Translated Copy of JURNAL RUDY [PDF]

Citation preview

1|



ANTIBACTERIAL ACTIVITY TEST OF TOBACCO LEAF EXTRACT (Nicotiana tabacum L.) AGAINST Mycobacterium Tuberculosis BACTERIA Muh. Fitra Ilyas, Muh. Rusdi, Rudy Adyaksyah Pharmacy Study Program, Faculty of Medicine and Health Sciences, Alauddin Makassar Islamic University Email: [email protected] ABSTRACT



Research on the extraction of antituberculosis from n-hexane extract, ethyl acetate, 96% ethanol and water on tobacco leaves (Nicotiana tabacum L.). This study aims to determine the inhibitory effect of n-hexane, ethyl acetate, ethanol 96% extract andwater tobacco leaf on tuberculosis-causing bacteria and compounds that have the potential to be antituberculosis. Extract n-hexan, ethyl acetate, ethanol 96% and water from the leaves of tobacco leaves (Nicotiana tabacum L) has the potential to inhibit the growth of Mycobacterium tuberculosis through tests performed by the method of MODS as well as classes of compounds contained in extracts which have an inhibitory effect against Mycobacterium tuberculosis . The sample was extracted by a multilevel extraction method using three solvents namely nhexane extract, ethyl acetate, 96% ethanol. Then proceed to the reflux method with water solvent. Then the results of the extract were tested for inhibition of the growth ofbacteria Mycobacterium tuberculosis H37Rvusing the MODS method. And the identification of compound groups is carried out. The result of the inhibition of the growth of Mycobacteriumtuberculosis showed that an extract of n-hexan, ethyl acetate, ethanol 96% and water from the leaves of tobacco leaves (Nicotiana tabacum L) with each at a concentration of 2000 ppm showed activity antituberculosis, evidenced from the number of cord fewer after being observed under a microscope. which shows the inhibitory effect seen from the small number of cord that looks after observed in the microscope, while the extract of n-hexan, ethyl acetate, ethanol 96% and water at a concentration of 400 ppm and 800 ppm, 1200 ppm and 1600 ppm did not show any inhibition against Mycobacteriumtuberculosis is seen from a large number of cords and approaches when compared to negative controls. After that identification of compounds is carried out. The group of compounds contained inextract tobacco leaf (Nicotiana tabacum L) after identification of groups namely n-hexan extract containing flavanoid compounds, ethyl acetate extract containing steroid and phenolic compounds, ethanol 96% containing alkaloids, flavonoids, terpenoids and phenolic compounds , while the water extract contains alkaloids and phenolics. Keywords : Tobacco Leaves, n-hexane Extract, Ethyl Acetate Extract, 96% Ethanol Extract, Water Extract, Mycobacterium tuberculosis,MODEL INTRODUCTION



Tuberculosis (TB) is a contagious infectious disease that is a problem in the world. Tuberculosis is the 9th cause of death worldwide and a major cause of infectious agents. In 2016 an estimated 10.4 million people suffered from tuberculosis, 90% of adults, 65% of men, and 10% were accompanied by HIV. Globally, the death rate falls 3% annually. Tuberculosis sufferers fall by around 2% per year and 16% die from tuberculosis. Most deaths from tuberculosis can be prevented by early diagnosis and appropriate treatment.



Millions of people are diagnosed and successfully treated every year (53 million, 2000 - 2016), but there are still many gaps in detection and treatment (WHO, 2017). Tuberculosis sufferers who experience resistance become a new threat that continues. In 2016, there were around 600,000 new cases that had been resistant to Rifampicin, while this drug was the most effective first-line drug. In addition there are 490,000 multidrug resistant cases (MDR TB) (WHO, 2017).



Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



2|



Some of the reasons above encourage researchers to test the compound group and test the antituberculosis activity of tobacco leaf extract (Nicotiana tabacum L.) against the bacteria Mycobacterium tuberculosis RESEARCH METHODOLOGY



Tools and Materials The tools used are autoclave (Hirayama), maceration vessel, chamber (Lamag), center glass, separating funnel, incubator (memmert), beaker, watch glass, measuring cup, Laminan Air Flower (LAF) (ESCO), refrigerator (Modena), bowl, microscope, analytic balance, vortex mixer, micro pipette (Socorex), 24 well plates, rotary evaporator (Heidolph), iron spoons, analytical scales (Kern), and vials The material used is Distilled water (Aquadestillata), Aluminum Chloride, Pure culture (Mycobacterium tuberculosis), Tobacco leaves (Nicotiana tabacum L. ), DMSO, Ethanol, Ethyl Acetate, Filter paper, Middlebrook 7H9, N-hexane, OADC Nutrient (oxalid acid, albumin, destrosa, and catalase), Nutrient PANTA, Reagent (Liebermann-Buc hard, FeCl3, Dragendroff) Work procedures Preparation of Samples The research sample used was tobacco leaf (Nicotiana tabacum L.). Sampling is done at 09.00 WITA. The leaves used are good leaves, not damaged, not moldy and not yellow or too old. The sample that has been taken is then wet sorted to separate the sample from dirt or other foreign material. Then the sample is washed with clean water to remove soil or other impurities that are attached to the leaves. After that the samples are chopped into small pieces, then dried by aerating, protected from sunlight and then processed to become simplicia. Extraction with Multilevel method Samples of tobacco plant leaves (Nicotiana tabacum L) maserationthat have been dried are weighed as much as 800 g for multilevel maceration using 3 solvents, namely n-hexane, ethyl acetate, ethanol 96%, samples inserted into a maceration vessel then the sample soaked with solvent n-hexane as much as 8000 ml. The maceration container is closed



and stored for 2 x 24 hours in a place which is protected by direct sunlight while occasionally stirring, then filtered, separated from pulp and filtrate. The pulp is macerated again with the same number of dancers, carried out 3 times. The liquid n-hexane extract obtained was then concentrated using a rotary evaporator until a thick n-hexane extract was obtained. The pulp is aerated to remove residual solvents. The pulp was macerated again using an ethyl acetate solvent of 8000 ml, the same procedure was carried out to obtain an ethyl acetate extract. Then macerated using 96% ethanol as much as 8,000 ml, the same procedure was carried out until 96% ethanol extract was obtained. Furthermore, the refluxed sample Reflux Method after the multilevel maceration process was completed, then the last method was reflux using distilled water solvent for 4 hours. After that the sample is filtered and separated from the pulp and filtrate. The extract obtained is then evaporated. Each extract that has been dried is used to be tested on Mycobacterium tuberculosis. RESULTS AND DISCUSSION



Research Results From the extraction of 800 g of tobacco leaf simplicia (Nicotiana tabacum L) using a multilevel maceration method with n-hexane, ethyl acetate, 96% ethanol and water. Table 1. Results of extraction oftobacco leaves Simplisia Weight % 800 g ofExtract HexRendame n 22.4 g 2.8% Ethyl Acetate



21.4 g



2.67%



Ethanol 96%



39.5 g



4.94%



Water



20.2 g



2.52%



Test Results Leaf extract Tobacco (Nicotiana tabacum L) Againstbacteria Mycobacterium tuberculosis extracts for antibacterial activity test n-hexane, ethyl acetate, and ethanol 96% and water and then tested on the bacterium Mycobacterium tuberculosis strain H37RV.The purpose of this



Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



3|



screening test is to find out extracts activethat can inhibit microbial growth by observing bacterial growth (cord) in the well in the plate with different concentrations, namely 400, 800, 1200,1600 and 2000 ppm. Table 2. Test Results antituberculosis n-hexan Leaf Extract Tobacco(Nicotianatabacum L) against the bacterium Mycobacterium tuberculosis extract N-hexan Treatment Information 1 2 Control Not inhibit Control + ++ ++ Inhibit concentration weak + + of 2000 ppm Inhibiting Concentration Does 1600 ppm 1200 ppm not Concentration inhibitDoes not inhibit 800 ppm Does not Concentration inhibit Concentration Does not of 400 ppm inhibit Table 3. Antituberculosis Test Results Ethyl Acetate Extract Tobacco Leaf (Nicotiana tabacum L) againstBacteria Mycobacterium tuberculosis Esk Treatment



