Efficacy of Merremia mammosa Hall Terpenoid Fraction againts Mycobacterium tuberculosis

 

Mangestuti Agil*, Herra Studiawan, Neny Purwitasari

Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia.

*Corresponding Author E-mail: mangestuti@ff.unair.ac.id

 

ABSTRACT:

Tuberculosis is still a pandemic disease in Indonesia. People in Madura Island, Indonesia, usually use the rhizome of Merremia mammosa Hall as anti-tuberculosis. In this study, the antibacterial activity of terpenoid fractions of Merremia mammosa rhizome against Mycobacterium tuberculosis has been studied using agar dilution method at a concentration of 200-25 µg/mL, incubated for 21 days in 7H10 Midlebrook medium. N-hexane extract of Merremia mammosa has been fractionated using ethyl acetate. The ethyl acetate fraction was subjected to vacuum column chromatography using gradient concentration of ethyl acetate-n hexane, and obtained 11 subfractions. There were two subfractions indicated the presence of terpenoid compounds. These subfractions were grouped and subjected to antimicrobial assay. Result showed that the terpenoid compounds have antituberculosis property with minimum inhibition concentration (MIC) value of 100µg/mL. The result was supported by acid -fast Zielh-neelsen stain, which indicated that at a concentration of 100 and 200 µg/ml of the subfractions, there were no growth of Mycobacterium tuberculosis visually detected. In conclusion, Merremia mammosa is potential to be developed as antituberculosis agent. The chemical compounds responsible for this activity are currently under investigation for further study.

 

KEYWORDS: Merremia mammosa Hall., Mycobaterium tuberculosis, terpenoid compounds.

 

 


INTRODUCTION:

Tuberculosis (TB) is a pandemic in Indonesia, It is an infectious disease caused by Mycobacterium tuberculosis and attack lungs as well as other body organs. Mycobacterium tuberculosis has special characteristics, which is resistant to acid in staining, and therefore is called acid resistant bacillus, dies quickly under direct sunlight, but can survive for several hours in dark and humid places. The bacteria can stay dormant in human tissues. According to the 2019 Global Tuberculosis Report of World Health Organization (WHO), there are 202 countries with a high prevalence of tuberculosis sufferers, in which 10 of them are Asian countries, with the highest prevalence being India (27%), China (9%) and Indonesia (8%). WHO estimates that there are around 10 million people infected with TB and 1.5 million died from this disease. WHO also stated, that in 2018 almost half of the world's TB sufferers (44%) lived in Southeast Asia1. In Indonesia there were 845.000 TB incidence in 2019, with 63,111 cases among children per year1.

 

The increase of tuberculosis cases is due to the high rate of primary and secondary resistances to tuberculosis drugs. Resistance to the drug is caused partly by the use of single tuberculosis drugs, inadequate combination of tuberculosis drugs, and irregular use of the drugs2. Many TB sufferers have to undergo re-treatment due to the neglect or termination of drugs, and relaps. TB control program in patients with sensitive TB germs requires 6 months of treatment, while resistant TB sufferers need very expensive second-line tuberculosis drugs. Such drugs are very toxic, have not been guaranteed of their cure rate, and have very long healing time3.

 

Considering the resistance and side effect caused by long-term use of TB drugs, scientific effort to the application of potential herbal medicines could become an alternative way to overcome such shortage. Practice of traditional medicine of Indonesia has long been conducted in the community as an effort to overcome various diseases, and therefore scientific efforts have to be initiated 4,5,6,7,8,9. Such efforts in the utilization of natural ingredients to overcome several diseases could support scientific data10,11, especially in a situation where 75-80% of the world population could not get clinical treatment to overcome the disease10,11. This shows the importance of traditional medicine, among others, in improving public health care. Some advantages of traditional medicine compared to modern medicine, among others, are relatively low side effects, and have been proven its health benefits empirically. Generally, medicinal plants contain many chemical substances work synergistically to exert certain actions, while a single plant can exert more than one pharmacological effects. Another advantage in using natural-based ingredients for producing drugs is its availability in providing plant materials, caused especially by the biodiversity of developing countries like Indonesia10,11.

