- Email: [email protected]
Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis
Journal Pre-proof Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis Nihong Lu, Yongrui Yang, Jun Liu, Jie Li, Bing Ouyang, Jiawei Xia, Yingrong Du PII:
S0882-4010(19)31924-2
DOI:
https://doi.org/10.1016/j.micpath.2020.103971
Reference:
YMPAT 103971
To appear in:
Microbial Pathogenesis
Received Date: 3 November 2019 Revised Date:
31 December 2019
Accepted Date: 6 January 2020
Please cite this article as: Lu N, Yang Y, Liu J, Li J, Ouyang B, Xia J, Du Y, Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.103971. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis.
Nihong Lu, Yongrui Yang, Jun Liu, Jie Li, Bing Ouyang, Jiawei Xia, Yingrong Du*
Department of Respiratory and Critical Care, Kunming Third People's Hospital ，Kunmin， Yunnan ， China ， 650041
*Corresponding author Yingrong Du ， Department of Respiratory and Critical Care, Kunming Third People's HospitalKunmin， Yunnan ， China ， 650041 Tel & Fax: 0086-0871-63513914, Email: [email protected]
Abstract Tuberculosis is a devastating disease responsible for approximately 1.5 million deaths annually especially in developing countries. Although there is recommended and standard treatment for tuberculosis but the non-adherence of the patients to the lengthy treatment, adverse effects of the drugs and the emergence of multi-drug resistant strains hurdles the management of this devastating disease. This study examined the anti-mycobacterial activity of a plant derived triterpenoid, sophoradiol, against the drug-resistant strains of Mycobacterium tuberculosis and also in murine model of tuberculosis. The results showed that sophoradiol exhibits remarkable activity against the H37RV strain with an MIC of 8.5 µg/mL. The MIC of sophoradiol against the drug resistant strains of M. tuberculosis (CX1 to CX5) ranged from 9 to 16 µg/mL. Additionally, sophoradiol exhibited a bactericidal activity against H37RV strain with MBC equal to 2X MIC. Drug interaction studies showed that sophoradiol exhibits additive interaction with isoniazid and synergistic interaction with rifampicin. In the mice model of tuberculosis, sophoradiol also exhibited remarkable efficacy. Finally, cytotoxicity assays showed that sophoradiol exhibits negligible toxicity against the normal human breast cell lines. Taken together, it is concluded that sophoradiol may prove beneficial lead molecule for the management of tuberculosis.
Keywords: Tuberculosis; Triterpenoids; Sophoradiol; Mycobacterium; Drug resistance.
Introduction Among infectious diseases, tuberculosis is one of the devastating diseases causing huge number of deaths every year world over. Annually more than 1.5 million mortalities are caused by tuberculosis world over. Because of inefficient diagnostic assays and treatment strategies, tuberculosis causes more damage in developing countries [1]. It has also been reported that 1/3 of the HIV related deaths are caused by tuberculosis. As per the reports, 2 billion people are infected by Mycobacterium tuberculosis globally [2]. The emergence of drug resistance strains has further worsened the problem. M. tuberculosis resistant to rifampicin and isoniazid (two of the major first line anti-tuberculosis drugs) have also been reported [3]. Additionally, multidrug resistant strains of M. tuberculosis (MDR-TB) have also been reported. MDR-TB is tuberculosis due to organisms which exhibits high-level resistance to both isoniazid and rifampicin, with or without resistance to other anti-TB drugs [4]. Lengthy treatment and adverse effects of tuberculosis drugs form major hurdles in the tuberculosis management. Because of the failure of the current treatment strategies, there is compelling need to discover new chemical scaffolds with strong anti-mycobacterial properties [5]. Triterpenes constitute a huge and diverse group of plant secondary metabolites. They have been reported to
exhibit enormous pharmacological potential [6]. Anticancer and antimicrobial activities have been attributed to triterpenoids [7]. Against this background this study was designed to investigate the anti-mycobacterial activity of sophoradiol against the drug resistant-mycobacterium tuberculosis as well as in the murine model of tuberculosis. The results showed that sophoradiol exhibited strong activity against the H37RV strain as well the drug resistant isolates of mycobacterium. Sophoradiol also exhibited potent anti-mycobacterium activity in murine models suggesting that sophoradiol may prove beneficial lead molecule for the development of chemotherapy for tuberculosis Materials and Methods Minimum
inhibitory
concentration
(MIC)
and
Minimum
bactericidal
concentration (MBC). The MIC of sophoradiol (98% pure by HPC obtained from Sigma Aldrich) against M. tuberculosis H37RV and other resistant strains was evaluated by methodology as described earlier [8]. MBC of sophoradiol was evaluated by diluting the concentrations above MIC to sub-MIC levels and platting them onto Middle brook 7H11 agar plates. The plates were then subjected to incubation at 37 °C and the bacterial counts were determined 3 weeks post-incubation. MBC was taken as the lowest dose of sophoradiol that killed 99% of mycobacterial population. In vitro kill curve assay