Controls Control + Concentration of 2000 ppm Concentration of 1600 ppm 1200 ppm



EthylAcet ate 1 2 -



-



++



++



+



+



-



-



-



-



Description



Does not inhibit Inhibits Inhibits weak Does not inhibitDoes not inhibit Does not inhibit



Table 4. Antituberculosis Test Results Ethanol Extract 96% Tobacco Leaf (Nicotiana tabacum L) against Bacteria Mycobacterium tuberculosis Esktrak Ethanol Treatment Remarks 96% 1 2 Controls Does not inhibit Control + ++ ++ Inhibits Concentration Inhibits weak + + of 2000 ppm Concentration Does of 1600 ppm 1200 ppm not inhibitDoes not inhibit Concentration No inhibiting 800 ppm Concentration concentration Not hamper of 400 ppm Table 5. Test Results antituberculosis Water Extract Tobacco leaves(Nicotianatabacum L) against the bacterium Mycobacterium tuberculosis extract Water treatment Description 1 2 ControlsNot inhibit Control + ++ ++ inhibit concentration Inhibits weak + + of 2000 ppm Concentration Does of 1600 ppm Concentration not of 1200 ppm inhibitDoes not inhibit Concentration Does not of 800 ppm inhibit Concentration Does not 400 ppm inhibit



Concentration ofConcentrati on of 800 ppm Concentration Not inhibiting of 400 ppm Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



4|



Description: : There is growth of Mycobacterium tuberculosis (many) + : There is growth (Few) ++ : No growth of Mycobacterium tuberculosis Control + : Isoniazid Control - :bacteria Mycobacterium tuberculosis As for h The results obtained after incubation for 7 days showed that the negative control (-) with the treatment of + 50 µl DMSO + 950 µl media + Mycobacterium tuberculosis contained bacterial growth which was mostly seen in the microscope observation, for positive control (+) with treatment + 50 µl ddH2O + drug isoniazid + 950 mL of media + Mycobacterium tuberculosis there is no bacterial growth at all. The extract which can inhibit the growth of Mycobacterium tuberculosis is the four extracts, namely n-hexane extract, ethyl acetate, and 96% ethanol and water each at a concentration of 2000 ppm. The extract showed a strong enough inhibition on the growth of Mycobacterium tuberculosis seen from the appearance of the microscope with the results that the inhibition of bacterial growth was indicated by the number of cords which was less than the four extracts namely n-hexane extract, ethyl acetate and 96% ethanol and water at the concentration of 400 ppm, 800 ppm, 1200 ppm and 1600 ppm did not have any inhibition at all against the bacteria Mycobacterium tuberculosis so that the best inhibition of growth of Mycobacterium tuberculosis was found in each extract at a concentration of 2000 ppm. The inhibition power is seen in the presence of cord growth. Phytochemical Screening Test Results Based on phytochemical screening tests on tobacco leaf extract, the results obtained can be seen in the table as follows.



Table 6. phytochemical screening test Tobacco leaf extract(Nicotianatabacum L) Extract Nhexane



Compoun d Alkaloids



Reagent



results



Dragendorf



Yellow (-) Terpenoids LB Yellow / Steroids (-) Phenolic FeCl3 Orange (-) Flavanoid AlCl3 Yellow (+) Ethyl Alkaloids Dragendorf Green (acetate ) / Steroids LB Green (+) Steroids Phenolic FeCl3 Black(+ ) Flavanoid AlCl3 Clear () Ethanol Alkaloid Dragendorf Orange( 96% +) Terpenoid / LB Brown Steroids (+) Phenolic FeCl3 Black (+) Flavanoid AlCl3 Yellow (+) Water Alkaloid Dragendorf Orange (+) Terpenoid / LB Yellow Steroid (-) Phenolic FeCl3 Black (+) Flavanoid AlCl3 Brown (-) Result of identification of compound compounds extracts of tobacco leaves ( Nicotiana tabacum L) at 2000 ppm concentration those with inhibitory activity against the bacteria Mycobacterium tuberculosis contain compounds as follows, n-hexan extract contains flavanoid compounds, ethyl acetate extracts contain steroid and phenolic compounds, ethanol extract 96% contains alkaloid compounds, flavanoids, terpenoids, and



Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



5|



phenolics, while water extracts contain alkaloids and phenolics. CONCLUSION



The extract n-hexan, ethyl acetate, ethanol 96% and water from the leaves of tobacco leaves (Nicotiana tabacum L) has the potential to inhibit the growth of Mycobacterium tuberculosis through tests performed by the method of MODS. Extract n-hexan, ethyl acetate, ethanol 96% and water from the leaves of tobacco leaves (Nicotiana tabacum L) with each at a concentration of 2000 ppm showed activity antituberculosis almost the same in the test on the inhibition of Mycobacteriumtuberculosis,is not evident from the number of cords more little after being observed under a microscope. While at concentrations of 400 ppm, 800 ppm 1200 ppm and 1600 ppm in each extract there was no inhibition of the bacteria Mycobacterium tuberculosis. The group of compounds contained inextract tobacco leaf (Nicotiana tabacum L) after identification of groups namely n-hexan extract containing flavanoid compounds, ethyl acetate extract containing steroid and phenolic compounds, ethanol 96% containing alkaloids, flavonoids, terpenoids and phenolic compounds , while the water extract contains alkaloids and phenolics. REFERENCES



Agoes, G. Teknologi Bahan Alam. Bandung: ITB Press. 2007. Andareto, O. Apotik Herbal di Sekitar Anda, Jakarta: Pustaka Ilmu Semesta. 2015. Atun, S. Metode isolasi dan identifikasi stuktur senyawa organik bahan alam. Jurnal Konservasi Cagar Budaya Borobudur. 2014. Caviedes L, Moore DA. Introducing MODS: a low-cost, low-tech tool for highperformance detection of tuberculosis and multidrug resistant tuberculosis. Indian J Med Microbiol. 2007.



Dewi, S., Winarsih, S., dan Natasha, D.P., Uji Efektivitas Kulit Buah Delima (Punica Granatum) Sebagai Antimikroba Staphylococcus aureus Penyebab Infeksi Kulit dan Jaringan Lunak di Rumah Sakit dan Komunitas Secara In Vitro. Malang : Universitas Brawijaya. 2011. Dini, et al. The Potential Role Of Garlic (Allium Sativum) Against The Multi-Drug Resistant Tuberculosis Pandemic: A Review. Institut Superiore di Sanita: Italy. 2011. Dirjen POM. Pengawasan Obat dan Makanan Direktorat Pengawasan Obat Tradisional. Jakarta. 2000. Harborne, J.B. Metode Fitokimia Penuntun Cara Modern Menganalisis Tumbuhan. Penerbit ITB. Bandung. 1987. Hardy, Fleming’s Life Saving Discovery, Hardy Diagnostics, Santa Maria. 2011. Hermanto, S., L. Sugoro, Ikmalia. Profil Protein Escherichia coli Hasil Inakvitasi Iradiasi Gamma Sebagai Bahan Vaksin Mastitis. Jurnal. Pusat Aplikasi Tenaga Isotop dan Radiasi – BATAN. 2012. Joseph M Nguta et al. Antimycobacterial and cytotoxic activity of selected medicinal plant extract. University of Ghana. Ghana. 2016 Kee, Joyce L., & Evelyn R. Hayes (Ed. Yasmin Asih, Skp.). Farmakologi. Pendekatan Proses Keperawatan. Penerbit Buku Kedokteran EGC. Jakarta. 1996. Kemenkes RI, Profil Kesehatan Indonesia 2016. Jakarta: Bakti Husada. 2017. Kemenkes RI. Laporan Hasil Riset Kesehatan Dasar (Riskesdas) Indonesia tahun 2013. Jakarta : Badan Penelitian dan Pengembangan Kesehatan Kemenkes RI. 2013.



Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



6|



Kemenkes RI. Pengendalian Penyakit dan Penyehatan Lingkungan. Jakarta; Kementerian Kesehatan Republik Indonesia. 2014. Kemenkes, R.I. Petunjuk Teknis Manajemen TB Anak. Jakarta: Kementerian Kesehatan, Direktorat Jenderal Pengendalian Penyakit dan Penyehatan Lingkungan. 2013 Kementrian Agama RI. Al-Qur’an dan Terjemahnya. Bandung: CV. Penerbit Diponegoro, 2013.



Leba, Maria Aloisia Uron. Ekstraksi Dan Real Kromatografi. Deepublish: Yogyakarta. 2017. Listyanto. Budidaya Tanaman Tembakau (Nicotiana tabacum L.) Menggunakan Pupuk Hayati Bio P 2000 Z. PT. Alam Lestari Maju Indonesia. 2010. Martindale. The Complete Drug Reference Thirty Sixth Edition,: Pharmaceutical Press. London. 2009. Murwani Sri. Dasar- Dasar Mikrobiologi Veteriner. Universitas Brawijaya Press.Malang.2015 Nursalam Hamzah, dkk. Aktivitas Inhibisi Pertumbuhan Mycobacterium Tuberculosis dan Plasmodium Falciparum dari Ekstral Metanol Daun Botto-Botto (Chromolaena odorata Linn). Fakultas Kedokteran Dan Ilmu Kesehatan. Universitas Islam Negeri Alauddin Makassar. 2017. Rahul Shivaji et al. Comparative Study Of Antimicrobial Compounds Extracted From Leaves Of Nicotiana Tabacum.Departement of Microbiologi. India. 2015



Rusli, M. S, dkk. Antibacterial activity of temanggung tobacco extract variety Gadjah Kemloko. institute pertanian bogor: Bogor. 2011. Sudjadi. Metode Pemisahan Edisi Pertama. Yogyakarta : Kansius. 1988. Suharmiati dan Herti. Khasiat Dan Manfaat Daun Dewa Dan Sambung Nyawa. Agromedia Pustaka: Jakarta. 2003. Supardi S, Susyanty AL. Penggunaan obat tradisional dalam upaya pengobatan sendiri di Indonesia (Analisis Data Susenas 2007). Buletin Penelitian Kesehatan. 2010. Suriyai



Mohamad et al. Antituberculosis potential of some ethnobotanically selected Malaysian plants. School of Biological Sciences. Malaysia. 2010



Susilowati, E. Y. identifikasi Nikotin dari Daun Tembakau (Nicotiana tabacum) kering dan uji efektifitas ekstrak Daun Tembakau sebagai insektisida penggerak batang padi (Scirpopaga Innonata). Semarang: Universitas negeri semarang. 2006. Tiwari, P. B. Phytochemical Screenig and Extraction : A Review. International Pharmaceutical Science. Internationale Pharmaceutica Sciencia, 1(1), 98-106. 2011. Tresnaasih, Nanan. Aktivitas Anti-Tuberkulosis Ekstrak Larut Etil Asetat Daun Kenikir (Cosmos Caudatus H.B.K) Secara In Vitro. Universitas Gadjah Mada: Yogyakarta. 2017. WHO. Global Tuberculosis Report. World Health Organization. 2017



Ramadhani, Ikha. Uji Aktivitas Antituberkulosis Fraksi Dan Ekstrak Etil Asetat Batang Brotowali (Tinospora crispa (L.) Yahya, Rithoa. Karakteristik Mikrobiologis Dan Miers) Secara In Vitro. Universitas Aktivitas Antimikroba Susu Kuda Gadjah Mada: Yogyakarta. 2015. Fermentasi Koumiss Terhadap Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)



7|



Salmonella typhimurium Dan Mycobacterium tuberculosis. Institut Pertanian Bogor: Bogor. 2012



Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria Mycobacterium Tuberculosis (Rudy Adyaksyah)