key: cord-1046833-ba4vah3k
authors: Maddahi, Seyedeh Zahra; Jokar, Assie; Kamalinejad, Mohammad; Behnampur, Nasser
title: The efficacy of Jujube syrup on the prevention of drug‐induced hepatotoxicity in pulmonary tuberculosis patients: A pilot randomized double‐blind placebo‐controlled clinical trial
date: 2021-12-22
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.902
sha: 2f0041750809fc9eacf6b6a5e6dc1bed1d001b84
doc_id: 1046833
cord_uid: ba4vah3k

Liver injury is the most common complication of anti‐tuberculosis drugs that can cause significant problems. The study aimed to determine the effectiveness of Jujube syrup on the prevention of antituberculosis drug‐induced hepatotoxicity (DIH). This pilot randomized double‐blind study was conducted based on a placebo‐controlled design in patients with tuberculosis (TB). The patients were divided into two groups based on the block random allocation method and received 10 cc of jujube or placebo syrup per day. The liver enzyme levels were assessed as primary outcomes, and the severity of cough, anorexia, and nausea along with the quality of life (QOL) was assessed as secondary outcomes. Finally, eight and nine patients in the jujube and placebo groups completed the study, respectively. In the second week of the study, 27.3% of the patients in the placebo group developed hepatotoxicity. Moreover, there was no liver toxicity in the jujube group. This difference between the two groups was statistically significant (p = .037). Furthermore, the severity of cough in patients in the jujube group decreased significantly during weeks 2 and 4. The QOL significantly improved in the jujube group, compared to the placebo group. This study suggested that Jujube syrup could prevent anti‐TB DIH. It can also improve the severity of cough and the QOL in pulmonary TB patients.

Checklist" that is attached. Sciences, Tehran, Iran. It should be noted that after washing and drying, this fruit was ready to prepare the medicine.

The total amount of phenolic compounds was measured using the Folin-Ciocalteu method (as a reagent), and Gallic acid was used as the standard phenolic compound for measurements. The results were then expressed in milligram of Gallic acid per gram of extract.

Folin-Ciocalteu is one of the most common methods for measuring phenolic compounds. The basis of this method is the reduction of Folin reagent by phenolic compounds in an alkaline environment and the formation of a blue complex that shows the maximum absorption at a wavelength of 765 nm. 19 For this purpose, one ml of Gallic acid solution in different dilutions (20, 40, 60, 80 , and 100 μg/ml) was mixed with 5 ml of diluted Folin-Ciocalteu reagent in a ratio of 1:10 and incubated at room temperature. After 10 min, 4 ml of 7.5 mg/ml sodium carbonate solution was added, and the final mixture was incubated at room temperature for 30 min away from light. After this period, the uptake of each sample of Gallic acid was measured at 765 wavelengths.

This experiment was repeated three times for each of the gallic acid dilutions. Following that, based on the obtained absorption information, the gallic acid calibration curve was drawn as a linear absorption curve against concentration. The same method was used for the sample; however, instead of 1 ml of gallic acid, 1 ml of aqueous extract of 1.25 mg/ml was used, and the total phenolic content was calculated using the standard curve of Gallic acid. As a result, due to the reaction of the extract with the Folin Cicalto reagent and based on its comparison with standard gallic acid solutions and according to the linear equation obtained from the standard gallic acid curve, 7.7 μg/dl was obtained in 5 cc of syrup.

In this study, the spectrophotometer method was used along with an aluminum chloride solution as a reagent and routine, as well as a standard flavonoid compound for assays. Calorimetric technique was used for flavonoids estimation. The method of aluminum chloride colorimetric is that aluminum chloride forms acid stable complexes with the C-4 keto group and the C-3 or C-5 hydroxyl group of flavones and flavonols, which have the highest absorption at 415 nm. 20 For this purpose, 2.5 ml of routine solution in different dilutions (20, 40, 60, 80 , and 100 μg/ml) with 2.5 ml of an ethanol solution of aluminum chloride (20 mg/ml) was incubated at room temperature. After 40 min, the absorbance of each sample was measured at 415 nm. This experiment was repeated three times for each of the routine dilutions, and a routine calibration curve (absorption line curve against concentration) was drawn based on the obtained absorption information. The same method was performed for jujube aqueous extract; however, routine extracts with concentrations of 1.25 and 2.5 mg/ml were used in this study. The test was negative and the concentration of flavonoids was zero.

