DA NANG UNIVERSITY OF SCIENCE TECHNOLOGY - DEPARTMENT OF

VEGETABLE CELL TECHNOLOGY 

APPLICATION OF PLANT TISSUE CULTURING

SECOND  COMPOSITION PRODUCTION
 High in vitro production of ant-canceric indole alkaloids
from periwinkle (Catharanthus roseus) tissue culture

TEAM 7 - CLASS 19.49

INSTRUCTOR: PHD NGUYEN HOANG TRUNG HIEU 
MEMBERS:
LE THI KIM NGAN 
DOAN THANH NGUYEN
NGO DANG NHAN
VO VAN THIEN
HA THI THUY TRANG 

DA NANG, APRIL 28, 2023 1

  Contents

SECONDARY VINCA ALKALOID CULTURE AND

01 COMPOUND 02 AKALOID  03 EXTRACTION PROCEDURE

04 ANALYSIS TLC 05 ANALYSIS HPLC 06 RESULT

2
01
SECONDARY
COMPOUND

3
 Concept

 Secondary compounds are substances that have

no direct function in the processes of assimilation,
respiration, transport, enhancement, and plant
growth.
 The primary function of secondary compounds is
to protect plants against pathogens.

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Classify

Phenolic

Secondary
Terpene
compound

Alkaloid

5
 Classify
 Terpene

 Terpenes or isoprenoids are among the most diverse classes of secondary

compounds, mainly of plant origin, including flavoring agents, antibiotics, plant
and animal hormones, membrane lipids, insect repellents, and more.
 All terpenoids are synthesized through the condensation of isoprene units
(C5). 6
 Classify
 Phenolic
 Contains at least one aromatic ring
attached to one or more hydroxyl groups. 
 More than 8000 phenol structures have
been found.
 Classification based on the number and
arrangement of carbon atoms consists of
2 subgroups:
o  Flavonoid 
o  Phi flavonoid

7
 Classify
 Akaloid
 Crystalline alkaloids are nitrogenous
compounds that can be extracted using an
acidic solution.
 Structure: In addition to carbon, hydrogen and
nitrogen can contain oxygen, sulfur and rarely
other elements such as chlorine, bromine and
phosphorus.
 Alkaloids are produced by many organisms,
such as bacteria, fungi, and animals, but mainly
from plants.

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Role

9
 02
VINCA ALKALOID

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Concept
 A family of dimer-like indole compounds,
extracted or semi-synthetically from the
periwinkle Catharanthus roseus
 In particular, two types of alkaloids, vincristine
and vinblastine, have anti-cancer activity
 Their mechanism of cytotoxic action is to inhibit
microtubule formation by binding to the protein
tubulin, which stops the cell cycle at the mitosis
stage.

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 Research history

1961 1988 2018

It was first isolated from Endo et al. have established a Pliankong et al. added chitosan
periwinkle trees process for culturing a to Catharanthus roseus cell
periwinkle cell suspension suspension culture medium

1966 2008

Carew tried plant tissue Ramani and Jayabaskaran used UV-B light
culture technology to stimulate an increase in catharanthine
and vindoline levels.

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ADVANTAGES & DISADVANTAGES

Advantages Disadvantage

Affects all types of rapidly dividing cells,

Prevent the duplication of
making it essential for very specific
cancer cells
drug uses.

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 03
ALKALOID CULTURE AND
EXTRACTION PROCEDURE

15
ALKALOID CULTURE AND EXTRACTION PROCEDURE

Catharanthus Homogenized
roseus

Sterilized by
Alkaloids
hypochlorite isolation
5%

Cultured on
MS agar Acid phase
medium (with seperation
NAA and Kin )

Chloroform
Alkaloid
phase
Extracts
seperation

Crushing
(3-4g scar Extracts
tissue)
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Sample acquisition and processing

Tissue culture using petiole segments

(2cm) from six-week-old C. roseus
seedlings.

Petioles are disinfected with 5%

hypochlorite for 45 minutes, rinsed with
disinfected distilled water.

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 Cell-tissue culture
• The sample fragments will be cultured on MS agar media,
supplemented with different concentrations of NAA and kinetin.