Around 10 ml of media was taken in 50 ml centrifuge tubes containing sophoradiol at the doses of 1, 4 and 8X MICs. These tubes were subjected to inoculation with M. tuberculosis H37RV at 105 CFU/mL. This was followed by incubation at 37 ○C. Untreated control was also processed using same protocol. From each sample, aliquots were harvested at 0, 4, 16 and 20 days of time intervals. These aliquots were serially diluted and subjected to platting on Middlebrook 7H11 agar plates, followed by incubation at 37 ○C for 21 days for viability count. In vitro drug-drug interaction studies In vitro drug interactions of sophoradiol were evaluated by the methodology as described previously. The Fractional inhibitory concentrations (FICs) were estimated as per the formula
+
=
ℎ
The fractional inhibitory index (ΣFIC) was determined as FIC of compound X + FIC of compound Y. An FICs of > 0.5 was taken as synergistic, FICs of > 4.0 as antagonism, and in between the two was taken as additive interaction In vivo study Experimental Animals Seven week old BALB/C mice weighing about 25 ± 2 g were used in the study. The rats were given free access to pellet diet and water. The mice were also
procured from the animal centre of the same university and kept in sterile stainless steel cages in a dark and night period of 12 hours each, 22 ○C temperature and around 50% relative humidity. The animal protocols for the study were approved by animal ethical committee of the institute. Acute toxicity study and mice models For acute toxicity OCDE 2001 [9] guidelines were followed. After overnight fasting, they were administrated with pure drinking water. This was followed by random grouping of the mice into 3 groups consisting of six mice each. While as the control group was administrated with water, the other two groups were administrated with 500 and 1000 kg of body weight. Thereafter, the mice were subjected to fasting for 3 h. For the next two weeks the animals were monitored for any toxicity or death. At the end of 14 days the body weight of the mice was determined Treatment groups The mice were divided randomly into 5 groups. Group 1 contained 6 mice that were treated with vehicle only (CMC). Group 2 were treated with RIF at the dosage of 10 mg/kg body weight, group 4 animals were treated with sophoradiol at the dosage of 75 mg/kg body weight and group 5 animals were treated with rifampicin and sophoradiol combination. Treatment was initiated on day 3 post-infection and continued for 10 days. Three mice in the control group were sacrificed at the start of experiment to determine the baseline CFU values in the lungs of mice.
Cytotoxicity assay For cytotoxicity assay, the breast cell lines (CMMT, MB157 and CL-S1) were treated with 0 to 200 µM concentrations of sophoradiol. Around 10µl of culture medium from each well was replaced with 10 µl of 5% 3-(4, 5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and again an incubation of 4 h at 37oC was given. Thereafter, 150 µl of DMSO was added to each well for dissolving the formazan crystals. Subsequently, absorbance at 570 nm was recorded with microplate photometer. Statistical analysis Data were presented as mean ± SD. Statistical significance and IC50 values were analyzed by GraphPad Prism Demo, Version 5 (GraphPad Software, San Diego, California, USA). Student’s t test (For comparison between two samples) and one way ANOVA followed by Tukey’s test (For comparisons between more than two samples) were used for statistical analysis. *P< 0.01, **P< 0.001, ***P< 0.0001 were considered as the significant difference Results MIC and MBC of sophoradiol against normal and drug resistant strains of M. tuberculosis The MIC of Sophoradiol and the anti-TB drugs against the H37RV strain and the drug resistant isolates was examined by broth micro-dilution methodology. The results showed that the MIC of Sophoradiol to be 8.5 µg/ml against the H37RV strain which was slightly higher than the MICs of rifampicin (2 µg/m)
and isoniazid (1 µg/ml) (Table 1). The MIC of sophoradiol was also examined against the five different drug-resistant isolates of M. tuberculosis. The results showed that the MIC of sophoradiol varied from 9 to 16 µg/ml with lowest MIC of 9 µg/ml against the CX3 and highest of 16 µg/ml against the CX1 strains (Table 2). Next, the viable count methodology was employed to evaluate the MBC of sophoradiol and it was found that sophoradiol exhibits an MBC of 16 µg/ml. At MBC, sophoradiol decreased the bacterial load by 3.5 Log10 Sophoradiol shows interaction with tuberculosis drugs To gain insights about the interaction of sophoradiol with the standard antituberculosis drugs, the checkerboard assay was employed. Thereafter, the fractional inhibitory index (ΣFIC) was determined for each concentration of sophoradiol used. In combination with isoniazid, sophoradiol showed two fold improvements in MIC and the ΣFIC became 1 indicative of the additive interaction between sophoradiol and isoniazid. Nonetheless, in combination with rifampicin a four-fold enhancement was observed in MIC suggestive of synergism. To assess the effects of time and concentration on the activity of sophoradiol, a kill curve study was carried out where in M. tuberculosis was treated with varied concentrations of sophoradiol in 16 days. The results showed sophoradiol to be bacteriostatic as no significant alteration was observed on the bacterial load over the period of 16 days. However, when 2X or 4X was used the bacterial load diminished by 3 log10 time dependently suggestive of the time
