Archivio Italiano di Urologia e Andrologia 2017; 89, 2134 ORIGINAL PAPER The role of an herbal agent in treatment for Escherichia coli induced bacterial cyctitis in rats Murat Tuken 1, Mustafa Zafer Temiz 1, Emrah Yuruk 1, Asuman Orcun Kaptanagasi 2, Kayhan Basak 3, Fehmi Narter 4, Ahmet Yaser Muslumanoglu 1, Kemal Sarica 4 1 Bagcilar Training & Research Hospital, Dept. of Urology, Istanbul, Turkey; 2 Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Biochemistry, Istanbul, Turkey; 3 Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Pathology, Istanbul, Turkey; 4 Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Urology, Istanbul, Turkey. Objectives: The aim of this study is to evalu- ate the effects of the herbal agent in the pre- vention and treatment of bacterial cystitis in a rat model. Material and Methods: A total of twenty-eight male Sprague- Dawley rats were divided into four groups. Group-1 constituted the control group (operated and normal saline injected into the bladder, received only drinking water for 7 days); Group-2 con- stituted the no-treatment group (operated, E.coli J96 strain injected into the bladder, received only drinking water for 7 days); Group-3 constituted the short-term treatment (operated, E.coli J96 strain injected into the bladder, received the herbal agent added into drinking water for 7 days) and Group-4 con- stituted the long-term treatment (operated, E. coli J96 strain injected into the bladder, received herbal agent added into drinking water for 14 days). At the end of the pre-defined treat- ment periods of duration, the rats were sacrificed, urine sam- ples collected from the bladder for culture and bladders were harvested for histopathological evaluation. Urine culture results and histopathological findings were comparatively evaluated between the groups. Results: Urine cultures were positive for implanted E. coli strains in 0%, 85.7%, 42.8% and 0% of rats in Group 1, Group 2, Group 3 and Group 4, respectively (p = 0.001). Although histopathological evaluation revealed increased vascular dila- tion in the bladder specimens obtained from Group 2 and Group 3 (p = 0.028) no significant difference was noticed in level of inflammation (p = 0.610), edema (p = 0.754) and thick- ness of uroepithelium (p = 0.138). Conclusion: While long term (14 days) treatment with an herbal agent added into the drinking water resulted in complete clearance of urine from E. coli; shorter application of the agent revealed partial clearance. Further clinical studies are needed to support our results. KEY WORDS: Antibiotics; Bacterial cystitis; Herbal agent. Submitted 3 February 2017; Accepted 3 April 2017 Summary No conflict of interest declared. use, related antimicrobial resistance and the spread of bacterial resistant strains make the problem more serious than ever. Like other infectious diseases, the early and proper treatment of cystitis decreases the incidence of disease related morbidity and this situation necessitates the initiation of an empirical antimicrobial treatment in the majority of cases (2). On the other hand, with this approach, the antimicrobial resistance patterns of the hospital and the country are generally ignored by the responsible physicians. Administration of an inappropri- ate empirical antimicrobial therapy may eventually result in the accelerated rates of antimicrobial resistance (3). Moreover, these agents are also being used for the antimicrobial prophylaxis in the prevention of urinary tract infections particularly in patients with anatomical abnormalities or increased tendency for serious urinary tract infections. However, the long term prophylactic use of antimicrobial agents has the potential risk of not only the drug resistance but also the development of drug related adverse events. Taking all these facts and difficulties in antimicrobial treatment of infections into account, natural remedies composed of herbal agents have been used as an alterna- tive treatment to antimicrobial medications in an attempt to maintain similar efficacy and lower the side effects. Studies with such agents did show that some of these herbal remedies may treat UTI by several favorable effects. Furthermore, some of them may also be helpful in the prevention of future recurrent attacks (4). In present study we aimed to evaluate the potential pro- tective effects of ‘Tutukon’ application in the management of bacterial cystitis in rat model. MATERIAL AND METHODS Study design After approval by the Local Ethical Committee of Bagcilar Training and Research Hospital, twenty-eight male Sprague-Dawley rats weighting approximately 300-350 g were randomly divided into 4 groups. A 12/12-hour day&night cycle animal housing cage and ad libitum access to food