Effects of Anti Mycobacterium tuberculosis Nanoherbal Legundy Leaf Extract (Vitex trifolia)

TYPE OF ARTICLE: Research BackgrounAbstract: Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Indonesia ranks 2 largest in the world after India. Resistance to anti-tuberculosis drugs is one of the difficulties in treating tuberculosis. This research aim to determine the effectiveness of legundi (Vitex trifolia) leaf extract nanoherbal in killing Mycobacterium tuberculosis. This study was conducted with an experimental design using M. tuberculosis colonies taken from 25 samples of aspirate of tuberculosis patients with lymph nodes and then given the ethanol extract of legundi leaves (70% and 90%) with the process of making nanoparticle extracts. The evaluation of the observations was in the form of measuring the inhibition zone for bacterial growth which were categorized as weak (<5mm), moderate (6-10mm), strong (11-20 mm) and very strong (> 21mm). The results of the study from 6 repetitions found that the inhibition zone category was 0% weak, 39% moderate, strong 52.3%, and very strong 8.7%. Comparison of the concentration of legundi leaf extract to the diameter of the bacterial growth inhibitory zone was analyzed using the Kruskal Wallis test, which found a significant relationship with p value = 0.000 (<0.05). Legundi's leaf extract can be used as an antibacterial agent for M. tuberculosis.

Abstrak: Tuberkulosis (TB) adalah penyakit infeksi menular yang disebabkan oleh Mycobacterium tuberculosis. Indonesia menempati urutan ke-2 terbesar didunia setelah India. Resistensi terhadap Obat Anti Tuberkulosis menajdi salah satu kesulitan pengobatan Tuberkulosis. Penelitian ini bertujuan untuk mengetahui efektivitas nanoherbal ekstrak daun legundi (Vitex trifolia) dalam membunuh Mycobacterium tuberculosis. Penelitian ini dilakukan dengan rancangan eksperimental menggunakan koloni Mycobacterium tuberculosis yang diambil dari aspirat pasien tuberkulosis kelenjar getah bening sebanyak 25 sampel kemudian diberikan ekstrak etanol daun legundi (konsentrasi 70% dan 90 %) dengan proses pembuatan ekstrak nanopartikel. Evaluasi hasil pengamatan berupa pengukuran zona hambat pertumbuhan bakteri yang dikategorikan lemah (<5mm), sedang (6-10mm), kuat (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) ) in 2017 on each 100 thousand populations of Indonesia found 138 people  diagnosed TB cases by health workers and 64 people with smear-positive. In Medan, based on the 2016 Medan City Health Profile, total number of TB cases was 6418 cases, the number of new TB cases with positive of Acid Resistant Bacteria (ARB) examinations was found 2829 cases and TB cases in children 0-14 years 231 cases. Although pulmonary TB is the most prevalent, extrapulmonary TB is also an important clinical problem. Based on epidemiology extrapulmonary TB constitutes 15-20% of all TB cases, of which TB lymphadenitis is the most prevalent form (35% of all extrapulmonary TB ). Primary infection occurs after a person inhales M. tuberculosis, after passing through the mucociliary barrier of the airway, the TB bacilli will reach the alveoli. The germs spread through the lymph flow to the hilar lymph nodes causing a local lymphangitis process. 1 2 3 4 The relatively long time for TB treatment (6 -8 months) is the reason TB patients find it difficult to recover because TB patients drop out of drugs after feeling healthy even though the treatment process has not been completed. Antituberculosis drugs (OAT) which is used consisting of isoniazid (H), Ethambutol (E), Pyrazinamide (Z), Rifampicin (R) and streptomycin (S ). The use of OAT itself has side effects. Isoniazid has the effect of peripheral neuropathy and hepatitis, Ethambutol additions have the effect of loss of vision which progressively because of retrobulbar neuritis, Pirazinamid has the effect of liver damage (hepatotoxicity) and arthralgia, Rifampicin effect on gastro-intestinal tract, such as nausea, loss of appetite and mild abdominal pain, diarrhea sometimes arise, and streptomisin has become hypersensitive and skin effect occurs hearing loss. 2 5 6 Because of the many side effects, multiple resistance and frequent failure of tuberculosis treatment, several studies have examined alternative tuberculosis treatment options. Herbal medicine is more easily accepted by the community because besides being familiar with the community, it is cheaper and easier to obtain. One of the plants in Indonesia that is accepted by the community is legundi. So far, there have been many studies that reveal the benefits and properties of this legundian plant both from the leaves, stems, fruits and seeds, especially their effects as antibacterial, antifungal, insecticide, anticancer, analgesic, tracheospasmolytic, allergy and antipyretic. 6 7 Legundi leaves contain essential oils which are composed of sesquiterpenes, terpenoids, ester compounds; alkaloids (vitrisin, flavonoid glycosides (artemetin and 7 desmethyl artemetin) and nonflavonoid components friedelin, ß-sitosterol, glucosides and hydro-carbon compounds (Listiawati, 2010). Flavonoids and essential oils are phenolic class compounds that will kill bacteria by coagulating or denaturing the protoplasm of the cell protoplasm, or causing cell lysis by changing the structure of the cell membrane resulting in leakage of cell contents. While the saponin mechanism is thought to damage the lipid membrane so that it can penetrate the cell wall and kill bacteria. 8 Legundi leaves are proven to be antibacterial for Staphylococcus aureus and Eschericia coli with a minimum kill rate of 20%. Research conducted by Lubis HML in 2016 proved that giving legundian fruit at a dose of 0.5 g/kg / day and 1 g/kg/day for 2 weeks can reduce the size of skin tumors due to the flavonoid content of these plants. Research in vitro have been developed which are often taken from sputum specimens but were taken from lymph node aspirates cervical lymph undiscovered related literature. For this reason, the effect of legundi leaf extract will be assessed in the treatment of tuberculous lymphadenitis so that the use of synthetic drugs can be avoided. 9 10