Initially, 100 g of dried jujube fruit without seeds (pulp) were added to 500 cc of distilled water and boiled on a lamp flame in the laboratory for 10 min. After cooling, the content of the container was mixed thoroughly, and it was then strained using a suitable filter. In the next stage, the water in the extract was evaporated using a Bainmarie to obtain a dry extract. Therefore, 15 g of dry extract was obtained from 100 g of dried jujube. As a result, 10% of jujube syrup was obtained using the USP34 simple syrup formula with 66.7% sugar concentration. 

The statistical population included all new cases of pulmonary TB, living in Gorgan, Aliabad, Azadshahr, Ramian, and Gonbad in Golestan province, Iran. The patients were selected from urban and rural health centers affiliated to the Golestan University of Medical Sciences. Patients who met the following criteria were enrolled to the study: those were willing to participate with the study and gave written consent forms, age range of 18-68 years, diagnosed with pulmonary tuberculosis by an infectious disease specialist or a physician and planned to receive a standard antituberculosis treatment regime.

The participants were excluded if they met the following criteria: active liver and kidney diseases, pregnant or lactating women, patients with elevated serum alanine aminotransferase enzyme (ALT) and aspartate aminotransferase (AST) (more than 3 times of upper normal limit), the occurrence of any severe drug side effects in the course of the study, and consumption of less than half of the medicine for any reason.

Based on the values obtained from a pilot study at the level of α = .05 and β = .20, the sample size was determined at 24 cases in each group based on the average comparison formula.

The random allocation of participants based on computerized-system with a block of two and four was performed by a statistician who was not involved in recruitment of patients. The randomization list was then sealed in an opaque envelope. A research assistant opened envelops and treatments were assigned to patients who met the inclusion criteria, accordingly. All of medicines were obtained from the Faculty of Pharmacy, numbered, and coded with a label and only a statistician knew about the details of the study drugs that the patients received. As a result, the outcome assessors were blind. The eligible participants were randomly allocated to the jujube syrup or placebo group in a 1:1 ratio. Jujube syrups and placebo were similar in appearance (in glass bottle of the same shape and size) and drug concentration. In addition, the remaining were hidden from the study executors and participants until the end of data analysis (double-blind).

The patients with newly pulmonary TB were registered after diagnosis in the TB register system. The first-line anti-TB drugs, including Isoniazid (5 mg/kg/day), rifampin (10 mg/kg/day), ethambutol (15 mg/kg/day), and pyrazinamide (25 mg/kg/day), were administered once a day at fasting time. The jujube syrup group was given a dose of 10 cc per day (5 cc twice daily), 20 min after breakfast and 20 min after dinner. The patients were assigned to 2 groups as soon as the diagnosis of TB was confirmed by a TB physician and were routinely treated with anti-TB by DOTs according to the national TB guideline and in dosage calculated according to the patient's body weight.

The amount of syrup consumed was evaluated weekly on the days of follow-up to check the patient's compliance and adherence.

Furthermore, the side effects were recorded daily by DOTs executives. After the initiation of the study, as well as at the end of weeks two and four, the patients were referred for clinical evaluation and sampling of liver enzymes; then they were visited by the researcher.

In addition to the daily care, participants were evaluated by telephone every week for the incidence and severity of drug side effects or any other complications. Although the required information was explained orally, all necessary advice about the amount of consumption, duration of treatment, and how to use the medicine were provided to patients in written notes. In order to match and create similar behaviors in all participants in this study, the protocol of how to take the drug was reviewed occasionally. Moreover, the patients were given the necessary training and they were asked to contact the researcher by telephone if they had any questions or problems during the treatment period.