dependent activity of sophoradiol. The 8X MIC was found to be the most effective dose of sophoradiol as remarkable reduction of bacterial load was reported at this concentration. Acute toxicity study No behavioural change was reported for any animal when sophoradiol was administrated at 5000 or 1000 mg/kg dosage. Additionally, none of the mice died during the study. Cervical dislocation was carried out at the 14th day to sacrifice the mice for microscopic pathological analysis which showed no apparent alterations in the anatomy of the animal liver. Hence, LD50 of sophoradiol was taken as >1000 mg/kg of the body weight. Sophoradiol exhibits anti-mycobacterial activity in vivo In the mice model of tuberculosis, sophoradiol exhibited remarkable efficacy. Sophoradiol when administrated orally at the dosage of 75 mg/kg body weight suppressed the rise in CFU of the lungs of the mice. This was in contrary to the mice which received only the vehicle and exhibited a steep increase in bacterial load of the lungs. Additionally, in combination with rifampicin, sophoradiol showed efficient activity causing 2.5log10 decrease in the CFUs. Sophoradiol is non-cytotoxic The MTT assay was carried out to investigate the cytotoxicity of sophoradiol against the three normal human breast cell lines (CMMT, MB 157, CL-S1). Sophoradiol was administrated to these cell lines at the doses ranging from 0 to 200 µM. The results showed that sophoradiol exhibits negligible cytotoxic
effects on all these cell lines as evident from the IC50 values of 140, 125 and 150 µg/ml against CMMT, MB 157 and CL-S1 cell lines respectively. Discussion The development of effective treatment regimes for tuberculosis demands the discovery of novel and potent lead molecules, their evaluation followed by semi-synthetic approaches to develop more effective and viable drugs [10]. Moreover, the cytotoxicity of the molecules should always be taken into consideration as the drug may exhibit significant adverse effects on human body. Human beings have been using and consuming plants and plant derived compounds since times immemorial. The human body has evolved in a way that most of the compounds present in edible plants do not cause any harm [11]. Therefore, it is belived that the drugs of plant origin may have no or comparatively lower adverse effects. As such scientists across the world are isolating compounds from plants and even from microbes and subsequently evaluating their anti-mycobacterium activity [12].
Triterpenoids are plant
derived metabolites which plants synthesize for their defense against pathogens and diseases [13]. Furthermore, researches have proved that triterpenoids have remarkable
pharmacological
potential.
Sophoradiol
is
very
important
triterpenoid extracted from different plant species and has shown some medicinal properties [14]. However, there is not a single study that reports the anti-mycobacterial activity of sophoradiol. Thus this study is the first report on the anti-mycobacterial activity of sophoradiol. This study revealed the MIC of
sophoradiol against the H37RV strain to be 8.5 µg/ml and that of the standard anti-TB drugs rifampicin and isoniazid to be 2 and 1 µg/ml respectively. Previous studies have also shown similar MICs for these anti-TB drugs confirming the accuracy of our assays [15]. Additionally, from the MBC values it may be inferred that sophoradiol exhibits bactericidal activity against the H37RV strain at MIC of 2X. The development of drug resistant strains of mycobacterium has imposed great challenges in the treatment of tuberculosis [16] but sophoradiol showed fairly good anti-mycobacterial activity against the drug resistant isolates. These results suggest that sophoradiol may prove an effective lead molecule which can be manipulated via semi-synthetic approaches to synthesize sophoradiol derivatives that can target the drug resistant strains of mycobacterium more effectively. Since, sophoradiol exhibited good anti-mycobacterium activity in vitro, we also attempted to investigate its activity in mice model of tuberculosis. Interestingly, sophoradiol showed potent activity in vivo. Conclusion Taken together, the results of the present study indicated that sophoradiol exhibits strong anti-mycobacterial activity against the drug resistant isolates of M. tuberculosis. Additionally, it also exhibit remarkable efficacy in vivo. More importantly, sophoradiol exhibits very low cytotoxicity against the normal breast cells indicating that it may be utilised as lead molecule in the development of anti-tuberculosis drugs.
