My Biochemistry research
- 1. Anti Salmonella typhi activities of non aqueous camelia sinensis crude extract Tiurma PT Simanjuntak,STP M.Si Lecturer biochemistry
- 2. OUTLINE Materials and methods Result and analysis Summary Introductions
- 3. Introductions typoid fever caused by Salmonella typhi . paratypoid fever caused by Salmonella paratyphi A,B,C,D,E Salmonella typimurium from mouse and causing typimurium fever similarly with typoid fever . Typoid fever causing deadly people over 600.000 from 17 million cases every year. Drug discovery promising a new drugs alternative.
- 4. Salmonella Typhi Founded in 1880 causing Tifoid fever ( pagositosis ) Every each 10 9 colonies on 1 ml suspency posibilities 95% sickness. Every each 10 5 colonies on 1 ml suspency posibilities 25 % sickness. Amplifications using PCR on 0,4 kb for identifications
- 5. Natural product Material Camelia sinensis sp. Harvesting from 2 years New drugs alternative having polyphenols . Processing became green tea , oolong tea and black tea
- 6. Drug discovery Acquisition ( raw material, field colections, culture colections ) Extractions Primary screens Isolation and Characterisation secondary screens structural elusidation pre-clinical development
- 7. On going research Crude extract from green tea being extraction by etanol p . a and screening activity antibacterial S.typhi Crude extract from green tea being extraction by using water. Screening activity anti bacterial S. typii and also activity antibacterial S. paratypii A,B,C,D,E. S. typimurium and E.coli antibiotic resisten. result Diameter inhibition area S. typii from ethanolic crude extract of green tea about : 33,3 % and 40 %.From water fractions 39,5 % and 44,7 %. Diameter inhibitions area S.paratypii is 10 mm and 14,5 mm. There is also diameter inhibitions to S. typimurium and E.Coli antibiotic resisten
- 8. Questions How is the activity of green tea crude extract against S.typhi in other solvent? Expecially by using n-heksan,benzena,diklometan,etil asetat.
- 9. Methodology 50 g green tea + 175 mL water as solvent Extract green tea ( brown ) agar difussion method Crude extract green tea (dry) Crude extract green tea (liquid) With n -heksan agar difussion method extraction with soklet Drying with vacuum konsentrator on 30 C Re-solvent again screens extractions
- 10. Fasa n -heksan Fasa water Extractions with benzen Activity antibacterial Fasa water Fasa benzen Extractions with diklorometan Fasa water Fasa diklorometan Fasa water Fasa etil asetat Extractions with etil asetat Activity antibacterial Activity antibacterial Activity antibacterial 200 uL dried (fraksi water 1 ) Drying and resolvent again Drying and resolvent 200 uL dried (fraksi water 2) 200 uL dried (fraksi water 3 ) 200 uL dried (fraksi water 4 ) Drying and resolvent Dried and resolvent again
- 11. Result and analysis green tea (50 g) Soxletation 175 with water as solvent for10 hours Crude extract (50 ml) Crude extract dried (2,186 g) Liquid Crude extract Dried 60 °C, 2 days Resolvent again with water Green tea crude extract (200 l) Liquid Crude extract (800 l) Partitions n-heksan (1:1) …………… ... dried
- 12. continue Fraksi water (0,051 g) Fraksi heksan (0,002 g) Partitions with 600 ml benzen p.a (1:1) Fraksi water (0,038 g) Fraksi benzen (0,004 g) Fraksi water (0,077 g) Fraksi water (0,077 g) Partitions with 400 ml diklorometan (1:1) Partitions with 200 ml etil asetat (1:1) Fraksi diklorometan (0,003 g) Fraksi etil asetat (0,003 g) Screening with agar diffusion method and coloni counting plate method
- 13. Crude extract green tea with partitions by n -heksan, benzen, diklorometan dan etil asetat
- 14. Result from activity anti S.thyphi by Agar Difussion methods B A C D A : 10 ul crude extract green tea 54,65mg/ ml B : 10 ul liquid extract green tea C : 10 ul aquadest D: 10 ul ampisilin 100 mg/ml Diameter inhibitions area of extract green tea. Ampicilin as positive control Aquadest as negative control
- 15. Diameter Inhibition area note : a : crude extract green tea from water 54,65 mg/ml b: crude extract green tea in other solvent 54,65 mg/ml c: kontrol - d: kontrol + H B D E
- 16. Result from coloni counting plate
- 17. Analysis 54.65 54,65 54,65 54,65 54,65 0,5 1 2 1 2 83 420 550 320 940 Control 1140 Result and analysis from crude extract green tea Crude extract green tea Consentration (mg/ml) Diameter inhibitions (mm) Total colonies Fractions water Fractions water 1 Fractions water 2 Fractions water 3 Fractions water 4
- 18. Analysis 54,65 54,65 54,65 54,65 0 1 0 1 500 0 2 0 Analysis and result from green tea non aqueous solvent Extractions Consentrations (mg/ml) Diameter inhibi tions (mm) Total coloni N-heksan benzena diklorometan Etil asetat
- 19. Summary Diameter inhibitions area using agar diffusion showed O mm,1 mm, O mm,1 mm for green tea extract using non water as solvent with concentrations 54,65 mg/ml Diameter inhibitions area 2 mm for green tea crude extract using water. The counting plate experiment showed a decrease in the total number of bacteria colonies. (7,2 %) A traditionals drugs for activity anti Salmonella Typhi provide a new alternative,expeciallly by using water as solvent. This methode are simple, cheaper,easy and also could give other benefits to phytopharmaca science
- 20. Perkebunan teh desa mekar sari Subang sources of green tea.