 

Empirically, people of Sampang District in Madura Island of Indonesia have utilized Merremia mammosa Hall (Convolvulaceae) tubers to treat tuberculosis12 and helped them to control the disease. A study reported by Anda Kriselina, showed that normal hexane extract of the tubers has antibacterial activity against Staphylococcus aureus13. Previous studies have shown that normal hexane and water extracts of Merremia mammosa tuber can inhibit the growth of M. tuberculosis bacteria14. Our previous study also suggested that this plant is rich in terpenoid compounds14. Terpenoid, showed moderate to significant biological activity againts M.tuberculosis15.

 

In this research, separation of active compounds and activity test were conducted. Crude extract was separated using fractionation and column chromatography methods. Fractions contained terpenoid compounds were tested for their activity against Mycobacterium tuberculosis.

 

MATERIALS AND METHODS:

Plant material:

Tubers of Merremia mammosa Hall have been taken from Sumenep, Madura, Indonesia. Determination was carried out at Purwodadi Botanical Garden, Pasuruan, Indonesia.

 

Chemicals and equipments:

Chemicals are pure  n-hexane (Merck), pure ethyl acetate (Merck), pure Rifampicin (Sigma, Germany), dimethyl sulfoxide (DMSO) (Merck), sulfuric acid, Ziehl neelsen stain, middlebrook agar 7H9, middlebrook agar 7H10, laminar air flow cabinet, temperature incubator ± 37 °C, vortex (Labinco L46), ose needles, autoclaves and glasswares.

 

Figure 1. Photograph of Merremia mammosa Hall.

 

Material preparation:

Tubers were washed, sliced very thinly, and dried under direct sunlight between 6:00 and 10:00 am to avoid over exposure that could damage thermolabile chemical contents of the tubers. Dried tubers were grinded into fine powder and ready for further extraction.

 

Material extraction:

Merremia mammosa Hall tuber powder was macerated with n-hexane, and the residue was macerated with ethyl acetate to extract semi-polar compounds. Both n-hexane and ethyl acetate extracts were concentrated with a vacuum rotary evaporator to obtain thick extracts. The ethyl acetate extract was then fractionated with vacuum column chromatography method using n-hexane and ethyl acetate (100:0; 90:10 to 0:100) gradient solvents as much as 100 mL each. From the vacuum column, 11 subfractions were obtained which were separated further on a thin layer chromatography (TLC) plate and eluted using mixtures of n-hexane: ethyl acetate (3.5:7.5) as the mobile phase. TLC plates were sprayed using sulfuric acid anisaldehyde to identify terpenoid compounds. The terpenoid contents of the subfractions showed pink to purple spots. All terpenoid- containing sub fractions were combined and tested using Mycobacterium tuberculosis antimicrobial assay. The assay was conducted in triplicate.

 

Antibacterial activity test:

The bacterium used was Mycobacterium tuberculosis H37RV ATCC 27294 which was sensitive to antibiotic ethambutol, and obtained from bacterial supplies of Tuberculosis Laboratory, Institute of Tropical Disease, Universitas Airlangga. The antibacterial activity test was conducted at the Tuberculosis Laboratory, Institute of Tropical Diseases, Universitas Airlangga, Surabaya, Indonesia The test groups were positive control, negative control, and extracts. Rifampin was used as a positive control. DMSO solution in distilled water was used as negative control. Extracts as test solutions were made from a mixture of terpenoid- containing sub fractions, with a concentration of 200, 100, 50, and 25 ppm, and 1% DMSO and distilled water were used as solvents.  Solid agar media (Middlebrook 7H10 medium, supplemented with nutrients such as glycerol, oleic acid, albumin, and dextrose) that contained test solutions and bacteria were incubated for 21 days. Every 7 days the growth of the colony of bacteria was observed, and on the 21st day the colony was taken for identification of the bacteria using Zielh-neelsen staining.

 

RESULTS AND DISCUSSION:

Results of n-hexane extract:

20 kg of Merremia mammosa Hall tubers, were dried and grinded, and 1,225 grams of fine powder  were obtained. The powder was then extracted gradually with n-hexane and ethyl acetate, and obtained 86.5 grams and 23.7 grams of n-hexane and ethyl acetate extract respectively. Ethyl acetate extract was then subjected to vacuum column chromatography using gradient solvent of hexane and ethyl acetate.