Acknowledgement We acknowledge funding support from Project of Yunnan Science and Technology Department (No: 2018FH001-077).
References 1. Barry CE, III, et al. 2009. The spectrum of latent tuberculosis: rethinkingthe biology and intervention strategies. Nat. Rev. Microbiol. 7:845– 855. 2. Rodrıguez JC, Ruiz M, Lopez M, Royo G. In vitro activity of moxifloxacin,
levofloxacin,
gatifloxacin
and
linezolid
against
Mycobacterium tuberculosis. Inter J antimicrob agents. 2002; 20: 464-7. 3. Lew W, Pai M, Oxlade O, Martin D, Menzies D. Initial drug resistance and tuberculosis treatment outcomes: systematic review and metaanalysis. Ann Internal Med 2008; 149: 123-34. 4. Zhang Y, Heym B, Allen B et al. (1992) The catalase-peroxidase gene and isoniazid resistance in M. tuberculosis. Nature, 358, 591–3. 5. Andries K, Verhasselt P, Guillemont J, Göhlmann HW, Neefs JM, Winkler H, Van Gestel J, Timmerman P, Zhu M, Lee E, Williams P. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.Science.Science. 2005;307:223.
6. Huang M, Lu JJ, Huang MQ, Bao JL, Chen XP, Wang YT. Terpenoids: natural products for cancer therapy. Expert opinion on investigational drugs. 2012 Dec 1;21(12):1801-18. 7. Rufino-Palomares EE, Pérez-Jiménez A, J Reyes-Zurita F, GarcíaSalguero L, Mokhtari K, Herrera-Merchán A, P Medina P, Peragón J, A Lupianez J. Anti-cancer and anti-angiogenic properties of various natural pentacyclic tri-terpenoids and some of their chemical derivatives. Current Organic Chemistry. 2015 May 1;19(10):919-47. 8. Wiradharma N, Khoe U, Hauser CA, Seow SV, Zhang S, Yang Y-Y. Synthetic cationic amphiphilic a-helical peptides as antimicrobial agents. Biomaterials 2011;32:2204e12. 9. Zeiger E, haseman JK, galding J, Caspary WA, Michael Resnick ST, Stasiewicz BE, robert M. Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science.1987; 236: 933-41. 10.Connolly LE, Edelstein PH, Ramakrishnan L. 2007. Why is long-term therapy required to cure tuberculosis? PLoS Med. 4:e120. 11.Springob K, Kutchan TM. Introduction to the different classes of natural products. InPlant-Derived Natural Products 2009 (pp. 3-50). Springer, New York, NY. 12.Griffin SG, Wyllie SG, Markham JL, Leach DN. The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour and Fragrance Journal. 1999 Sep;14(5):322-32.
13.Zhou L, Zhang Y, Gapter LA, Ling H, Agarwal R, Ng KY. Cytotoxic and anti-oxidant activities of lanostane-type triterpenes isolated from Poria cocos. Chemical and Pharmaceutical Bulletin. 2008 Oct 1;56(10):145962. 14.Kinjo J, Hirakawa T, Tsuchihashi R, Nagao T, Okawa M, Nohara T, Okabe H. Hepatoprotective constituents in plants 14. Effects of soyasapogenol B, sophoradiol, and their glucuronides on the cytotoxicity of
tert-butyl
hydroperoxide
to
HepG2
cells.
Biological
and
Pharmaceutical Bulletin. 2003;26(9):1357-60. 15.Falzari K, Zhu Z, Pan D, Liu H, Hongmanee P, Franzblau SG. In vitro and in vivo activities of macrolide derivatives against Mycobacterium tuberculosis.Antimicrob Agents chemother 2005 49:1447-54. 16.Dhar N, McKinney JD. 2010. Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice. Proc. Natl. Acad. Sci. U. S. A. 107:12275–12280.