MATERIALS AND METHOD
Legundi leaves were from Yogyakarta city and identified in the Laboratory of Herbarium Medanense (Meda), University of North Sumatra. Prepare legundi nano leaf extract by washing legundi leaves with water, draining then drying in an oven at 40 o C. Dry materials are pollinated using nano technology using a High Energy Ballmill tool and making legundy leaf ethanol extract consisting of a concentration of 70 % and 90%. 11 Mycobacterium tuberculosis colonies were taken from aspirates of tuberculosis lymphadenitis patients from the Laboratory of Anatomical Pathology, Faculty of Medicine, Muhammadiyah University of North Sumatra. The result shows that 5 from 25 samples were positive level 1 (1+), 8 samples were positive level 2 (2+), and 12 samples were positive level 3 (3+) (Figure 1, 2 and 3). 12 The culture process of M. tuberculosis colonies using Lowenstein-Jensen medium as much as 150 petri dishes for 6 repetitions of the treatment. Evaluation of the observations in the form of bacterial growth inhibition zone categorized as weak if inhibition zone diameter ≤ 5mm, 6-10mm in diameter when the medium category, a category stronger as the diameter of 11-20 mm, the category of very strong when diameter ≥ 21 mm. Statistical analysis of the results inhibition zone diameter with statistical Kruskal Wallis analysis. 13

RESULT
The results of anti-Mycobacterium tuberculosis examination for nanoherbal leaves of legundi (V. trifolia ) with a concentration of 70% and 90% , are shown in Table 1 and Table 2. In the examination of the inhibition zone measurement for the growth of M. tuberculosis, it was found that 39% had a moderate inhibition zone, 52.3% had a strong inhibition zone, 8.7% had a very strong inhibition zone (Figure 4, 5 and 6). The results of the inhibition zone diameter had analyzed using the Kruskal Wallis test found a significant difference with a p value = 0.00 (<0.05). Furthemore the Mann Whitney test shows that there is a significant difference

DISCUSSION
Of the many traditional plants that are useful for the treatment, Legundi (V. trifolia) is a plant that is not so popularly used in Asia, especially in Indonesia un tuk treatment of tuberculosis. Vitex trifolia (Family-Verbenaceae) is an aromatic tree, which can grow up to 4m and is found from the foothills of the Himalayas to the south throughout most of India. Legundi's leaf extract is used as an anti-cancer, while the fruit is good for amenorrhea. Vitex trifolia flowers mixed with honey are used in cases of fever accompanied by vomiting and dehydration. The methanol extract of this plant has been reported to have strong antioxidant activity. Legundi leaves are proven to be antibacterial for Staphylococcus aureus and Eschericia coli with a minimum blood content of 20%. 3 9 14 The content of flavonoids and essential oils from legundi leaves works to kill bacteria by coagulating or denaturing cell protoplasmic proteins, or causing cell lysis by changing the structure of the cell membrane resulting in leakage of cell contents. From the experiments conducted, it was proven that legundian leaves can inhibit the development of M. tuberculosis bacteria by observing the improvement of the inhibition zone at all the concentrations of legundian leaf extract examined. In the early stages of this bacterial infection the macrophages will engulf the bacteria and form granulomas. Mycobacterium tuberculosis is an inactive state, actively replicates but is killed by the immune response, or is metabolically altered by a limited replicative cycle, making OAT treatment more difficult. The action of flavonoids and essential oils that remove cell contents including the M. tuberculosis bacillus makes it easier and more adequate for the action of legundian leaves. 5 8 15 Immunopathology regarding the development of tuberculosis is very intensively carried out and requires deeper research, especially in terms of the development of very potential traditional medicines. From the results of this research, it is proven that legundian leaf extract can be used as an antimicrobial against M. tuberculosis and can be continued for further research on tuberculosis immunotherapy.

CONCLUSION
The results of the study from 6 repetitions found that the inhibition zone category of legundy leaf extract was 0% weak, 39% moderate, strong 52.3%, and very strong 8.7%. Comparison of the concentration of legundi leaf extract to the diameter of the bacterial growth inhibitory zone was analyzed using the Kruskal Wallis test, which found a significant relationship with p value = 0.000 (<0.05). Legundi's leaf extract can be used as an antibacterial agent for Mycobacterium tuberculosis.