and water were maintained to all rats DOI: 10.4081/aiua.2017.2.134 INTRODUCTION Being responsible for 95% of all symptomatic urinary tract infections (UTI), uncomplicated cystitis is the most common type of UTI (1). Although it can be treated in an effective manner with proper antibiotic selection, cys- titis is now accepted as a major public health issue due to its high prevalence. Moreover, the antimicrobial mis- Tuken_Stesura Seveso 20/06/17 09:35 Pagina 134 135Archivio Italiano di Urologia e Andrologia 2017; 89, 2 Treatment of cystitis with an herbal agent throughout the study. Group-1 constituted the control group (0.6 ml sterile saline injected into the bladder and fed with normal drinking water for 7 days). Group-2 con- stituted the no-treatment group (0.3 ml of sterile saline and 0.3 ml of Escherichia coli [E. coli] J96 strain with a dose of 108 colony forming units [CFU] injected into the bladder and fed with normal drinking water for 7 days). Group-3 and Group-4 represented the short and long term treatment groups, while rats in both groups received 0.3 ml of sterile saline and 0.3 ml of E. coli J96 strain with a dose of 108 CFU injected into the bladder and fed with 15 ml/day of Tutukon. Tutukon was applied by adding it into the drinking water for 7 and 14 days, in the last two groups respectively. At the end of study, the urine sam- ples were collected for culture and the bladders were har- vested for pathological evaluation. Operative technique Following 6-hours of fasting, a single intramuscular injec- tion of 60 mg/kg katemine and 10 mg/kg xylazine was applied to anesthetize the rats. Loss of reflex response to pinching of paws was used to control the efficacy of anes- thesia. Thenafter, the abdominal wall of each rat was shaved with electric clippers and the skin was cleansed with 10% povidine iodine (Poviodine, Dioagnokim Inc., Istanbul, Turkey). After a 1.5 to 2 cm lower abdominal wall incision, the abdominal wall muscles were separated with blunt dissection. The urinary bladders were isolated and exposed. Using a 2 ml syringe, the urine inside the blad- der was removed and the predefined amount of sterile saline or E. coli was injected into the bladder. After the replacement of the bladder to its original location, the abdominal muscles were approximated using 3/0 absorbable polyglactine (Vicryl, Ethicaon Inc., Somerville, NJ, USA) and the skin was closed with 2/0 silk (Perma- hand Silk, Ethicon Inc., Somerville, NJ, USA). The wounds were cleansed using 10% povidine iodine. At the end of the predefined period, the rats in each group were anesthetized using 60 mg/kg katemine and 10 mg/kg xylazine and the bladders were exposed applying the same technique used at the beginning of the study. Using a 1ml syringe, the urine in the bladder was aspirated in a sterile condition and sent for culture. The bladder was then har- vested for further pathological examination. While the harvested bladders were fixed in 4% neutral formaldehyde, embedded in paraffin blocks and 4-6μm sections were stained with haematoxylin and eosin, theurine samples were kept and transferred at 4°C and cultured for microbiological evaluation. The rats were then sacrificed. Herbal agent Tutukon (Laboratorio Miquel Y Garriga, S.A., Barcelona, Spain) is a plant based herbal agent compromised with a fixed dose of essential fatty acids, flavonoid quercatin, polysaccharides, rosamarinic acid, boldin and flavonglico- zides. It has been used as Resolutivo Regium for its antiox- idant, anti-inflammatory, diuretic, spasmolytic, antibac- terial and nephro-protective effects. The drug is available as hidrolate form in bottles of 600 ml. The recommended adult dose is 45 ml three times daily. Every 100 ml of the drug is composed aqueous distillate of the dried parts of 570 mg Enguisetum arvensis stem, 330 mg of whole plant of Spergularia rubra, 280 mg of Peumus boldus leaves, 170 mg of flowers of Opuntia ficus indica, 170 mg of flowers of Sideritis angustifolia, 170 mg of Rozmarinus officinales leaves, 170 mg of Cynodon dactylon rhizomes and 170 mg of Melissa officinalis leaves. Microbiological evaluation Urine cultures were performed by inoculating the 0.01 ml of rat urine aerobically at 35°C for 24 h on to Mac Conkey agars and streaking the entire plate surface to obtain quantitative colony counts. Histopathological examination All examinations were performed by a single experienced genitourinary pathologist. All samples were examined under the light-microscopy and level of edema (none, mild, moderate and severe), vascular congestion (none, mild, moderate and severe), level of inflammation (none, mild, moderate and severe), changes in transitional epithelium (fibrosis, calcification, mitosis, dysplasia), thickness