STUDIES ON THE CELL BIOMASS PRODUCTION PROCEDURE OF YEW TREE (TAXUS WALLICHIANA ZUCC) FOR EXTRACTING ANTICANCER COMPONENTS Speciality: Pharmaceutical technology Code: 62 73 01 01 Full name: Vu Binh Duong Full name of supervisor: 1. Phan Dinh Chau, Dr.Sc., Prof. 2. Trieu Duy Diet, PhD. Educational foundation: Military Medical University INTRODUCTION Vietnamese yew tree (Taxus wallichiana Zucc.) is a rare – valuable herb distributed mainly in Hymalaya mountainous regions. In Vietnam, a small number of yew trees are found in central highland in Dalat, Lam Dong province. There are several anti-cancer/cancer-inhibitory compounds in Yew tree such as paclitaxel (Taxol), cephalomannin or pro-compounds used for semi-synthesis to produce anti-cancer drugs such as: baccatin III, 10-deacetyl baccatin III However, yew tree is a slow growing species; meanwhile the content of bioactive compounds is very low. Thus, the raw material for natural resource is not enough for the increasing demands of treatment. To overcome this drawback, together with natural breeding and growing, the plant cellbiomass is the new, promising direction to produce active compounds from herbs in general and yew tree as well. The palnt cell biomass is the cell culture in sterilized conditions in test tube or big tank/flask, to produce the cellmass for extraction of active compounds. To contribute to the production of raw material for paclitaxel from Vietnamese yew tree using plant cell biomass technology, the dissertation “Study on the yew tree cell (Taxus wallichiana Zucc.) bio-mass production procedure to extract anti-cancer active compounds” was carried out to: 1. To build the cell bio-mass production procedure at laboratory scale. 1 2. To determine the chemical composition, extract, isolate some main active compounds and establish the institutional standard for raw materials of yew tree cell biomass. The significance of the dissertation The yew tree cell bio-mass production procedure for the first time has been established in Vietnam (Taxus wallichiana Zucc.). The influent factors for the growth rate and active compound content has been evaluated, contribute to produce stable, sustainable and effective raw materials. For the first time in Vietnam, the chemical composition in yew tree cell biomass was investigated. The extraction procedure as well as the institutional standards of raw material was established to produce raw active compounds for anti-cancer drugs. The new contribution of dissertation The yew tree (Taxus wallichiana Zucc.) cell bio-mass production procedure has been done at laboratory scale. The optimized media composition, culture conditions for the best cell mass growth with highest content of paclitaxel has been established. At the same time, the harvest procedure for yew tree cell biomass has been studied as material for extraction of anti-cancer compounds. Chemical composition groups in yew tree cell biomass has been determined. 9 main compounds has been extracted, isolated and elucidated with taxane – frame derivative compounds. The simultaneous determination of paclitaxel and baccatin III has been developed and validated in yew tree cell biomass. From the yew tree cellmass, the extraction and purification procedures has been developed to produce paclitaxel. The content of paclitaxel product is up to 98.07% in purity; meets the standard criteria of USP 30. The institutional standards of yew tree cell biomass has been built in accordance with USP 30 and Vietnamese Pharmacopoeia IV including the 2 specifications: appearance, water content, impurity, qualitative, quantitative criteria. The strutuure of dissertation: inclduing 4 chapter, with 137 pages devided to following parts: introduction (2 pages), review of related research (28 pages), material and methods (11 pages), results (62 pages), discussion (31 pages) and conclusion (2 pages), perspectives (1 pages), references (with 148 references: 15 Vietnamese references, 133 english references) and appendix. Chapter 1: REVIEW OF RELATED RESEARCHES Review of related researches has 3 parts: - General information and researches about Vietnamese yew tree (Taxus wallichiana Zucc.): species characteristics, distributions, chemical compoisitions and bioactivity. - General information about the plant cell biomass technology: concepts, plant cell biomass procedures and influent factors – general knowledge of plant cell biomass technology to produce yew tree cellmass for paclitaxel production, the measure to increase the content of active compounds, quantitation and extraction of paclitaxel from yew tree cellmass. Chapter 2: MATERIALS AND METHODS 2.1. MATERIALS, REAGENTS Young branch of 10-year-old yew tree (Taxus wallichiana Zucc.) collected in Dalat, Lam Dong in April 2008, certified by the Institute of Ecology and Biorecource –Vietnam Accademy of Science and Technology. The specialized reagents met the criteria for plant cell culture. The specialized instruments used in cell culture and analysis. 2.2. METHODS 2.2.1. Develop the yew tree cellmass production procedure The yew tree plant cell biomass production procedure was carried out in accordance with the principles of R. M. Enaksha, G. Parc, R.M. Cusido. The young branches of yew tree were sterilized, cut into small slice, place 3 onto agar medium to create callus. Maintain the callus on the agar medium to produce the undifferentiable cells. After that, transfer the cells to liquid medium on a shaker. At this stage, the suitable time, pH, temperature and medium composition is needed to be determined. At the same time, the suitable elicitors were investigated. Then the cultivation was scaled up in 5- lit-bioreactor with the optimized/investigated conditions. The cell biomass was then harvested, the chemical composition was investigated, extracted, isolated and structure determined, the institutional standard of raw material and active compounds was established. 2.2.2. Investigate the chemical composition, extract and isolate main compounds, establish the institutional standard of raw material of yew tree cell biomass 2.2.2.1. Study on qualitative and quantitative analysis of active compounds in yew tree cellmass - Qualitative analysis of bio-active compound groups using characteristic chemical reaction and qualitative analysis of paclitaxel and baccatin III using HPLC. - Develop and validate the simultaneous determination of paclitaxel and baccatin III in the cell biomass using HPLC. 2.2.2.2. Study on the extraction, isolation and structure determination of some main compounds in the cellmass Extract compounds in cell biomass using ultrasonication method; purify these compounds using reverse and normal phase column chromatography. Structure determination of these isolated compounds based on the data of UV, IR, ESI-MS, NMR: 1 H, 13 C-NMR, DEPT 90, DEPT 135, HSQC, HMBC, COSY và NOESY. 2.2.2.3. Study on the extraction, purification of paclitaxel from yew tree cell biomass The extraction of paclitaxel in yew tree cellmass was carried out using ultrasonication. Purification of this compounds using the sequential 4 precipitation and crystallization methods and reverse phase column chromatography. In this investigation process, the factors that influence on the extraction yield and content of paclitaxel in each step were investigated. 2.2.2.4. Establish the institutional standard of yew tree cellmass Establish the institutional standard of yew tree cellmass in accordance to the methods in Vietnamese Pharmacopoeia IV and USP 30. 2.2.2.5. Quality control of paclitaxel Carry out the quantitative analysis of paclitaxel according to the method in paclitaxel section in USP 30. 