Blood samples were drawn to evaluate liver enzymes as the primary outcome at the beginning of the study, as well as the end of weeks 2 and 4. In total, 2 cc of blood samples were transported to the laboratories, and then after serum separation with cold chain were sent to Gorgan Health Center laboratory as soon as possible, so that all samples were measured in a common laboratory and with the same kits using a BS-480 (Mindray) auto-analyzer (manufactured by Pars Azmun, Iran). The Visual Analogue Scale (VAS) was completed on day 0 and at the end of weeks 2 and 4 for evaluating cough, anorexia, and nausea, with scores of 0 and 10 indicating "no sign" and "severe signs," respectively. The validity of the VAS was assessed by an expert pharmacist. Moreover, the standard QOL form (SF36) with confirmed validity and reliability in Iran was completed by the researcher on day 0 and at the end of week four. 21 Hepatotoxicity was defined as follows: 1-elevated serum ALT or AST more than 3 times of upper normal range with one of the symptoms of nausea and vomiting, anorexia, weakness, pain in the right upper quadrant of the abdomen and jaundice; or 2-elevated serum ALT or AST more than 5 times of upper normal range without clinical symptoms. In either case, the patient was referred to an infectious disease specialist. If the doctor stopped taking anti-TB drugs, the syrups would be stopped. The jujube syrup or placebo would be started again simultaneously with the re-use of anti-tuberculosis drugs.

The data were analyzed in SPSS software (version 18) and R software. Subsequently, the quantitative and qualitative variables were described by mean ± SD, as well as frequency distribution tables and graphs, respectively. Moreover, the normality of quantitative trait distribution and the homogeneity of the variance-covariance matrix were assessed using the Shapiro-Wilk test and the Mauchly's Test of Sphericity, respectively. Repeated analysis of variance was also used to evaluate the effect of medicine, time, and drug-time interaction. Due to the random assignment of individuals into two groups, repeated measures analysis of covariance was utilized to adjust the effect of intervening variables. A p-value of less than .05 was considered statistically significant.

In total, 82 patients were evaluated regarding the eligibility criteria, and 48 cases were excluded from the study due to the lack of complete admission requirements (n = 41) and decline to participate in the study (n = 7). Thirty-four patients were enrolled to the study. In the first follow-up, which was performed during the first two weeks of the study, 12 patients were excluded from the study. In the jujube group, six cases were excluded from the study due to COVID-19 (n = 4), migration (n = 1), and medication discontinuation (due to side effects) (n = 1). Similarly, six cases in the placebo group were excluded from the study due to COVID-19 (n = 3), migration (n = 2), and unwillingness to continue the study (n = 1). Out of 11 patients who were analyzed at the end of the second week in two groups, three spectively. According to Cohen, the former is classified as a large effect size, and the latter is regarded as the medium effect size.

Cough severity was evaluated as a secondary outcome with VAS during weeks two and four of the study. The trend of changes in mean cough is shown in Figure 4 . The mean severity of coughs in the week two (0.046) and four (0.024) was reduced significantly in the jujube group. Gastrointestinal symptoms, including nausea and anorexia, were also evaluated as other secondary outcomes by the VAS scale; however, no significant difference was observed between the two groups in this regard. The QOL measured by the SF36 questionnaire at the end of the first month was improved in the jujube group compared to the placebo, which was statistically significant (p = .032). This level of significance was observed in the physical dimension (p = .01); however, the psychological dimension was close to the significance level (p = .096). The comparison of the mean of the secondary outcomes between the 2 groups is given in Table 3 .

During the study, no significant adverse reactions were found regarding the consumption of the jujube syrup. In addition, no patients ceased jujube treatment because of its adverse reactions.

Furthermore, serious adverse effects related to anti-TB drugs were not noted in this study. Additionally, no case of acute hepatic failure or fulminant hepatitis was detected in this study. There were no significant differences between the two groups regarding the incidence of mild adverse effects (nausea, itching, anorexia, and heartburn).

The DIH is an important complication of anti-TB drugs that can occur during the first two months of treatment, especially when isoniazid, rifampin, and ethambutol are used concomitantly. The incidence rate The effectiveness of jujube was determined by comparing ALT and AST levels, as well as the incidence of hepatic toxicity in the jujube and placebo groups. Endpoint analysis was performed at the end of week 4, since most enzymatic changes occurred in the first month of treatment during the studies. 22 The results of study conducted by Baghaei and Tabarsi reported that the highest probability of hepatotoxicity occurred during the first two weeks, as observed in this study. 22, 23 In the present study, none of the patients were hepatotoxic at the end of the first month, and the mean enzyme levels in the placebo group increased as expected due to anti-TB drugs during the first two weeks of treatment. Moreover, no increase was observed in the enzyme levels in the jujube group. However, it was found that this difference between the two groups was significant regarding the levels of AST enzyme, and it was not significant in terms of ALT.