Figures
Figure 1. Kill curve of sophoradiol against M. tuberculosis H37Rv. M. tuberculosis H37Rv was treated with varied doses of sophoradiol for different time intervals and bacterial counts were determined on Middlebrook agar plates. All experiments were carried out in triplicates.
Figure 2: Efficacy of sophoradiol in acute murine model of TB. Control mice were euthanized on day 3 post infection; all other groups were euthanized after 12 days including last 2 days of wash out period. P values for comparison of two groups were determined by 2-tailed Student’s t test (* P< 0.05; ** P< 0.01; *** P< 0.001).
Figure 3: Cytotoxicity of sophoradiol on different normal breast cell lines as determined by MTT assay. The experiments were performed in triplicate and expressed as mean ± SD.
Table 1: MIC of sophoradiol and standard first line anti-tuberculosis drugs. Drug
MIC (µg/ml)
Sophoradiol
8.5
Isoniazid
2
Rifampicin
1
Table 2: MIC of sophoradiol against the drug resistant strains of tuberculosis. S. No. 1 2 3 4 5
Drug resistant strain CX1 CX2 CX3 CX4 CX5
MIC (µg/mL) 16 12 9 14 9.5
Table 3. Interaction of sophoradiol with Isoniazid and Rifampicin drugs against M. tuberculosis H37RV
Drug/drug
MIC (µg/ml)
FIC
ΣFIC
Remarks
combination Alone
Combination
8.5
4.25
0.5
Isoniazid
1
0.5
0.5
Rifampicin
2
0.25
0.125
Sophoradiol
8.5
1.05
0.125
Sophoradiol
1
0.25
Additive
Synergism
Highlights • Sophoradiol inhibited the growth of drug resistant strains of M. tuberculosis • Sophoradiol exhibits additive interaction with isoniazid and synergistic with rifampicin. • Sophoradiol exhibits remarkable efficacy in murine models. • Sophoradiol exhibits negligible toxicity against the normal human breast cells
S0882-4010(19)31924-2
DOI:
https://doi.org/10.1016/j.micpath.2020.103971
Reference:
YMPAT 103971
To appear in:
Microbial Pathogenesis
Received Date: 3 November 2019 Revised Date:
31 December 2019
Accepted Date: 6 January 2020
Please cite this article as: Lu N, Yang Y, Liu J, Li J, Ouyang B, Xia J, Du Y, Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.103971. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Sophoradiol inhibits the growth of drug resistant Mycobacterium tuberculosis in vitro and murine models of tuberculosis.
Nihong Lu, Yongrui Yang, Jun Liu, Jie Li, Bing Ouyang, Jiawei Xia, Yingrong Du*
Department of Respiratory and Critical Care, Kunming Third People's Hospital ，Kunmin， Yunnan ， China ， 650041
*Corresponding author Yingrong Du ， Department of Respiratory and Critical Care, Kunming Third People's HospitalKunmin， Yunnan ， China ， 650041 Tel & Fax: 0086-0871-63513914, Email: [email protected]
Abstract Tuberculosis is a devastating disease responsible for approximately 1.5 million deaths annually especially in developing countries. Although there is recommended and standard treatment for tuberculosis but the non-adherence of the patients to the lengthy treatment, adverse effects of the drugs and the emergence of multi-drug resistant strains hurdles the management of this devastating disease. This study examined the anti-mycobacterial activity of a plant derived triterpenoid, sophoradiol, against the drug-resistant strains of Mycobacterium tuberculosis and also in murine model of tuberculosis. The results showed that sophoradiol exhibits remarkable activity against the H37RV strain with an MIC of 8.5 µg/mL. The MIC of sophoradiol against the drug resistant strains of M. tuberculosis (CX1 to CX5) ranged from 9 to 16 µg/mL. Additionally, sophoradiol exhibited a bactericidal activity against H37RV strain with MBC equal to 2X MIC. Drug interaction studies showed that sophoradiol exhibits additive interaction with isoniazid and synergistic interaction with rifampicin. In the mice model of tuberculosis, sophoradiol also exhibited remarkable efficacy. Finally, cytotoxicity assays showed that sophoradiol exhibits negligible toxicity against the normal human breast cell lines. Taken together, it is concluded that sophoradiol may prove beneficial lead molecule for the management of tuberculosis.
Keywords: Tuberculosis; Triterpenoids; Sophoradiol; Mycobacterium; Drug resistance.