 

Vacuum column chromatography:

The vacuum chromatography column obtained 11 subfractions, and were further subjected to thin layer chromatography. Subfraction no 1 obtained during separation with hexane:ethyl acetate = 100:0,  and subfraction no 2 obtained during separation with hexane:ethyl acetate = 90:10 showed pink to purple spots  with  Rf values of  0.69 (a), 0.54(b), 0.39(c) and 0.09 (d). Both subfractions were combined for anti-tuberculosis activity test (Figure 2).


 

Figure 2.  TLC of subfractions obtained from vacuum column chromatography of  tubers of Merremia mammosa Hall.

(Mobile phase n-hexane: ethyl acetate= 3.5:7.5, spray reagent:  Sulfuric acid anisaldehid)

 


Anti-tuberculosis activity test results:

Anti-tuberculosis activity test was incubated for 21 days in petri dishes. Bacterial growth were observed in negative control, extracts of 25 ppm and 50 ppm groups.  But the colonies were clearly observed in the negative control group. Bacteria were taken and identified with Ziehl Neelsen stain. Table 1 showed the test result on the 21th day of the test, and ZN stain were shown in Figure 3.

 

Table 1. Bacterial Growth on the 21st of Anti-Tuberculosis Test

Group

Growth

Average Number of Colonies

Rifampicin

no growth

0

Negative Control

growth

17

Extract  (test solution )25 ppm

growth

14

Extract (test solution) 50 ppm

growth

11

Extract (test solution)100 ppm

no growth

0

Extract (test solution)200 ppm

no growth

0

 

Table 2. Microscopic Examination of Bacterial Growth with ZN Stain

Test Solutions and Concentrations

Growth

IA/25 ppm

+++++

II A/50 ppm

++

III A/100 ppm

-

IVB/200 ppm

-

 

Figure 3. Observation of bacterial growth at day 21. The pink-purple coccus (arrows) showed the presence of bacterial colonies

 

Concentration of the subfraction  were  200-25 µg/mL based on the preliminary research conducted by the author14.

From the result, it was shown that the minimum inhibitory concentration of the test solution was 100 ppm.

This method is also possible to be used in microbiological studies for soluble or insoluble compounds15.

 

The microbe used is Mycobacterium tuberculosis whose cell walls are rich in lipids, making it difficult to penetrate extracts that are dispersed in water. Since it was difficult to homogenize Mycobacterium tuberculosis in 7H9 midllebrook media, glass beads were added and then vortexed to make them more homogeneous. The microbial inoculum used was compared to the Mc Farland 1.0 standard and then diluted to obtain 105 CFU/mL of bacterial colonies. Below 105 CFU/mL microbial growth was not clearly visible,  whereas above 105 CFU/mL bacterial inhibition can not be observed clearly because of too many bacteria found. The positive control used is rifampicin as a standard drug for Mycobacterium tuberculosis infection.

 

Observation on the 3rd week of the test at concentrations of 200 and 100 ppm showed no bacterial growth based on the fact that visually there was no Mycobacterium tuberculosis detected microscopically with Zielh-neelsen stain. Zielh-neelsen staining is carried out to identify acid-resistant bacteria, mainly Mycobacteria, such as M.tuberculosis and M.leprae. It is sensitive and a low-cost method.

 

Phytochemical tests detected several compounds of Merremia mammosa Hall tubers such as polyphenols, terpenoids and triterpenoids, which showed inhibitory activity against Mycobacterium tuberculosis. Some plants with terpenoid contents and showed antituberculosis activity have been known, including Byrsonima crassa17, and Juniperus communis L. (Cuppressaceae)18.

 

It was expected, that, next study  should be arranged to isolate the active compounds with antituberculosis property, that can be developed as a new herbal medicine against tuberculosis by taking into account its safety and effectiveness aspects.

 

CONCLUSION:

Results from this study showed that the subfractions of Merremia mammosa Hall tubers contained a mixture of terpenoid compounds able to inhibit the growth of Mycobacterium tuberculosis at a minimum inhibitory concentration of 100 ppm.

 

ACKNOWLEDGEMENT:

Authors would like to thank the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      https://www.who.int/tb/publications/global_report/tb19_Report_country_profiles_15October2019.pdf?ua=1 accessed in 21 April 2020.