In line with these results, the study of Luangchosiri et al., which aimed to "determine the effect of silymarin for the prevention of anti-TB drug-induced liver injury", revealed that the maximum ALT level during 4 weeks after initiation of anti-TB treatments was Many studies suggested that anti-TB drugs cause hepatotoxicity or liver injury mainly due to excess free radical formation.

Antioxidants are compounds that inhibit the activity of free radicals and prevent their oxidative attacks. Additionally, they protect the body's cells from the destructive effects of these compounds by inactivating them. The mechanism of these antioxidants includes the prevention of the extension of oxidation chain reactions by hydrogen atoms transfer to the formed free radicals. Among antioxidants, phenolic compounds are the most effective because they are good hydrogen donors. 24 According to studies, the antioxidant activity of different parts of herbs depends on the total amount of their phenolic compounds. 25 Considering that the total phenol content of the jujube syrup was 7.7 micrograms per deciliter in 5 cc of syrup, the hepatoprotective effects of jujube can be attributed to its phenolic compounds.

Phenols are formed in response to environmental stresses in plants, and due to their hydroxyl groups, they can act as electrons or hydrogen donors and neutralize free radicals. 24 In this study, the mean severity of coughs in weeks 2 and 4 was statistically and significantly reduced in the jujube group compared to the placebo group. New studies have also confirmed the antitussive effects of jujube in children and also in dry cough. 26, 27 The

Ziziphus jujube contains abundant tannins, which have an effect on the pharyngeal-laryngeal mucosa and modulate them to decrease exudation, it provides a protection against the contact of any antigen (pathogen/allergen), and tannins also have local antimicrobial action which annihilates the microbes coming in contact. Thereby, tannins

can justify the antitussive effects of jujube. 27 Although the improvement in constipation was not a consequence of the current study, 18% of the patients explicitly reported an improvement in defecation status in the jujube group, which appears to be due to the abundant mucilage in the jujube. According to the results of several studies, it is recommended to focus more on this issue in future studies. 28 In general, QOL is expected to increase in patients after treatment initiation and progression of the recovery process. 

Regarding the strengths of the study, one can refer to the novelty, existence of a control group, and provision of a new method to prevent the drug-induced liver enzyme disorders in TB patients. The multicenter nature of this study meant that none of our patients were out of the same center at the same time and did not visit each other. This results in the randomization and blending without the slightest problem. Another advantage of this study was the administration of the drug in the syrup form, which was well received by the patients since it was not the same as all TB drugs that are in tablets.

The advantages of the syrup (Oral liquid dosage forms) include ease of swallowing in patients with dysphagia and elderly people; moreover, the dosage can be adjusted easily. 29

The limitation of this study was the small sample size of subjects in both groups. We were unable to recruit tuberculosis patients to the expected number from sample size calculation due to coronavirus epidemic and safety reasons. Accordingly, the research procedure had to be terminated, and the study was conducted as a pilot.

According to the results of this study, 10 cc of jujube syrup per day for four weeks can play a preventive role against hepatotoxicity caused by anti-TB drugs without any significant side effects. It seems that the presence of phenols and the antioxidant properties of jujube can be one of the main mechanisms of liver protection. It can also be useful in controlling the cough in TB patients and improving their QOL. However, a study with larger sample size is required to confirm the results of this study.

The authors thank to all the staff in the health centers and the officials of the laboratories located at Gorgan, Ramian, Azadshahr, Aliabad, and Gonbad in Iran. Moreover, deepest gratitude goes to all the TB officials of the urban centers and the health workers of the rural health houses who had the necessary cooperation with the researcher to achieve the best plan. The authors sincerely thank all the TB patients and their family members who participated in conducting this study. This study was extracted from a Ph.D. dissertation sponsored by Mazandaran University of Medical Sciences, Sari, Iran.

All authors have made significant contributions to formation of concept, design of the study, the collection, analysis and interpretation of data, final conclusion, writing of the manuscript and the response to the reviewer's comments. All authors have seen and approved the final version of the article. And they all agreed to be accountable to all aspects of the work.

The authors declare no conflicts of interest.

The data are not publicly available due to privacy, legal, and ethical restrictions. 

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