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.
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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
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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
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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

3-2
<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

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.
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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

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

,
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
4-2
< 0.05 p
4-3
< 0.05 p
4-5
>0.05
Using the saccharose concentration at 30 g/l, the cell growth rate was
the highest, cellmass weight reached 51.61 g/l and growth rate reached 5.14
times (p<0.05).
3.1.3.7. Study on the selection of suitable elicitors
a. Selection of suitable elicitors
The addition of elicitors including MJ, JA, acid caffeic, acid ferulic at
the concentration of 150 µM and yeast extract at the concentration of
100µg/g at the day of 12 in culture cycle. After 3 day of culture, the results
showed in the table 3.21
Table 3.21: Influence of elicitor on the growth rate and content of
paclitaxel in cellmass (n=10)
No Elicitor types Cellmass weight (g) Paclitaxel (mg/l)
1 Control 52.41±2.43 6.48±0.17 p
3-1
< 0.05
2 JA 36.83±2.10 8.84±0.21 p
3-2
< 0.05
3 MJ 38.98±1.98 9.39±0.20 *
4 Acid ferulic 45.22±2.21 7.53±0.14 p
3-4
< 0.05
5 Acid caffeic 46.39±1.86 7.64±0.18 p

Paclitaxel (mg/l)
1 Control 48.31±2.34 6.49±0.31 p
6-1
< 0.05
2 1 44.37±2.12 7.21±0.26 p
6-2
< 0.05
3 2 44.23±1.97 8.75±0.21 p
6-3
< 0.05
4 3 41.45±2.04 10.39±0.37 p
6-4
< 0.05
5 4 42.98±2.02 11.06±0.28 p
6-5
< 0.05
6 5 42.37±2.17 12.32±0.12 *
7 6 39.65±1.98 9.93±0.27 p
6-7
< 0.05
After 5 days exposed with MJ, the content of paclitaxel in cellmass
reached the highest 12.32 mg/l (p<0.05).
* Study on the exposed time point with elicitor
Bảng 3.24: Influence of the exposed time point between cells and MJ to the
growth rate and content of paclitaxel (n=10)
No
Exposed time
point (day No
th
)

Table 3.25: Influence of the saccharose addition time (n=10)
No
Time point to add
saccharose
(day
th
)
Dried cellmass
(g)
Paclitaxel
(mg/l)
1 Control 48.23±2.11 6.51±0.09 p
5-1
<0.05
2 No addition 42.28±1.57 12.45±0.19 p
5-2
<0.05
3 2 38.25±2.09 9.72±0.29 p
5-3
<0.05
4 6 42.41±1.89 11.26±0.36 p
5-4
<0.05
5 10 42.57±1.54 15.10±0.42 *
6 14 44.06±2.08 12.47±0.33 p
5-6
<0.05
When the addition of saccharose to culture medium at the day of 10,
content of paclitaxel reached highest at 15.1 mg/l (với p<0.05).
3.1.4. The culture results in 5-litter bioreactor system

- Cont: 35 days, 24
0
C, in darknees
Callus induction
Culture in
suspension medium
- SH + NAA (3mg/l), BAP (2mg/l), saccharose (30g/l).
- Cont: rate mass of 20%; t
0
=24
0
C; pH=5,6; stir 130r/m,
14days
Callus/Agar medium
Cell mass/ Bioreactor
- Filter
- Wash 3 times with water
- Dry 40 -60
0
C/10h
Harvest cellmass
Dried cell mass
- SH + NAA (3mg/l), BAP (2mg/l), saccharose (30g/l+20g/l
after 10days culture), MJ (100µM) added at 12th
- Cont: rate of mass 20%; t
0
=24
0
C; pH=5,6; stir 60 r/m, O2
30%; 17days