- 21. Thank you
- 23. DNA amplifications from chromosome S.typhi 1419 517 396 214 75 65 Dna kromosom s.typhi kontrol positif DNA kromosom S typhi yang hidup dalam ekstrak 0.4 kb pb PUC/HinfI
- 27. Metoda koloni hitung Media luria bertani padat Media LB padat + ekstrak teh 20 ul Media LB + ekstrak teh hasil partisi benzena 20 ul dan diklorometan + 30 ul bakteri + 30 ul bakteri + 30 ul bakteri Masing-masing di spread dan di inkubasi 37OC Hitung jumlah koloni
- 28. Tabel.Berat kering ekstrak yang diperoleh dalam pelarut selain air. Tabel. Berat kering ekstrak yang diperoleh dalam pelarut air 0,077 200 L Fraksi air 4 0,077 200 L Fraksi air 3 0,038 200 L Fraksi air 2 0,051 200 L Fraksi air 1 0,075 200 L Fraksi air Berat kering yang diperoleh (gr) Volume yang dikeringkan suhu 30 o C Ekstrak teh 0,003 150 L Fraksi etil asetat 0,003 300 L Fraksi diklorometan 0,004 550 L Fraksi benzen 0,002 750 L Fraksi n-heksan Berat kering yang diperoleh (gr) Volume yang dikeringkan suhu 30 o C Ekstrak pelarut teh
- 29. Taksonomi Camelia Sinensis Divisio : Spermatophyta Sub-divisi : Angiospermae Kelas : Dicotyledonae Bangsa : Guttiferales Suku : Theaceae Marga : Camelia
- 32. Koloni hitung
- 33. Klasifikasi Salmonella typhi Genus : Salmonella Famili : Enterobacteriaceae Ordo : Eubacteriales Kelas : Schizomycetes Divisi : Protophyta
- 34. Analisis porsentase koloni hitung pada pelarut tanpa ekstrak 100 1140 KONTROL 0,87 10 Etilasetat 3,5 40 diklorometan 12,63 144 Benzen 80,52 918 n-heksan % koloni hidup Jumlah koloni hidup Ekstrak teh
- 35. Analisis porsentase koloni hitung pada ampicilin dalam berbagai konsentrasi 100 450 0 66,6 300 25 40 180 50 28,57 120 75 7,1 32 100 % koloni hidup Jumlah koloni hidup Konsentrasi (mg/ml)
- 36. Analisis porsentase koloni hitung pada pelarut 100 1140 Kontrol 0 0 Etilasetat 0,17 2 Diklorometan 0 0 Benzen 43,85 500 N-heksan % koloni hidup Jumlah koloni hidup Ekstrak pelarut
- 37. Kepolaran Pelarut Organik Lazim untuk fraksinasi N-Heksan pelarut paling non polar terhadap air,konstanta dielektrik =1.88 Benzena ,pelarut non polar terhadap air,konstanta dielektrik = 2,28 Diklorometan pelarut semi polar, konstanta dielektrik=5,9 Etil Asetat pelarut semi polar, konstanta dielektrik= 6,02
- 38. Analisis porsentase koloni hitung pada fraksi air 82,45 940 Fraksi air4 28,07 320 Fraksi air3 48,24 550 Fraksi air2 36,84 420 Fraksi air1 7,2 83 Fraksi air % koloni hidup Jumlah koloni hidup Ekstrak teh