally, the supernatant was recovered and stored at -4°C until it is used.
Determination of in vivo antibacterial activity
Animal: Eighteen male Wistar rats (50-70 g) aged between 8 and 10 weeks mice were purchased from the Pasteur Institute (Tunisia) to perform all in vivo experiments. They were kept in a temperature-controlled room under a 12 h light 12 h dark cycle. Animals had free access to commercial solid food and water ad libitum and were acclimatized for at least 1 week prior to beginning the experiments. All mice experiments in this study were approved by the Bizerte University Animal Ethics Committee in accordance with the guidelines of the Tunisia Council on Animal Care.
Preparation of bacteria: Salmonella typhimurium (ATCC 14028) was used in this study. This strain was purchased from Institute Pasteur Tunis, stored at -80°C in glycerol stocks and used as required during different experiments.
In vivo assay using mice: Mice were divided into the following groups: Control (CON), Salmonella-infected (SI) and Salmonella-infected+UU-extract (SIUU). Each group contained six mice. The growth inhibition of the test organisms in mice was then determined by monitoring S. typhimurium in the feces of the mice. Briefly, S. typhimurium was grown overnight in Luria-Bertani broth (Difco), centrifuged, washed in phosphate-buffered saline (PBS) and then diluted into 20% sucrose solution to achieve a final concentration of 1×105 CFU mL–1. The SI and SIUU groups were then inoculated using gavage needle orally with 0.1 mL of already prepared bacterial suspension. Each day, 1 h after infection, 2 mL of UU aqueous extract were orally administered to all animals of the SIUU group (using gavage needle), whereas CON and SI animals were not. Fecal samples were then collected at 0, 1, 2, 3, 4, 5, 6 and 7 days after the bacterial suspensions were administered and the numbers of the bacteria per gram of feces were determined. Aliquots (100 μL) of fecal suspensions were serially diluted in PBS and then plated on duplicate Salmonella-Shigella agar plates (Difco), which were subsequently incubated overnight at 37°C. Typical colonies were counted using the method of Lee et al.13 on plates that contained between 30 and 300 colonies, after which confirmation of S. typhimurium was performed by a PCR assay using a previously described method14. At day 4 post-infection, the mice were sacrificed and tissue specimens of the liver, spleen and intestine organs were transferred to 10% buffered neutral formalin for histopathologic examinations and then processed using standard procedures. Sections of paraffin-embedded tissues were then stained with hematoxylin and eosin.
Determination of in vivo antioxidant activity
Experimental procedure: Hydrogen peroxide (H2O2) is a selectively toxic chemical agent. The H2O2 induced Reactive Oxygen Species (ROS) and/or a decrease the antioxidant defense mechanisms15. The ROS include free radicals. However, the increase in ROS and free radicals secretion was revealed to be an important cause among different biochemical manifestations in various diseases16.
Stress was induced in male Wistar rats (50-70 g) by a single intraperitoneal injection according to Donnini et al.17 of hydrogen peroxide (H2O2) at a dose of 1 mmol L–1 in 0.5 mL PBS. The animals were grouped into three groups containing six animals in each group. The first group served as control, the second group was administered H2O2 by intraperitoneal injection (negative control). The animals of the 2nd and 3rd groups were given dose of H2O2 at 1 mmol L–1 until the 14th day and the 3rd group was administered the aqueous extract of UU via oral route at 3-5 mg kg–1 b.wt., for 14 days. The dose was selected on the basis of the LD50 at the equivalent of up to 2 g dried drug kg–1 b.wt.3. Some of rats in first group were treated with physiological saline, daily for 14 days. They were housed at University Animal House in standard conditions and fed with standard diet with water ad libitum. At the end of experimental period, animals were sacrificed and the liver, spleen and small intestine were isolated to prepare homogenate.
Markers of oxidative stress: Animals were sacrificed and tissue was collected and then washed with ice-cold saline, weighed and minced, 10% homogenate was prepared in 0.15 M ice-cold KCl for TBARS (thiobarbituric acid-reactive substances), a marker for lipid peroxidation was estimated with the method of Ohkawa et al.18 and protein was determined according to the method reported by Lowry et al.19. Measurements of glutathione were performed according to the method of Ellman20 and superoxide dismutase concentration was determined according to the method of Marklund21, using a teflon tissue homogenizer.
Tissue processing: Liver, spleen and intestine were flushed with chilled 1.15% (w/v) KCl solution. A 10% (w/v) homogenate was prepared in 50 mM phosphate buffer, pH 7.4 and centrifuged at 8000×g for 15 min at 4°C. Experiment were carried out according to the method described by Sen et al.22. The supernatant so obtained were used for the estimation of lipid peroxidation (MDA), glutathione (GSH) and superoxide dismutase (SOD). The protein content was determined by the method of Lowry et al.19, using bovine serum albumin as the standard.