2.2.3. Data analysis The data analysis was carried out using the statistical methods in Microsoft excel. Chapter 3. RESULTS 3.1. DEVELOP THE YEW TREE CELLMASS PRODUCTION PROCEDURE 3.1.1. Yew tree callus induction 3.1.1.1. Study on the media for callus induction Table 3.5: The media selection results No Medium Number of samples Callus weight (mg) p 1 MS 50 14.3 ± 1.5 p 3-1 <0.05 2 SH 50 20.1 ± 2.1 p 3-2 <0.05 3 B5 50 24.5 ± 2.2 * 4 White 50 12.9 ± 1.5 p 3-4 <0.05 When the B5 medium was used, the growth rate of callus is higher than those with other media (p< 0.05). 3.1.1.2. Influence of culture time The culture time optimization results showed that: callus weight was highest at the 35 th day. After this time, the callus weight gradually reduced. 5 (ngày) 3.1.1.3. Influence of growth hormone * Influence of growth hormone types Table 3.6: Influence of growth hormone type on the development of callus (n=50) No Batches Callus weight (mg) 1 2,4-D + kinetin 17.8 ± 1.4 2 2,4-D + BAP 19.8 ± 2.7 3 NAA + kinetin 24.8 ± 2.5 4 NAA + BAP 21.2 ± 1.8 p 3-1 <0.05 p 3-2 <0.05 p 3-4 <0.05 In the combination of NAA (1.0 mg/l) and kinetin (0.1 mg/l) medium, the growth rate of cellmass is 3 time higher than those of 3 remained combination (p<0.05). * Influence of NAA concentration Table 3.7. Influence of NAA concentration to the growth of callus (n=50) No NAA concentration (mg/l) Callus weight (mg) 1 0.5 23.6 ± 2.1 2 1.0 24.1 ± 2.3 3 1.5 25.0 ± 1.8 4 2.0 26.8 ± 1.9 5 2.5 25.6 ± 1.7 p 4-1 < 0.05; P 4-2 < 0.05 p 4-3 < 0.05; p 4-5 > 0.05 When the kinetin concentration was increased, the cell mass weight was increased and got the maximum at the NAA concentration of 2.0 mg/l (p<0.05). However, if the kinetin concentration continuously increased, the cellmass however was decreased. * Influence of kinetin concentration Table 3.8. Influence of kinetin concentration on the development of callus (n=50) No Kinetin conc. (mg/l) Callus weight (mg) 6 1 0.1 24.5 ± 1.8 2 0.2 27.9 ± 2.3 3 0.3 26.1 ± 1.9 4 0.4 25.9 ± 2.4 5 0.5 26.2 ± 2.2 p p 1-2 <0.05; p 2-3 >0.05 ; p 2-4 >0.05; p 2-5 >0.05 The most suitable concentration of kinetin is 0.2 mg/l. When the concentration of kinetin is higher than 0.2 mg/l, the callus weight is reduced. 3.1.2. Mentainance of yew tree callus on agar medium 3.1.2.1. Influence of media and the number of transferation The investigated results for the mentanence of callus on 2 media SH and B5 showed that: on B5 medium, the growth of callus was slow and easily differentiated into buds and root. Meanwhile, on SH medium, the growth of yew tree callus was much higher. Thus the SH medium was selected for further study in callus mentanence of yew tree. Table 3.9: The characteristic of yew tree cellmass after several transferation on SH medium Number of transferation Cell appearance Differentiable Growth rate (times) No 1 Hard Differentiate into buds 1.87 No 2 Hard, some cells was soft Differentitated into callus 2.25 No 3 Soft undifferentiated 2.78 No 4 soft, friable undifferentiated 3.51 No 5 soft, friable undifferentiated 3.72 After 5 times of transferation on SH medium, the callus growth rate is stable (reach 3.72 times compared with inoculant) and undifferentiated into tissue and organs. 7 3.1.2.2. Influence of saccharose concentrations Influence of saccharose concentration to the development of yew tree callus was presented in table 3.10. Table 3.10: Influence of concentration on the development of callus (n=10) No Conc. of saccharose (g/l) Weight (g) Growth rate (times) 1 10 1.16 ± 0.15 2.48 2 15 1.25 ± 0.13 2.65 3 20 1.75 ± 0.16 3.72 4 25 1.54 ± 0.13 3.29 5 30 1.26 ± 0.11 2.68 5 35 1.22 ± 0.14 2.60 7 40 1.12 ± 0.13 2.38 At the saccharose concentration of 20 g/l, the growth rate of callus is the highest (p<0.05), the growth rate reach 3.72 times. 