Introduction Among infectious diseases, tuberculosis is one of the devastating diseases causing huge number of deaths every year world over. Annually more than 1.5 million mortalities are caused by tuberculosis world over. Because of inefficient diagnostic assays and treatment strategies, tuberculosis causes more damage in developing countries [1]. It has also been reported that 1/3 of the HIV related deaths are caused by tuberculosis. As per the reports, 2 billion people are infected by Mycobacterium tuberculosis globally [2]. The emergence of drug resistance strains has further worsened the problem. M. tuberculosis resistant to rifampicin and isoniazid (two of the major first line anti-tuberculosis drugs) have also been reported [3]. Additionally, multidrug resistant strains of M. tuberculosis (MDR-TB) have also been reported. MDR-TB is tuberculosis due to organisms which exhibits high-level resistance to both isoniazid and rifampicin, with or without resistance to other anti-TB drugs [4]. Lengthy treatment and adverse effects of tuberculosis drugs form major hurdles in the tuberculosis management. Because of the failure of the current treatment strategies, there is compelling need to discover new chemical scaffolds with strong anti-mycobacterial properties [5]. Triterpenes constitute a huge and diverse group of plant secondary metabolites. They have been reported to
exhibit enormous pharmacological potential [6]. Anticancer and antimicrobial activities have been attributed to triterpenoids [7]. Against this background this study was designed to investigate the anti-mycobacterial activity of sophoradiol against the drug resistant-mycobacterium tuberculosis as well as in the murine model of tuberculosis. The results showed that sophoradiol exhibited strong activity against the H37RV strain as well the drug resistant isolates of mycobacterium. Sophoradiol also exhibited potent anti-mycobacterium activity in murine models suggesting that sophoradiol may prove beneficial lead molecule for the development of chemotherapy for tuberculosis Materials and Methods Minimum
inhibitory
concentration
(MIC)
and
Minimum
bactericidal
concentration (MBC). The MIC of sophoradiol (98% pure by HPC obtained from Sigma Aldrich) against M. tuberculosis H37RV and other resistant strains was evaluated by methodology as described earlier [8]. MBC of sophoradiol was evaluated by diluting the concentrations above MIC to sub-MIC levels and platting them onto Middle brook 7H11 agar plates. The plates were then subjected to incubation at 37 °C and the bacterial counts were determined 3 weeks post-incubation. MBC was taken as the lowest dose of sophoradiol that killed 99% of mycobacterial population. In vitro kill curve assay
Around 10 ml of media was taken in 50 ml centrifuge tubes containing sophoradiol at the doses of 1, 4 and 8X MICs. These tubes were subjected to inoculation with M. tuberculosis H37RV at 105 CFU/mL. This was followed by incubation at 37 ○C. Untreated control was also processed using same protocol. From each sample, aliquots were harvested at 0, 4, 16 and 20 days of time intervals. These aliquots were serially diluted and subjected to platting on Middlebrook 7H11 agar plates, followed by incubation at 37 ○C for 21 days for viability count. In vitro drug-drug interaction studies In vitro drug interactions of sophoradiol were evaluated by the methodology as described previously. The Fractional inhibitory concentrations (FICs) were estimated as per the formula
+
=
ℎ
The fractional inhibitory index (ΣFIC) was determined as FIC of compound X + FIC of compound Y. An FICs of > 0.5 was taken as synergistic, FICs of > 4.0 as antagonism, and in between the two was taken as additive interaction In vivo study Experimental Animals Seven week old BALB/C mice weighing about 25 ± 2 g were used in the study. The rats were given free access to pellet diet and water. The mice were also
procured from the animal centre of the same university and kept in sterile stainless steel cages in a dark and night period of 12 hours each, 22 ○C temperature and around 50% relative humidity. The animal protocols for the study were approved by animal ethical committee of the institute. Acute toxicity study and mice models For acute toxicity OCDE 2001 [9] guidelines were followed. After overnight fasting, they were administrated with pure drinking water. This was followed by random grouping of the mice into 3 groups consisting of six mice each. While as the control group was administrated with water, the other two groups were administrated with 500 and 1000 kg of body weight. Thereafter, the mice were subjected to fasting for 3 h. For the next two weeks the animals were monitored for any toxicity or death. At the end of 14 days the body weight of the mice was determined Treatment groups The mice were divided randomly into 5 groups. Group 1 contained 6 mice that were treated with vehicle only (CMC). Group 2 were treated with RIF at the dosage of 10 mg/kg body weight, group 4 animals were treated with sophoradiol at the dosage of 75 mg/kg body weight and group 5 animals were treated with rifampicin and sophoradiol combination. Treatment was initiated on day 3 post-infection and continued for 10 days. Three mice in the control group were sacrificed at the start of experiment to determine the baseline CFU values in the lungs of mice.