of the epithelium (in millimeters) and epithe- lial cell layer (≤ 7 or > 7) were recorded. Statistical analysis Number Cruncher Statistical System 2007 Statistical Software (NCSS, LLC, Kaysville, UT, USA) was used for data analyses and statistical evaluation. The data were expressed as mean ± standard error of mean. Statistical comparisons between the groups were performed by the Kruskal-Wallis multiple comparison test and Dunn's multiple comparison test was used for comparisons of subgroups. Fisher's exact test and chi-square test were used for analysis of qualitative data. The differences were considered to be significant at a P value less than 0.05. RESULTS The pathologic evaluation of the bladder tissue samples with respect to the possible inflammatory changes induced by E. coli injection demonstrated a statistically significant difference regarding the degree of vascular dilation between the groups (p = 0.028) (Table 1). Dunn’s multi- ple comparison test revealed that degree of vascular dila- tion was lower in Group-1 when compared to Group-2 (p = 0.045), Group-3 (p = 0.048) and Group-4 (p = 0.044). On the other hand however, we were not able to note any significant difference with respect to the degree of edema, inflammation, the presence of fibrosis and the degree of calcification among the groups evaluated (Table 1). Although the uroepithelial thickness of the bladder was decreased in Group-2 rats; this difference was not statis- tically significant (p = 0.138) (Table 2). On the other hand, the number cell layer in bladder uroepithelium was significantly lower in Group-2 when compared with the Group-1 (p = 0.024), Group-3 (p = 0.024) and Group-4 (p = 0.045). Last but not least as an important parameter for urinary infection; the urine culture test results were negative for all rats of Group-1 and Group-4; while positive urine cultures were detected in 85.71% and 42.86% of rats in Group-2 and Group-3, respectively (p = 0.001). Tuken_Stesura Seveso 20/06/17 09:35 Pagina 135 Archivio Italiano di Urologia e Andrologia 2017; 89, 2 M. Tuken, M. Zafer Temiz, E. Yuruk, A. Orcun Kaptanagasi, K. Basak, F. Narter, A. Yaser Muslumanoglu, K. Sarica 136 DISCUSSION UTI, especially in the form of bacterial cystitis, is one of the most common bacterial infections, affecting the women more frequently than men. E. coli is the most common causative agent and histopathological findings of E. coli cystitis include infection induced interstitial edema, inflammatory cell infiltration and a decrease in the thickness of transitional epithelium of the bladder wall. Moreover, UTIs tend to recur. Recurrent UTI defines a condition in which the urinary tract is recur- rently infected with a pathogen causing inflammation. While E. coli is the predominant uropathogen responsi- ble for approximately 80% of recurrent disease, other causative organisms are Staphylococcus, Klebsiella, Enterobacter and Enterococci species (5-7). Although antibiotics are quite effective at providing clin- ical cure for UTIs in most of the cases, antibiotic resist- ance is globally increasing (8). There is a growing con- cern regarding antimicrobial resistance of pathogenic bacteria, particularly of E. coli with repeated antibiotic usage (9, 10). Although the resistance was initially described for agents limited to ampicillin, trimethoprim, sulphur-based antimicrobials or tetracyclines, it is now relevant to large families of agents including most ß-lac- tam antibiotics, aminoglycosides and fluoroquinolones (10, 11). Besides high resistance rates, antimicrobial agents also have side effects including disruption of the protective flora of the mouth, anal area, urethra and vagina, which results an increased risk of recurrent infections (12, 13). In addition, antibiotics can cause gen- eral adverse effects including palpitations, flushes, nausea, vomiting, diarrhoea, abdominal pain, rashes, headache and dizziness (13, 14). The presence of a nat- ural alternative that could prevent and treat UTI is preferable to any other treat- ment (8). Phytotherapeutic and herbal agents are well-studied alternatives to antimicrobial agents with documented efficacies. The most studied natural agent for UTI management is Vaccinium macrocarpon (Cranberry). Its benefical effects comes from hippuric acid content which acidifies the urine. It also has the potential anti- adhesive properties (5). Stothers enrolled 150 sexually active women for a one-year period in a randomized trial to evaluate the prophylactic effects of cranberry in the prevention of UTI. He showed that the cranberry prophylaxis resulted in a decrease in antibiotic use compared to placebo group and a statistically signifi- cant decrease in symptomatic UTI episodes (15). Haverkorn et al. also showed that 15 mL cranberry juice twice daily for one month resulted in decreased rates of bacteriuria (16). In a randomized, double-blind, placebo controlled trial including 153 elderly women revealed that usage of cranberry has resulted in sig- nificantly less bacteriuria with pyuria (17). A meta-analy- sis of 10 studies investigated the benefits of cranberry juice or tablets compared to a placebo control in 1049 patients susceptible to UTI. The study revealed that the cranberry products reduced the incidence of UTI by 35%, a statistically significant amount, over a 12-month period (18). Cranberry has been found to specifically inhibit hemagglutination of E. coli by expression of type 1 and P adhesin through the component compounds fructose and proanthocyanidins (19). Other natural treatment alternatives for UTI are Hydrastis canadensis (Golden seal or ground raspberry), Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape; Mahonia aquifolium), Berberis vulgaris (barberry), and Berberis aristata (tree turmeric). The common characteristic of these herbal agents is berberine content. Berberine is a plant alkaloid with a sig- nificant antimicrobial activity against a variety of organ- isms, including bacteria, viruses, fungi, protozoans, helminths, and Chlamydia. Cernakova et al. showed the growth inhibition effect of berberine on several bacteria, including both sensitive and resistant E. coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis strains (20). In an ex vivo/in vitro study, it has been demonstrated that pres- ence of 200 mcg/mL berberine sulfate in a culture medi- um consisting of a urinary pathogenic strain of E. coli iso- Table 1. Comparative evaluation of the pathologic findings of the rat bladders in all groups. Parameter Group-1 Group-2 Group-3 Group-4 p Vascular dilation None 4 (57.1%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0.028 Mild 1 (14.3%) 4 (57.1%) 2 (28.6%) 3 (42.9%) Moderate 2 (28.6%) 2 (28.6%) 3 (42.9%) 4 (57.1%) Severe 0 (0.0%) 1 (14.3%) 2 (28.6%) 0 (0.0%) Edema None 5 (71.4%) 3 (42.9%) 3 (42.9%) 3 (42.9%) 0.754 Mild 1 (14.3%) 2 (28.6%) 3 (42.9%) 2 (28.6%) Moderate 1 (14.3%) 1 (14.3%) 0 (0.0%) 2 (28.6%) Severe 0 (0.0%) 1 (14.3%) 1 (14.3%) 0 (0.0%) Inflammation None 5 (71.4%) 2 (28.6%) 3 (42.9%) 0 (0.0%) 0.610 Mild 1 (14.3%) 3 (42.9%) 3 (42.9%) 5 (71.4%) Moderate 0 (0.0%) 1 (14.3%) 1 (14.3%) 1 (14.3%) Severe 1 (14.3%) 1 (14.3%) 0 (0.0%) 1 (14.3%) Fibrosis Negative 7 (100%) 7 (100%) 7 (100%) 7 (100%) - Positive 0 0 0 0 Calcification Negative 7 (100%) 7 (100%) 7 (100%) 7 (100%) - Positive 0 0 0 0 Table 2. Histopathologic evaluation of the structural changes in the uroepithelium of rat bladders in all groups. Group-1 Group-2 Group-3 Group-4 p Thickness of uroepithelium (mm) 0.009 ± 0.025 0.063 ± 0.033 0.079 ± 0.03 0.096 ± 0.016 0.138 Number of cell layer in the uroepithelium 4.43 ± 0.79 2.86 ± 1.35 4.43 ± 0.98 4.29 ± 0.49 0.019 Tuken_Stesura Seveso 20/06/17 09:35 Pagina 136 137Archivio Italiano di Urologia e Andrologia 2017; 89, 2 Treatment of cystitis with an herbal agent lated from infected patients results in complete inhibi- tion of fimbrial synthesis (21). Arctostaphylos uva ursi (Bearberry) is another well studied herbal medicine for UTI. It has a direct antimicrobial effect due to its arbutin content which changes the bacte- rial cell surface characteristics. Arbutin is released in alka- line urine and for optimum treatment results, the urine pH should be alkaline (5). Turi et al. demonstrated that arbutin significantly increased the hydrophobicityof the bacterial cell surface of E. coli strains with decreasing the ability of bacteria to adhere to the host (22). Sharma et al. demonstrated that ethanol extract of some herbs such as Zingiber officinale, Punica granatum, Terminalia chebula, Ocimum sanctum, Cinnamomum cas- sia, Azadirachta indica and Ocimum sanctum have poten- tial antimicrobial effects against UTI pathogens such as E. faecalis, Gram-negative E. coli, K. pneumoniae and P. aeruginosa. The Authors suggested that plants are poten- tial sources of antimicrobial compounds (23). Many other herbals such as Barosma betulina (buchu), Apium graveolens (celery seed), Agrimonia eupatoria (agri- mony), Arctium lappa (burdock), Elymus repens (couch- grass), Hydrangea aborescens (hydrangea), Althea offici- nalis (marshmallow), Mentha piperita (peppermint) have been used successfully for treatment of UTI without comprehensive scientific researches (5). Tutukon is a medication composed of 8 different herbal ingredients including Equisetum arvensis, Spergularia rubra, Peumus boldus, Opuntia ficus indica, Sideritis angus- tifolia, Rosmarinus officinalis, Cynodon dactylon and Melissa officinalis with certain