3.1.2.3. Influence of culture temperature The investigated results on the influence of culture temperature to the development of yew tree callus showed that: at the temperature of 22-24 0 C the yew tree callus weight is higher than those of other groups (p<0.05), the growth rate reach 3.72 times. 3.1.2.4. Influence of culture medium pH The investigation of culture medium pH on the growth of yew tree callus showed that: in the medium with pH 5,6, the growth of cellmass was the highest (p < 0.05), the growth rate reaches 3.79 times. 3.1.3. The culture results in liquid medium 8 3.1.3.1. Influence of culture times Figure 3.7. The cellmass weight versus culture time curve The yew tree cellmass growth rate is increased with culture time, reached the maximum at the 14 th day. After that, the cellmass weight gradually reduced. 3.1.3.2. Influence of inoculum weight Table 3.14. Influence of inoculum ratio to the growth rate of cellmass No Inoculum ratio (%) Cellmass weight (g) (n=10) The growth rate (times) 1 10 17.25 ± 0.57 1.73 ± 0.06 2 15 24.59 ± 0.89 2.48 ± 0.08 3 20 28.29 ± 1.36 2.83 ± 0.07 4 25 28.78 ± 2.81 2.89 ± 0.09 5 30 25.02 ± 2.05 2.60 ± 0.11 p 3-1, 3-2, 3-5 < 0.05, p 3-4 >0.05 Using the inoculum ratio at 20% is the most suibtale for yew tree cell culture. hen the inoculum is 25% , the growth rate is not significantly changed (p 3- 4 >0.05), causing waste of sample. 3.1.3.3. Influence of medium pH The investigation results on the influences of medium pH showed that: at pH=5,6, the cell growth rate was the highest, the growth rate reached 2.83 times (p< 0.05). 9 3.1.3.4. Influence of culture temperature The investigation results on the influence of culture temperature to the cell growth rate showed that: at the temperature of 24 0 C, the growth rate of yew tree cellmass was the highest, the growth rate reaches 2.83 times (p<0.05). 3.1.3.5. Influence of growth hormones * Influence of BAP concentrations Table 3.18. Influence of BAP concentrations on the growth rate of cellmass (n=10) No Conc. of BAP (mg/l) Cellmass weight (g) Growth rate (times) 1 0.5 20.62 ± 1.41 2.06 ± 0.07 2 1.0 28.29 ± 1.36 2.82 ± 0.11 3 1.5 30.20 ± 1.61 3.02 ± 0.10 4 2.0 33.56 ± 1.60 3.27 ± 0.06 5 2.5 32.98 ± 1.33 3.28 ± 0.08 6 3.0 33.24 ± 1.63 3.30 ± 0.09 7 3.5 33.61 ± 1.27 3.34 ± 0.08 p 4-1 , 4-2 , 4-3 < 0.05, p 4-5 , 4-6 , 4-7 > 0.05 At the BAP concentration of 2.0 mg/l, the cellmass growth rate reached 3.27 times (p< 0.05). If the concentration of BAP > 2.0 mg/l, the growth rate was not significantly changed. * Influence of NAA concentration Table 3.19. Influence of NAA concentration on the development of cellmass No NAA conc.(mg/l) Cellmass weight (g) (n=10) Growth rate (times) 1 1.0 25.35 ± 1.51 2.53 ± 0.07 2 2.0 33.56 ± 1.60 3.27 ± 0.06 3 3.0 40.31 ± 1.84 4.01 ± 0.10 4 4.0 40.68 ± 1.46 4.05 ± 0.08 5 5.0 40.90 ± 1.79 4.07 ± 0.06 6 6.0 41.11 ± 1.71 4.09 ± 0.09 p 3-1 < 0.05 p 3-2 < 0.05 p 3-4 > 0.05 p 3-5 > 0.05 p 3-6 > 0.05 10 [...]... 12.32±0.12 * 7 6 39.65±1.98 9.93±0.27 p6-7< 0.05 After 5 days exposed with MJ, the content of paclitaxel in cellmass reached the highest 12.32 mg/l (p . introduction (2 pages), review of related research (28 pages), material and methods (11 pages), results (62 pages), discussion (31 pages) and conclusion (2 pages), perspectives (1 pages), references. 2α,5α,10β-triacetoxy-14β-(3-hydroxy-2-methyl)-butyryloxy-4(2 0), 11- taxadiene ( 4) và β-sitosterol ( 9) and four other compounds were identified 16 b c from the ethyl acetat fraction: 13-dehydroxy baccatin III ( 5), paclitaxel ( 6), ( +)- catechin. fractured : Taxuyunnanine C ( 1); 2α,5α,10β- triacetoxy - 14β - propionyloxy-4(2 0), 11-taxadiene ( 2); 2α,5α,10β-triacetoxy - 14β- (2-methyl) - butyryloxy-4(2 0), 11- taxadiene ( 3); 2α,5α,10β-triacetoxy-14β-(3-hydroxy-2-methyl)-butyryloxy-4(2 0), 11- taxadiene