AlMatar Manaf, Var Işıl, Kayar Begüm, Eker Emel, Kafkas Ebru, Zarifikhosroshahi Mozhgan, Köksal Fatih
Department of Biotechnology, Institute of Natural and Applied Sciences (Fen Bilimleri Enstitusu), Cukurova University, Adana, Turkey.
Department of Food Engineering, Agricultural Faculty, Cukurova University, Adana, Turkey.
Curr Pharm Biotechnol. 2019;20(4):317-326. doi: 10.2174/1389201020666190308130343.
The global rise of multi-drug resistant M. tuberculosis demands unconventional treatment to enhance the efficiency of current drugs. Punica granatum, which is known as pomegranate, is considered as a member of the Punicaceae family. Pomegranate, which is broadly documented for its activity against a wide spectrum of bacterial pathogens, deserves further scrutiny in this respect.
Within this scope, this study investigated the effect of fresh pomegranate juice (FPJ) on the antibacterial activity of anti-tuberculosis drugs (Rifampin (R) and Isoniazid (INH)) against MDR-TB clinical isolates. The drug resistance profiles in M. tuberculosis clinical isolates were determined by susceptibility test using BACTEC MGIT 960 system. Four concentrations of fresh pomegranate juice (FPJ) (5%, 10%, 15%, and 20%) were evaluated in combination with R and INH at a dose range of (1.0 µg/ml) and (0.1 µg/ml), respectively against the MDR-TB isolates by the BACTEC MGIT 960 system. Moreover, this study scrutinized individual phenolic compounds of FPJ by using highperformance liquid chromatography (HPLC). The total polyphenols (TP), total flavonoid (TF), total anthocyanins content (TAC), and the antioxidant capacity were also assessed in FPJ.
Synergistic effects were observed between R and INH with FPJ against all tested strains. However, combination therapy of rifampin was more effective than isoniazid one. Therefore, the combination of R and FPJ has been used against (27) MDR-TB clinical isolates. 5% of FPJ plus R (1.0 µg/ml) were found to suppress the growth of one isolates for first group (INH and R resistant). However, 5% of FPJ demonstrated no synergistic impact with R for second (SM, R and INH resistant) and third group (INH, EMB, R and SM resistant). Moreover, 10% of FPJ and R (1.0 μg/ml) inhibited the bacterial growth of three isolates of first group and two isolates and one isolate for second and third group, respectively. Remarkably, 15% of FPJ plus R (1.0 µg/ml) appeared to inhibit the growth of MDR-TB isolates for all tested groups indicating a strong synergistic effect. Regarding H37RV, the complete inhibition of the bacterial growth was found to occur at 15% and 20% concentrations of FPJ only. Minimum inhibitory concentration (MIC) of FPJ ranged from (4% to13%) for first group and from (10% to15%) for second and third group. Thus, FPJ at 15% inhibited 100% of bacteria for all tested isolates (MIC100% =15%). Phenolic compounds identified in FPJ were gallic acid, benzoic acid, syringic, folic acid, pelargonidin, naringin+ellagic acid, naringenin, chlorogenic acid, caffeic acid, catechin, myricetin, kaempferol, quercetin, cyanidin-3-glycoside, p-cummaric acid, ferulic acid, and rutin. Total phenolic (TP), total flavonoid (TF), and total anthocyanin (TA) content were 841.5 mg/L, 638.73 mg RE/L, and 47.43 mg/L, accordingly.
Overall, FPJ displayed synergistic effect with R against MDR-TB clinical isolates due to its high content of polyphenol and antioxidant capability.
全球耐多药结核分枝杆菌的增多需要非常规治疗方法来提高现有药物的疗效。石榴（Punica granatum）属于石榴科。石榴对多种细菌病原体具有活性，这一点已有广泛记载，因此在这方面值得进一步研究。
在此范围内，本研究调查了新鲜石榴汁（FPJ）对耐多药结核临床分离株的抗结核药物（利福平（R）和异烟肼（INH））抗菌活性的影响。使用BACTEC MGIT 960系统通过药敏试验确定结核分枝杆菌临床分离株的耐药谱。通过BACTEC MGIT 960系统分别以（1.0 µg/ml）和（0.1 µg/ml）的剂量范围，评估了四种浓度（5%、10%、15%和20%）的新鲜石榴汁（FPJ）与利福平和异烟肼联合对耐多药结核分离株的作用。此外，本研究使用高效液相色谱法（HPLC）对FPJ中的单个酚类化合物进行了分析。还评估了FPJ中的总多酚（TP）、总黄酮（TF）、总花青素含量（TAC）以及抗氧化能力。
观察到利福平和异烟肼与FPJ联合对所有测试菌株均有协同作用。然而，利福平联合治疗比异烟肼联合治疗更有效。因此，利福平与FPJ的联合用于对抗27株耐多药结核临床分离株。发现5%的FPJ加1.0 µg/ml的利福平可抑制第一组（对异烟肼和利福平耐药）中一株分离株的生长。然而，5%的FPJ与利福平联合对第二组（对链霉素、利福平和异烟肼耐药）和第三组（对异烟肼、乙胺丁醇、利福平和链霉素耐药）未显示协同作用。此外，10%的FPJ和1.0 μg/ml的利福平分别抑制了第一组的三株分离株、第二组的两株分离株和第三组的一株分离株的细菌生长。值得注意的是，15%的FPJ加1.0 µg/ml的利福平似乎对所有测试组的耐多药结核分离株的生长均有抑制作用，表明有很强的协同效应。对于H37RV，仅在15%和20%浓度的FPJ时发现细菌生长完全受到抑制。FPJ对第一组的最低抑菌浓度（MIC）范围为4%至13%，对第二组和第三组为10%至15%。因此，15%的FPJ对所有测试分离株均能抑制100%的细菌（MIC100% = 15%）。在FPJ中鉴定出的酚类化合物有没食子酸、苯甲酸、丁香酸、叶酸、天竺葵素、柚皮苷 + 鞣花酸、柚皮素、绿原酸、咖啡酸、儿茶素、杨梅素、山奈酚、槲皮素、矢车菊素 - 3 - 糖苷、对香豆酸、阿魏酸和芦丁。总酚（TP）、总黄酮（TF）和总花青素（TA）含量分别为841.5 mg/L、638.73 mg RE/L和47.43 mg/L。
总体而言，由于FPJ含有高含量的多酚和抗氧化能力，其与利福平联合对耐多药结核临床分离株显示出协同作用。
