Chemical, Biological and Comparative Clinical Evaluation of Entoban to Determine Safety and Efficacy For The Treatment of Chronic Diarrhea

Abstract

i ABSTRACT: Diarrhea is the third most frequent disease that affects people of all ages. In spite of the drop in global mortality rate, diarrhea still accounts for more than 2 million deaths per annum. About two-thirds of the total annual deaths in Pakistan of children under five are due to diarrhea. Different drugs are prescribed to treat the symptoms of chronic diarrhea whereas an empirical mode of treatment with antibiotics considered viable when the infection is elevated in the community. However, the resistance of antibiotic is responsible as the main factor for treatment failure. The adverse effects, inadequate accessibility of allopathic medicines and antibiotic resistance have led to the resurgence of plant based drugs as an alternate treatment option. Traditional herbal medicines have now been proven to be safe and effective and being utilized to cure many disorders, including GI ailments. Herbal dosage forms have been shown to heal acute as well as chronic diarrheal diseases. In current study, standardized coded polyherbal mixture was formulated in hard gelatin capsule and syrup. Various physicochemical parameters including physical appearance, weight variation and disintegration time were calculated for the capsule. Average weight of 20 capsules was between 450 mg and 550 mg (with a mean of 506 mg ± 10%). The maximum time for disintegration was 6 min. It was found that alkaloids and tanning agents in Entoban syrup and capsules were within the specified limits. The different physicochemical parameters of Entoban syrup were assessed. Entoban syrup revealed brown color, characteristic odor and sweet taste. The pH of Entoban syrup was 3.7 and specific gravity was 1.324. Entoban capsules and syrup were in agreement with the acceptable microbial limit. The prospective validation ii was executed to validate the manufacturing process of Entoban syrup and to make sure that it fulfills the predetermined specifications. To execute prospective process validation, critical process parameters were recognized, the protocol and reports were developed. Three consecutive batches of Entoban formulations were analyzed to reassure reproducibility of the results. It was found that the manufacturing process of syrup was reproducible for these batches and every parameter analyzed was in accordance with the specifications and validated according to the guiding principles stated in prospective process validation. An antimicrobial activity was evaluated against five gram negative bacterial cultures namely Salmonella enteric, Eschericia coli, Shigella dysenteriae, Pseudomonas aeruginosa, Vibrio cholera and one gram positive bacterial culture Staphylococcus aureus by agar well diffusion method. The prepared Entoban formulation inhibited the growth of these organisms. The stability study on Entoban syrup demonstrated no changes in all the tested physicochemical parameters during 24 hours, 48 hours and 72 hours. Entoban syrup and capsules have outstanding antioxidant ability with 8.5 and 10.3 μg/ml IC50 values respectively. The reducing ability of Entoban syrup and capsules increased in a dose dependent manner. It can be inferred that antioxidant activity could be helpful in slowing down the development of a variety of diseases of gastro intestinal tract associated with oxidative stress. Anti-inflammatory and anti-urease activities were determined on Entoban dosage form design to overcome H. pylori-associated inflammation. Anti-H.pylori and cell cytotoxic activity of Entoban syrup and capsule formulations was conducted by serial dilution method and cell survival assay, respectively. Anti-adhesion activity of Entoban was then evaluated. Entoban did iii not demonstrate anti-adhesion outcome against the cell co-culture of H. pylori. Additionally, Entoban syrup formulation suppressed H. pylori-induced IL-8 more as compared to capsule formulation. Entoban syrup and capsules revealed antiurease activity increased in a dose dependent way just like standard (Thiourea) using the indophenol method. The formulations have an excellent antiurease potential that can be used in the cure of different problems occurring due to urease enzymes. The Lipoxygenase inhibition activity of polyherbal formulation syrup and capsules increased in a dose dependent manner and revealed that formulations under test have good potential of lipoxygenase inhibition. The quantization of biomarkers gallic acid and berberine was explored in polyherbal formulation Entoban capsule and syrup. HPTLC was performed to evaluate the presence of gallic acid and berberine applying toulene–ethyl acetate–formic acid–methanol in ratio of 12:9:4:0.5 v/v and ethanol–water–formic acid in ratio of 90:9:1 v/v, as the mobile phase, respectively. The present standardization provides specific and accurate tool to develop qualifications for identity, transparency and reproducibility of biomarkers in Entoban formulations. Entoban medicinal plant syrup was analyzed for As, Cd, Pb and Hg by flame atomic absorption spectroscopy (FAAS). Contents of heavy metals in the examined samples were in the range: As (0.074–10.0 ppm); Cd (0.020–0.3 ppm); Pb (0.00–10.0 ppm) and Hg (0.00–1.0 ppm). Results were compared with permissible limit acceptability intake (AHPA). According to determined amounts of heavy metals, the investigated Entoban syrup samples were validated and considered safe for human consumption. iv In order to investigate the antidiarrheal activity albino mice were treated with Entoban at dosages of 2.5, 5, 10 mg/kg. For evaluation of acute toxicity the animals were administered orally with 1 or 5 g/kg of the Entoban capsule aqueous extract, maintained under standard laboratory conditions. Entoban was given in quantity of 50 mg/kg, 100 mg/kg and 200 mg/kg body weight for a period of 28 days for determining sub chronic oral toxicity. The data collected were summarized as mean ± SEM. Entoban showed significant