biological effects. The anti-inflam- matory effect is an important property of the drug (24). Equisetum arvensis has been traditionally used as a mild diuretic, anti-edematous, anti-inflammatory compound. It was also used for treating prostatitis, urinary inconti- nence and gonorrhea in the early 19th century (25, 26). Spergularia rubra is commonly used as diuretic and anti- septic agent for treating diseases related to the renal sys- tems in some regions (27, 28). Peumus boldus has strong inhibitory activities and strong antibacterial activity against Staphylococcus aureus strains and Streptococcus pyogenes due to its antimicrobic components including ethanol (29). Opuntia ficus indica commonly known as Nopal is called prickly pear cactusin the United States. Nopal is used for diabetes, hypercholesterolemia, obesi- ty, alcohol-induced hangover, colitis, diarrhea, benign prostatic hypertrophy (BPH) and atherosclerosis (30). Sideritis angustifolia is used to relieve the cramping that occurs commonly during menstruation, prepared alone or mixed with Sideritis foetens, in Spain (31). Rosmarinus officinalis has promising results in the case of urinary infections with Gram-positive bacteria and it is considerable alternative for the treatment of urinary infections (32). Cynodon dactylon possess antimicrobial, and antiviral activity and it has also been used to treat urinary tract infection, urinary calculi and prostatitis. Cynodon dactylon has been also used as an antidiabetic agent in traditional system of medicine in India (33). Melissa officinalis usually known as lemon balm is one of the oldest and it has been used traditionally to prepare tea in order to calming and anti-spasmolytic effects. Pharmacological investigations have shown that the most commonly known therapeutic properties of Melissa offic- inalis extract are sedative, carminative, antispasmodic, antibacterial, antiviral, anti-inflammatory, antioxidant, and neuroprotective (34). The present study shows that both short and long term oral administration of Tutukon has a potential effect against E. coli J96 strain induced bacterial cystitis while the effect is more prominent with the long term usage. The drug not only resulted in the eradication of patho- genic bacteria, but also increased the number of uroep- ithelial cell layer. Despite its beneficial effects on the uroepithelium, Tutukon treatment did not alter the inflammatory response of the bladder mucosa. In addi- tion, when comparing pyuria among the groups, the results clearly show that oral administration of Tutukon resulted in bacterial clearance. Application of oral Tutukon may be advantageous in situations where a high bacterial load exists in the urinary bladder. CONCLUSION Tutukon can be used as an alternative agent for the treat- ment of UTI. In addition to eradication of E. coli J96 strain, Tutukon also protects the uroepithelial cell layers. Further clinical trials are needed to evaluate the safety and efficacy of Tutukon treatment for management of UTI in humans. REFERENCES 1. Moura A, Nicolau A, Hooton T, Azeredo J. Antibiotherapy and pathogenesis of uncomplicated UTI: difficult relationships. J Appl Microbiol. 2009; 106:177. 2. Francesco MA, Ravizzola G, Peroni L, et al. Urinary tract infec- tions in Brescia, Italy: Etiology of uropathogens and antimicrobial resitance of common uropathogens. Med Sci Monit. 2007; 6:136. 3. Linhares I, Raposo T, Rodrigues A, Almeida A. 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Aerial parts of aque- ous extract of Cynodondactylon shows hypotensive effect in high fructose treated Wistar rats. Int J Res Pharm Biomed Sci International Journal of Research in Pharmaceutical and Biomedical Sciences. 2012; 3:585. 34. Hosseini R, Kaka G, Joghataei MT, et al. Assessment of neuro- protective properties of Melissa officinalis in combination with human umbilical cord blood stem cells after spinal cord injury. ASN Neuro. 2016; 8:1. Correspondence Murat Tuken, MD murattuken@hotmail.com Mustafa Zafer Temiz, MD dr_mustafazafertemiz@hotmail.com Emrah Yuruk, MD (Corresponding Author) emrah.yuruk@yahoo.com Ahmet Yaser Muslumanoglu, MD ymuslumanoglu56@hotmail.com Bagcilar Training & Research Hospital, Dept. of Urology Merkez M Mimar Sinan C 6. Sok. Bagcilar, Istanbul, Turkey Asuman Orcun Kaptanagasi, MD Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Biochemistry, Istanbul, Turkey asumanorcun@yahoo.com Kayhan Basak, MD Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Pathology, Istanbul, Turkey drkayhanbasak@yahoo.com Fehmi Narter, MD fehminarter66@gmail.com Kemal Sarica, MD saricakemal@gmail.com Kartal Lütfi Kirdar Training & Research Hospital, Dept. of Urology, Istanbul, Turkey Tuken_Stesura Seveso 20/06/17 09:35 Pagina 138