Statistical analysis: The results are expressed as Mean±SD of at least three sets of triplicate determinations for each data point. One-way ANOVA, Tukey and Dunnett tests were applied for analyzing the significance of difference between and among different groups.
RESULTS
In vivo antibacterial activity: The in vivo antibacterial activity of UU-extract was examined using a mouse S. typhimurium infection model. Briefly, mice were infected with 1×105 CFU of S. typhimurium SI, 1 h late.
The UU-extract was orally administered to the mice. Table 1 shows that treatment with the extract of UU was found to have marked effects on mortality and on the number of viable S. typhimurium recovered from feces. At day 1 post-infection, 10 mice in the SI and SIUU group did not shed viable S. typhimurium in feces, whereas the feces of mice in the SI group being found to contain bacteria at a concentration of 1×102 to 2×103 CFU g–1 and feces of mice in the SIUU group being found to contain bacteria at a concentration of 0-4.3×103 CFU g–1. In addition, at day 6 post-injection, one of the mice in the SIUU group had died, while all six mice in the SI group had succumbed.
Organ histopathologic changes: Salmonella typhimurium infected mice that did not receive the UU-extract were showed signs of histological damage in the liver, spleen and intestine. The central veins of the liver showed congestion with focal necrotic emboli-like materials.
| Table 1: | Effects of treatment with UU-extract on fecal shedding of S. typhimurium (CFU g–1) by mice |
| SI: Salmonella-infected, SIUU: Salmonella-infected+UU | |
| Table 2: | Level of MDA in liver, spleen and small intestine of control and experimental animals in each group |
| Values are expressed as Mean±SD (n = 6). ***Significantly different from control at p<0.001 | |
| Table 3: | Level of GSH in liver, spleen and small intestine of control and experimental animals in each group |
| Values are expressed as Mean±SD (n = 6), ***Significantly different from control at p<0.001 | |
In spleen, an extensive hemorrhagic necrosis was detected in the red pulp with multiple apoptotic bodies in the white pulp. In addition, destruction and atrophy with ischemic necrosis and edematous changes with polymorphonuclear leukocyte infiltration within the mucosal layers of the small intestine were evident. In contrast, clinical signs and histological damage were rarely observed in S. typhimurium-infected mice fed the extract of UU-extract (Fig. 1).
In vivo antioxidant activity: Effect of UU-extract on lipid peroxidation status: Table 2 represents the levels of lipid peroxidation (TBARS) in the liver, spleen and small intestine of control and experimental animals. A significant increase in the levels of TBARS was observed in the hydrogen peroxide (H2O2) alone treated animals (Group II) when compared with control animals (Group I). This was significantly reversed to near normal levels in Urtica urens (3 mg kg–1 b.wt.) treated animals (Group III). The UU-extract treated animals (Group III) did not show any significant variations when compared to control (Group I) animals. According to these observations UU-extract may induce the protection against the H2O2 induced oxidative stress by reducing the lipid peroxidation.
Effect of UU-extract on GSH status: Table 3 shows the level of non-enzymic antioxidant (GSH) in liver, spleen and small intestine of control as well as treated animals. The GSH status was found to be significantly lowered in H2O2 alone treated animals (Group II) when compared with control animals (Group I). The alterations of GSH-levels were reverted to nearly control values on the administration of UU-extract treated animals (groups III) when compared with (group I) animals. Animals intoxicate then treated with UU-extract (Group III) did not show any significant variations when compared to control (Group I) animals.
Effect of UU-extract on superoxide dismutase (SOD): Rats intoxicate with H2O2 had significantly low levels of SOD activity compared to the control rats. However, Animals intoxicate then treated with UU-extract (Group III) to show a significant (p<0.001) increase in SOD levels when compared with the control group (Table 4).
| Fig. 1(a-c): | Histopathological changes in organs in CON, SI and SIUU. (a) Liver (×200), (b) Spleen (×200) and (c) Small intestine (×200). CON-A control: Normal hepatocytes showing normal architecture with portal tried, showing portal veins, hepatic artery and vein. SI-A congestion and edematous changes within the central and portal veins of the liver and severe hemorrhagic necrosis was also observed within the red pulp of the (SI-B). In addition, destruction and atrophy with ischemic necrosis within the mucous layers of the small intestines where observed (SI-C). Urtica urens-fed mice (test group) infected with S. typhimurium. Histological damages in the above organs were rarely observed in these mice (SIUU) |
Organ histopathologic change: Group II animals were lethargic and showed signs of histological damage in the liver, spleen and small intestine. The central and portal veins of the liver showed congestion with focal necrotic emboli-like materials. The histological photomicrographs of the spleen sections are shown in Fig. 2. The congestion of the spleen tissue was showed in the H2O2-treated group, while no severe damages and lymph nodule proliferation of spleen tissue were observed in group III animals. In addition, destruction, atrophy and edematous changes with polymorphonuclear leukocyte infiltration within the mucous layers of the small intestines were observed. Conversely, clinical signs and histological damage were rarely observed in H2O2 intoxicated-mice fed with the UU-extract (Fig. 2).