inhibition of diarrhea in dose dependent manner. Entoban was not found to be the reason of death in albino mice at the specified doses of 1 g/kg or 5 g/kg. Toxicity indications including the loss of hair, mucus membrane (nasal), loss in weight, lacrimation, drowsiness, gait and tremors were also not observed. The study gave evidence of good tolerance of Entoban and the absence of detrimental effects on functional state of the vital organs of experimental animals in acute and sub chronic oral toxicity test. For the evaluation of the clinical safety and efficacy of Entoban for treating patients of chronic diarrhea, a controlled, randomized, multicenter clinical trial was conducted in Sharafi Goth hospital Korangi Karachi, Nawaz Salik Hospital in Rawalpindi and Victoria Hospital in Bahawalpur. The current trial enrolled 150 patients fulfilling the inclusion criteria, among them 95 were males and 55 were females. Among the total enrolled patients; 10 patients belonging to the test group and 7 of the control group did not receive the allocated treatment due to unknown reasons. Further 13 were dropped out during the treatment and 8 discontinued intervention due to side effects in control group. In test group, 15 were dropped out during the treatment and 4 discontinued intervention due to side effects. Overall 47 and 46 in control and test group completed the study. The trial was registered at http://www.ClinicalTrial.org, a service of the US v National Institutes of Health (registry No. NCT02642250). A block-randomization procedure, with a block size of 4, was adopted to assign participants either to treatment with allopathic therapy or with a phytomedicine-based formulation. Metronidazole tablets (Flagyl) in strength of 400 mg manufactured by Sanofi-aventis Pakistan limited was used in a control group for 7-10 days. The test group received Entoban capsule 400mg tds, every 8 hours for five days. The stool frequency was documented quantitatively, and semiquantitative factors including consistency of stool, abdominal pain, distention and incomplete evacuation were noted. Stool DR was noted at baseline and thereafter 2nd and 4th weeks of treatment. Adverse reactions were evaluated by patient history and physical assessment on daily basis every 3 days until the completion of study. The quantitative evaluation of daily bowel frequency was the primary outcome of the study and evaluation of clinical symptoms including consistency of stool, distention, abdominal pain and feeling of incomplete evacuation were the secondary outcome. Patients’ characteristic data was demonstrated as the mean ± standard deviation (SD). A χ2 test using a 2 × 2 contingency table was used to check for a statistically significant difference in the cure rate as well as in the proportions of other categorical variables between 2 treatment groups. A Wilcoxon signed-rank test was applied to analyze the intensity of symptoms at baseline (T0), after 2 weeks (T2) and 4 (T4) weeks of treatment, expressed through median values and interquartile ranges (IQRs) (p < 0.05 was considered significant). It has been found in current study that 39(84.78%) in test group and 37(78.72%) in control group showed complete improvement who completed the study. Participants in the test group exhibited a marked reduction in symptoms; the symptom score was decreased from 3 (maximum) to 1 (minimum) or 0 (absent) in most of participants. Participants in the test group with complete improvement exhibited significant decreases in overall GI symptoms from baseline (T0)—with a median of 8 and an IQR of 6 to 10, to week 2 (T2)—with a vi median of 3 and an IQR of 2 to 5, and to 1 month after treatment (T4)—with a median of 4 and an IQR of 3 to 6. There was a significant decrease in symptoms was observed for participants in the test group with no improvement, also from T0—with a median of 9 and an IQR of 6 to 10, to T2—with a median of 3 and an IQR of 2 to 5, and to T4—with a median of 4 and an IQR of 3 to 6. The intensity of individual symptoms in the test group was monitored and statistically significant improvement was recorded after treatment. Participants in control group with improvement exhibited a statistically significant reduction in the overall diarrheal symptom score, from T0—with a median of 9 and an IQR of 6 to 10, to T2—with a median of 4 and an IQR of 3 to 6, and toT4—with a median of 4 and an IQR of 3 to 7. No significant improvement in symptoms was observed, however, for the participants with no recovery, showing scores from T0—a median of 9 and an IQR of 6 to 10, to T2—a median of 6 and an IQR of 4 to 8, and to T4—a median of 8.5 and an IQR of 5 to 10. Patients in control group reported more side effects as compared to test (p value < 0.0001). Around 20% patient reported adverse effects in test group however in control group 55.31% reported adverse effects. The major adverse effects reported in control group were anorexia (14.89%), metallic taste (10.63%), dizziness (8.51%) and vomiting (4.25%). Among test group the major adverse effects reported were metallic taste (6.52%), anorexia and headache (4.34%). Entoban possesses considerable therapeutic efficacy for the treatment of chronic diarrhea and it is comparable with the standard conventional Metronidazole therapy. Entoban revealed high cure rates of chronic diarrhea with little or no side effects as compared to Metronidazole. Furthermore Entoban improves the well-being off over all sign and symptoms of diarrhea and has better compliance. vii Entoban, also exhibits strong anti-inflammatory activity against inflammation in gastric epithelial cells induced by H. pylori. Single poly herbal drug formulation with two modes of action against H. pylori can act as a double bladed sword ensuring complete suppression of H. pylori and its associated inflammation. Herbal drugs like Entoban are an excellent candidate for future in vivo and clinical studies, which are required in order to establish its definitive role as chemotherapeutic agent against H. pylori-induced gastric disease.