2.      Shruthi Ravimohan, Hardy Kornfeld, Drew Weissman, Gregory P. Bisson. 2018.Tuberculosis and lung damage: from epidemiology to pathophysiology European Respiratory Review  27 (147) 170077; DOI: 10.1183/16000617.0077-2017

3.      Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U., Salamat, M., & Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. Infection and drug resistance, 11, 1645–1658. https://doi.org/10.2147/IDR.S173867.

4.      Ansori ANM, Susilo RJK, Hayaza S, et al. Renoprotection by Garcinia mangostana L. pericarp extract in streptozotocin-induced diabetic mice. Iraqi Journal of Veterinary Science. 2019; 33(1): 13-19.

5.      Ansori ANM, Fadholly A, Hayaza S, et al. A review on medicinal properties of mangosteen (Garcinia mangostana L.). Research Journal of Pharmacy and Technology. 2020; 13(2): 974-982.

6.      Fadholly A, Ansori ANM, Jayanti S, et al. Cytotoxic effect of Allium cepa L. extract on human colon cancer (WiDr) cells: In vitro study. Research Journal of Pharmacy and Technology. 2019; 12(7): 3483-3486.

7.      Fadholly A, Ansori ANM, Proboningrat A, et al. Apoptosis of HeLa cells via caspase-3 expression induced by chitosan-based nanoparticles of Annona squamosa leaf extract: In vitro study. Indian Journal of Pharmaceutical Education and Research. 2020; 54(2): 416-21.

8.      Husen SA, Wahyuningsih SPA, Ansori ANM, et al. The effect okra (Abelmoschus esculentus Moench) pods extract on malondialdehyde and cholesterol level in STZ-induced diabetic mice. Ecology, Environment and Conservation. 2019; 25(April Suppl. Issue): S50-S56.

9.      Husen SA, Kalqutny SH, Ansori ANM, et al. Hepato-renal protective effects of Mangosteen (Garcinia mangostana L.) pericarp extract in streptozotocin-induced diabetic mice. Journal of Physics: Conference Series. 2020; 1445(1): 012018.

10.   Hayaza S, Istiqomah S, Susilo RJK, et al. Antidiabetic activity of ketapang (Terminalia catappa L.) leaves extract in streptozotocin-induced diabetic mice. Indian Veterinary Journal. 2019; 96(12): 11-13.

11.   Tacharina MR, Ansori ANM, Plumeriastuti H, et al. Beneficial effect of grinting grass (Cynodon dactylon) on the streptozotocin induced diabetes mellitus in the mice. Indian Veterinary Journal. 2020; 97(4).

12.   Kurniasih RT. The Anti-Inflammatory Effects of Ethanol Extract of Bidas Upas Bulbs (Merremia mammosa Hall.f.) Topically in Swiss Mice Strain Caragen-Induced. Thesis. Yogyakarta: Faculty of Pharmacy, Sanata Dharma University; 2014.

13.   Kriselina A. Test the Antibacterial Activity of Hexane and Methanol Extract from the Bidas Upas Bulbs (Merremia mammosa Hall). Thesis. Surabaya: Faculty of Pharmacy, Airlangga University; 1997.

14.   Mangestuti A, Noor E, Retno W, et al. Inhibition test of Mycobacterium tuberculosis from tubers upas bidas (Merremia mammosa Hall). Final Report of the Stranas-DP2M Dikti; 2010.

15.   Cantrell CL, Franzblau SG, Fischer NH. Antimycobacterial plant terpenoids.Planta Med. 2001 Nov;67(8):685-94. doi: 10.1055/s-2001-18365

16.   Andreas Romulo, Ervizal A. M. Zuhud, Johana Rondevaldova & Ladislav Kokoska (2018) Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine, Pharmaceutical Biology, 56:1, 287-293, DOI: 10.1080/13880209.2018.1462834

17.   Higuchi CT, Pavan FR, Sannomiya M, et al. Triterpenes and antitubercular activity of Byrsonima crassa. Química Nova. 2008; 31: 1719-1721.

18.   Gordien AY, Gray AI, Franzblau SG, et al. Antimycobacterial terpenoids from Juniperus communis L. (Cuppressaceae). Journal of Ethnopharmacology. 2009; 126(3): 500-505.

 

 

 

Received on 31.05.2020            Modified on 19.07.2020

Accepted on 21.08.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(12):6617-6620.

DOI: 10.52711/0974-360X.2021.01143