DISCUSSION
In the present study, Urtica urens-extract was screened for antibiotic activity against several pathogenic Salmonella serotypes.
| Fig. 2(a-c): | Histopathological changes in organs in CON, RI and RIUU. (a) Liver, (b) Spleen and (c) Small intestine, CON: Control rat, RI: Rats intoxicate with H2O2 alone (negative control), RIUU: Rats intoxicate with H2O2 then treated with aqueous extract of Urtica urens, (H and E X200) |
| Table 4: | Level of SOD in liver, spleen and small intestine of control and experimental animals in each group |
| Values are expressed as Mean±SD (n = 6). ***Significantly different from control at p<0.001 | |
The in vivo antibacterial assay revealed that the extract showed the effective inhibition of S. typhimurium growth and significantly reduced mice mortality (Table 1). Furthermore, clinical infection signs and histological damage were rarely observed in the SIUU-group (Fig. 1), whereas infected mice SI showed severe clinical signs and histological damage in the considered organs. This is the first report to describe the antibacterial activity of UU-extract against S. typhimurium. Based on this promising in vivo assay results it is clearly proved that UU-extract can be considered like a novel antimicrobial treatment for salmonellosis. The UU-extract has been reported only to show the antibacterial activity against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa delivery as reported by Leven et al.23. The antibacterial activity of UU-extract may be indicative of the presence of some metabolic toxins or broad-spectrum antibiotics. Several metabolites from herb species, such as, alkaloids, tannins, saponins and sterols have been previously associated with antimicrobial activity reported by Taguri et al.24. The major chemical constituents of Urtica urens are flavonoids, caffeoyl-esters, caffeic acid, scopoletin (cumarin), sitosterol (-3-O-glucoside), polysaccharides, fatty acids (e.g., 13-hydroxy-octadecadienoic acid), minerals (herba: up to 20% leaves: 1-5%) as discussed by Doukkali et al.25 And the aerial parts are rich in minerals and vitamins and Urtica urens was reported to show anti-food-borne pathogens.
Furthermore, antioxidant activity is one of the most intensively studied subjects in aqueous plant extract. In this study, the therapeutic effects of UU-extract were studied by examining the prevention of hydrogen peroxide induced stress in rats. The H2O2 is one of the most widely used toxicant for experimental induction of liver, spleen and intestine in laboratory animals.
Malondialdehyde is generated from the degradation of polyunsaturated lipids by ROS. It is one of the most frequently used indicators of lipid peroxidation26, in this study we have demonstrated that elevated levels of MDA in H2O2-induced rats were reduced after the treatment with UU-extract (Table 2).
Glutathione is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, it is noteworthy to cite the study of Swaroop and Ramasarma27. In the present study, significantly low GSH levels were observed in rats intoxicated with H2O2 as compared to the control. While UU-extract treatment showed significant increase above normal level. Thus, we noted that UU-extract may offer better antioxidant effect by scavenging free radicals and restoring the imbalance between oxidant/antioxidant homeostasis developed during stress condition (Table 3).
Antioxidant enzymes such as SOD have been shown vital to eliminate ROS. The SOD is the most important antioxidant enzyme that inhibit free radical formation and is usually used as biomarker to indicate ROS production28. The SOD is one of the important enzymes that scavenges superoxide radical
The results of this study were supported by similar observation in the others researchers30,31 that H2O2 was able to induce oxidative stress in these tissues. In case of H2O2-treated rats, strong modification in organ architecture and areas of hemorrhage and necrosis were seen. However, in the case of group III, the liver, spleen and small intestine were shown to retain normal architecture with few areas of hemorrhage (Fig. 2).
In this study, the results showed that UU-extract treatment prevented H2O2-induced stress in rats by strengthening the antioxidant defense system. Therefore, these results demonstrated that the UU-extract has protective function against H2O2 toxicity in rat liver, spleen and small intestine. Similar results have been reported for some other ethnobotanical fruits and herbs, in agreement with the analysis of Kim et al.32. The results of the present study may have very important implications for the chemopreventive potentials antibacterial and antioxidant profiles of aqueous extract of U. urens as a traditional herbal medicine.
CONCLUSION
This study may suggest new treatments in the curative of salmonellosis and oxidative stress reveals the importance of scientific research on miscellaneous plants with various medicinal properties. Further studies are required to evaluate the possible interactions of U. urens with therapeutic drugs and/or other dietary components in order to clarify its possible use as traditional medicinal herb.
ACKNOWLEDGEMENT
We thank Prof. Ben Attia Msaddek from Science of live department of Faculty of Science of Bizerta, for scientific discussion and advice.