analysis method showed high specificity, linearity, accuracy and precision,
stability.
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Figure 3.9. The yew tree cellmass procedure
Table 3.30. Gradient program
Time (min) ACN (%) H
2
O (%)
0 - 45 25 – 63 75 – 37
45 - 55 63 – 25 37 – 75
55 - 65 25 75
3.2.1.2. Qualitative determination of some groups of compounds in yew
tree cell biomass
The qualitative determination of bioactive compound groups in yew
tree cellmass using the characteristic chemical reactions showed that: the
yew tree cellmass contains several groups of active compounds such as
alcaloid, tanin, anthranoid, sterol, organic acid.
3.2.1.4. Quantitative analysis of paclitaxel and baccatin III in yew tree
cellmass
The quantitative analysis of paclitaxel and baccatin III in yew tree
cellmass by HPLC showed that the content of paclitaxel and baccatin III
were 0.0360% and 0.0061%, respectively. Compared with the newly
collected young branch yew tree and leaves, the content of paclitaxel was
3.5 times higher. However, the content of baccatin III was lower (p<0.05).
3.2.2. Extraction, fraction and identification bioactive compounds in
Taxol biomass
Nine compounds in Taxol biomass were fractured : Taxuyunnanine
C (1); 2α,5α,10β- triacetoxy - 14β - propionyloxy-4(20),11-taxadiene (2);
2α,5α,10β-triacetoxy - 14β- (2-methyl) - butyryloxy-4(20),11- taxadiene
(3); 2α,5α,10β-triacetoxy-14β-(3-hydroxy-2-methyl)-butyryloxy-4(20),11-

<0,05
Using MeOH for extracting paclitaxel in taxol biomass brings about the
highest productivity (97,30%) and the content of paclitaxel was 0,5%.
* Stage 2 (liquid-liquid extraction):
For using DCM for extracting paclitaxel in MeOH extract, the productivity
was 91,94%, and the quantity of paclitaxel was increased from 0,5% to
5,93%.
3.2.3.2. Purification of paclitaxel
* Results of eliminating impurities with activated carbon: with the ratio of
activated carbon to extract (3:1), the content ofpaclitaxel was highest
(7,81%) and the productivity was 88,68%.
* The rate of n-hexan using to purify paclitaxel (n=6)
Table 3.45. Results of using n-hexan to purify paclitaxel(n=6)
No
Ratio of
n-hexan
to DCM
Mass (g) Proportion
of
paclitaxel
(%)
Content of
paclitaxel
(mg)
Productivity
(%)
1 4 : 1 0,16 ± 0,02 27,10 ±
1,76
40,7±2,4 55,97 ± 4,19
2 6 : 1 0,19 ± 0,01 26,51 ±

of paclitaxel
(%)
The
content of
paclitaxel
(mg)
Productivity
(%)
1 2:8 89,4 ± 8,2 62,20 ±1,22 55,8±3,1 44,60 ±2,00
2 3:7 109,1 ± 6,9 60,03 ± 1,98 65,5±3,9 52,43 ±1,91
3 4:6 147,7 ± 9,4 60,46 ± 1,05 89,3±4,2 71,41 ± 1,45
4 5:5 179,2±10,7 50,78 ± 2,97 91,2±3,5 73,07± 1,25
5 6:4
231,7 ±
4,3
40,05 ± 2,05 92,8±2,9 74,22 ± 1,65
p p
3-4
<0,05 p
3-1, 3-2,3-4
<0,05
With the ratio H
2
O : MeOH (4 : 6), the productivity of the purification
was highest 71,41%, the proportion of paclitaxel was 60,46%.
* The purification of paclitaxel using the column chromatography
Table 3.47. Results of the purification of paclitaxel using column
chromatography
Sample
1

Productivity of each
stage (%)
Extraction with MeOH 0,50 ± 0,02 97,30 ± 0,56
Extraction with DCM 5,93 ± 0,25 91.94 ± 1,45
Elimination using activated
carbon
7,81 ± 0,18 88,68 ± 2,58
Precipitating in n-hexan 25,37 ± 1,12 82,43 ± 1,76
Precipitating in the mixture
MeOH: H
2
O
60,46 ± 1,05 71,41 ± 1,45
Using the column
chromatography 1
st
93,11 ± 1,74 91,97 ± 2,61
Using the column
chromatography 2
nd