Cytotoxicity assay For cytotoxicity assay, the breast cell lines (CMMT, MB157 and CL-S1) were treated with 0 to 200 µM concentrations of sophoradiol. Around 10µl of culture medium from each well was replaced with 10 µl of 5% 3-(4, 5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and again an incubation of 4 h at 37oC was given. Thereafter, 150 µl of DMSO was added to each well for dissolving the formazan crystals. Subsequently, absorbance at 570 nm was recorded with microplate photometer. Statistical analysis Data were presented as mean ± SD. Statistical significance and IC50 values were analyzed by GraphPad Prism Demo, Version 5 (GraphPad Software, San Diego, California, USA). Student’s t test (For comparison between two samples) and one way ANOVA followed by Tukey’s test (For comparisons between more than two samples) were used for statistical analysis. *P< 0.01, **P< 0.001, ***P< 0.0001 were considered as the significant difference Results MIC and MBC of sophoradiol against normal and drug resistant strains of M. tuberculosis The MIC of Sophoradiol and the anti-TB drugs against the H37RV strain and the drug resistant isolates was examined by broth micro-dilution methodology. The results showed that the MIC of Sophoradiol to be 8.5 µg/ml against the H37RV strain which was slightly higher than the MICs of rifampicin (2 µg/m)
and isoniazid (1 µg/ml) (Table 1). The MIC of sophoradiol was also examined against the five different drug-resistant isolates of M. tuberculosis. The results showed that the MIC of sophoradiol varied from 9 to 16 µg/ml with lowest MIC of 9 µg/ml against the CX3 and highest of 16 µg/ml against the CX1 strains (Table 2). Next, the viable count methodology was employed to evaluate the MBC of sophoradiol and it was found that sophoradiol exhibits an MBC of 16 µg/ml. At MBC, sophoradiol decreased the bacterial load by 3.5 Log10 Sophoradiol shows interaction with tuberculosis drugs To gain insights about the interaction of sophoradiol with the standard antituberculosis drugs, the checkerboard assay was employed. Thereafter, the fractional inhibitory index (ΣFIC) was determined for each concentration of sophoradiol used. In combination with isoniazid, sophoradiol showed two fold improvements in MIC and the ΣFIC became 1 indicative of the additive interaction between sophoradiol and isoniazid. Nonetheless, in combination with rifampicin a four-fold enhancement was observed in MIC suggestive of synergism. To assess the effects of time and concentration on the activity of sophoradiol, a kill curve study was carried out where in M. tuberculosis was treated with varied concentrations of sophoradiol in 16 days. The results showed sophoradiol to be bacteriostatic as no significant alteration was observed on the bacterial load over the period of 16 days. However, when 2X or 4X was used the bacterial load diminished by 3 log10 time dependently suggestive of the time
dependent activity of sophoradiol. The 8X MIC was found to be the most effective dose of sophoradiol as remarkable reduction of bacterial load was reported at this concentration. Acute toxicity study No behavioural change was reported for any animal when sophoradiol was administrated at 5000 or 1000 mg/kg dosage. Additionally, none of the mice died during the study. Cervical dislocation was carried out at the 14th day to sacrifice the mice for microscopic pathological analysis which showed no apparent alterations in the anatomy of the animal liver. Hence, LD50 of sophoradiol was taken as >1000 mg/kg of the body weight. Sophoradiol exhibits anti-mycobacterial activity in vivo In the mice model of tuberculosis, sophoradiol exhibited remarkable efficacy. Sophoradiol when administrated orally at the dosage of 75 mg/kg body weight suppressed the rise in CFU of the lungs of the mice. This was in contrary to the mice which received only the vehicle and exhibited a steep increase in bacterial load of the lungs. Additionally, in combination with rifampicin, sophoradiol showed efficient activity causing 2.5log10 decrease in the CFUs. Sophoradiol is non-cytotoxic The MTT assay was carried out to investigate the cytotoxicity of sophoradiol against the three normal human breast cell lines (CMMT, MB 157, CL-S1). Sophoradiol was administrated to these cell lines at the doses ranging from 0 to 200 µM. The results showed that sophoradiol exhibits negligible cytotoxic
effects on all these cell lines as evident from the IC50 values of 140, 125 and 150 µg/ml against CMMT, MB 157 and CL-S1 cell lines respectively. Discussion The development of effective treatment regimes for tuberculosis demands the discovery of novel and potent lead molecules, their evaluation followed by semi-synthetic approaches to develop more effective and viable drugs [10]. Moreover, the cytotoxicity of the molecules should always be taken into consideration as the drug may exhibit significant adverse effects on human body. Human beings have been using and consuming plants and plant derived compounds since times immemorial. The human body has evolved in a way that most of the compounds present in edible plants do not cause any harm [11]. Therefore, it is belived that the drugs of plant origin may have no or comparatively lower adverse effects. As such scientists across the world are isolating compounds from plants and even from microbes and subsequently evaluating their anti-mycobacterium activity [12].