98,07 ± 0,97 92,74 ± 3,09
The process 98,07 39,83
The total productivity of the extraction and purification of paclitaxel from
taxol biomass was 39,83%, and the content of paclitaxel in the product was
98,07%.
3.2.4. The standardisation of the material of taxol biomass and active
ingredients
3.2.4.1. The standardisation of the material of taxol biomass
* Form: dried, friable, brown biomass without fungi.

kinetin (0.2mg/l) was higher than that in the medium added other elicitors
at other concentration (p<0.05). The result was suitable with the result of
the R.M Enaksha study on Pacific taxol biomass.
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4.1.2. Maintaining taxol callus in agar medium
In SH medium added NAA (2.0mg/l), kinetin (0.2mg/l) and saccharose
20g/l, medium pH of 5.6, tempature of 24
0
C, after transfering 5 times the
biomass developed rapidly and became soft, friable and unable to
differentate a organ. The growth rate of biomass increased 3.81 times,
which indicated that the new developed cell adapted gradually with new
medium.
4.1.3. The taxol callus culture in liquid medium
4.1.3.1. Effect of culture conditions
* Period of culture in liquid medium
The reasonable time of taxol cell culture cycle in liquid medium was 14
days. This was suitable with the result of H.K.Choi’s study on T. chinensis
cell culture.
* Inoculum ratio
Using the inoculum ratio of 20% results in that growth rate increased 2.83
times. Therefore, this rate was appropriate. Compared with other study, this
rate was higher but the growth rate of biomass was rapid.
* Medium pH and tempareture of culture
For T.wallichiana cell culture, at the medium pH of 5.6 and temperature of
24
0
C the development rate of biomass was maximal and the growth rate of
biomass increased 2.83 times.
4.1.3.2. Effect of the components in culture medium

content of paclitaxel increased highest by 15.1 mg/l (increased
approximately 2.5 folds as much as the control sample). This result was
suitable with the H.Q. Wang‘s study on T.chinensis cell culture (saccharose
added on 7
th
day)
4.1.4. Taxol cell culture in 5-liter bioreactor
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After 8 batches of taxol cell culture in 5- liter bioreactor, the growth rate of
biomass increased 3.6 times and the content of paclitaxel inside cell was
12.92 mg/l and outside 1.23 mg/l. The growth rate of taxol biomass in this
study was higher than that in other study on taxol culture.
4.1.5. The process of harvesting taxol biomass
The result of the process of harvesting taxol biomass has shown that using
the method of normally filtering and washing with water 3-4 times
unchanges the content of palitaxel in biomass whereas the content of BAP
and NAA decreaed and is in the safe range recommended by the US
Department of protect environment.
4.2. INVESTIGATE CHEMICAL COMPOSITION, EXTRACTION,
ISOLATION, ESTABLISH INSTITUTIONAL STANDARD OF
YEW TREE CELLMASS
4.2.1. The chemical composition
4.2.1.1. Qualitative and quantitative analysis of chemical composition
* Qualitative analysis of bioactive compound group in yew tree cellmass
Qualitative analytical results of chemical composition in yew tree
cellmass showed that: yew tree cellmass contains chemical groups such as
alcaloid, anthranoid, sterol, tanin and organic acid. Compare with natural
yew tree, the yew tree cellmass contains almost all chemical groups,
however, it doesn’t contain flavonoid group.
* Qualitative and quantitative analysis of paclitaxel and baccatin III in yew

reverse column chromatography contained 98.07% paclitaxel. This result
was suitable with the S. H. Pyo’s study. The final product also was
acceptable to USP 30 in term of quantitative analysis.
4.2.3. Results of establishing manufacturer standards of taxol cellmass
The results of study have shown that taxol cellmass was acceptable to
general requirements of Vietnam pharmacopoeia IV (applied for medicinal
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