Triterpenoids are plant
derived metabolites which plants synthesize for their defense against pathogens and diseases [13]. Furthermore, researches have proved that triterpenoids have remarkable
pharmacological
potential.
Sophoradiol
is
very
important
triterpenoid extracted from different plant species and has shown some medicinal properties [14]. However, there is not a single study that reports the anti-mycobacterial activity of sophoradiol. Thus this study is the first report on the anti-mycobacterial activity of sophoradiol. This study revealed the MIC of
sophoradiol against the H37RV strain to be 8.5 µg/ml and that of the standard anti-TB drugs rifampicin and isoniazid to be 2 and 1 µg/ml respectively. Previous studies have also shown similar MICs for these anti-TB drugs confirming the accuracy of our assays [15]. Additionally, from the MBC values it may be inferred that sophoradiol exhibits bactericidal activity against the H37RV strain at MIC of 2X. The development of drug resistant strains of mycobacterium has imposed great challenges in the treatment of tuberculosis [16] but sophoradiol showed fairly good anti-mycobacterial activity against the drug resistant isolates. These results suggest that sophoradiol may prove an effective lead molecule which can be manipulated via semi-synthetic approaches to synthesize sophoradiol derivatives that can target the drug resistant strains of mycobacterium more effectively. Since, sophoradiol exhibited good anti-mycobacterium activity in vitro, we also attempted to investigate its activity in mice model of tuberculosis. Interestingly, sophoradiol showed potent activity in vivo. Conclusion Taken together, the results of the present study indicated that sophoradiol exhibits strong anti-mycobacterial activity against the drug resistant isolates of M. tuberculosis. Additionally, it also exhibit remarkable efficacy in vivo. More importantly, sophoradiol exhibits very low cytotoxicity against the normal breast cells indicating that it may be utilised as lead molecule in the development of anti-tuberculosis drugs.
Acknowledgement We acknowledge funding support from Project of Yunnan Science and Technology Department (No: 2018FH001-077).
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Figures
Figure 1. Kill curve of sophoradiol against M. tuberculosis H37Rv. M. tuberculosis H37Rv was treated with varied doses of sophoradiol for different time intervals and bacterial counts were determined on Middlebrook agar plates. All experiments were carried out in triplicates.
Figure 2: Efficacy of sophoradiol in acute murine model of TB. Control mice were euthanized on day 3 post infection; all other groups were euthanized after 12 days including last 2 days of wash out period. P values for comparison of two groups were determined by 2-tailed Student’s t test (* P< 0.05; ** P< 0.01; *** P< 0.001).
Figure 3: Cytotoxicity of sophoradiol on different normal breast cell lines as determined by MTT assay. The experiments were performed in triplicate and expressed as mean ± SD.
Table 1: MIC of sophoradiol and standard first line anti-tuberculosis drugs. Drug
MIC (µg/ml)
Sophoradiol
8.5
Isoniazid
2
Rifampicin
1
Table 2: MIC of sophoradiol against the drug resistant strains of tuberculosis. S. No. 1 2 3 4 5
Drug resistant strain CX1 CX2 CX3 CX4 CX5
MIC (µg/mL) 16 12 9 14 9.5
Table 3. Interaction of sophoradiol with Isoniazid and Rifampicin drugs against M. tuberculosis H37RV
Drug/drug
MIC (µg/ml)
FIC
ΣFIC
Remarks
combination Alone
Combination
8.5
4.25
0.5
Isoniazid
1
0.5
0.5
Rifampicin
2
0.25
0.125
Sophoradiol
8.5
1.05
0.125
Sophoradiol
1
0.25
Additive
Synergism
Highlights • Sophoradiol inhibited the growth of drug resistant strains of M. tuberculosis • Sophoradiol exhibits additive interaction with isoniazid and synergistic with rifampicin. • Sophoradiol exhibits remarkable efficacy in murine models. • Sophoradiol exhibits negligible toxicity against the normal human breast cells