• The cultures will be placed in a growth chamber: dark for the first 2 weeks
and illuminated 24 hours for 4 weeks at 35°C.  After 6 weeks, scar tissue
and roots are formed. 
• The roots, scar tissue and root scar tissue of N.1 and N.14 were
transplanted in MS N.1 and MS0 media, with a lighting time of 24 hours.
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 Alkaloid Extracts
- For 3-5g of scar tissue, the roots are ground, then homogenized in 60-100ml
ethanol, 1 minute. Place in a room with a temperature of 60°C for 10 minutes.
- The solution will be filtered. After filtration, the ethanol extract of the alkaloid will be
rotated by a vacuum evaporator at 60°C and sent to isolate.
- Stages of Alkaloid Isolation:
• Using sulfuric acid (5%) dissolve the powder in a cannon. Add the Diethelic Ether.
Phase separation and retention of the acid phase. 
• Neutralize the acid phase with NaOH 10N (pH=10). Then put 60-100ml of chloroform
in a decanting jar. Some organic solvent-loving substances will enter chloroform, the
rest will be in the aqueous phase. 
• Taking the Chlorofomic phase to concentrate with a device she rotates vacuum at
60°C.  The resulting alkaloid extracts will dissolve in 1ml of ethanol.
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04
ANALYSIS TLC

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 Thin-layer Chromatography (TLC) analysis
 TLC solvent systems routinely used was
ethyacetate: ethanol (4:1) and TLC plates were
formed by silica gel G60 (Merck).

 Alkaloids were identified using TLC and the

color reaction with Ceric Ammonium Sulfate
(CAS) and Ultra violet (UV) detection (=254
and 366 nm)

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Thin-layer Chromatography (TLC) analysis

Table 1 : Retention factor (RF) and standards alkaloids color

reaction by TLC analysis. 

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 05
ANALYSIS HPLC

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 High-performance liquid chromatographyanalysis
b. HPLC
•Using Shimatzu LC-6A chromatography
•This method using a programmed isocratic
eluents made of acetonitrile
and ammonium carbonate buffer pH 7.3 (1:1) 
•In HPLC analyses, for obtaining carefully
results, the 20 µl alkaloids were injected
three times: without standard alkaloids, mixed
with standard alkaloids and standard alkaloids
without extracts.
•Alkaloids were detected by retention time
at 254 nm. 
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 High-performance liquid chromatography
analysis

Retention time and concentration for some standards

alkaloids by HPLC analyze

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06
Result

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 Result
a. Callus and root induction 
• After six week incubation, callus and roots were
producby petiole segments in the presence 0.1, 5, 10
and 20 mg/l of NAA supplemented with 0.1, 5, 10 and
20 mg/l of Kin.
• Calluses were produced in all the
treatments but in 0.1 mg/l of NAA with 0.1 mg/l of
Kin (N.1) was low (15%). 

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 Result
a. Callus and root induction 
•Root, callus and root-callus from
N.1 and N.14 were subcultured in
N.1 and MS0 media; these grew
and produced new callus and roots.​
•Callus and roots
were regenerated in N. 18, 19, 20,
21 and 23 of medium 2 but only
callus in N.17 and root in N.18 of
medium 2 were produced.
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 Result
b. TLC results
•Result of TLC showed the pattern of vindoline (vin), catharanthine (cat) and
ajmalicine (aic) of roots and callus from MS media supplemented with 0.1 mg/l
of
NAA and 5, 10, and 20 mg/l of Kin, were similar to the pattern of petiole of
intact plant​.

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 Result
b. TLC results
•The result of TLC showed the pattern of vindoline, catharanthine
and ajmalicine of roots and callus from the Callus (Ca), Callus-Rooting (CaR),
Petiole-Rooting (PR) and Root (R) of N.17- 23 were similar to the pattern of
petiole of intact plant.

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 Result
c. HPLC results
•The HPLC analysis showed that the pattern of mobility and characters of
vindoline, catharanthine, ajmalicine, vincristine (vcr) and serpentine (ser) of
new roots of petioles in 0.1 mg/l of NAA is similar to the intact plant petioles
but with lower levels of each.

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 Result
c. HPLC results
•The C. roseus leaves contain the demerit alkaloids such as vincristine
and vinblastine in concentrations of 0.0004 to 0.0003% dry weight (d.w) which
find use in cancer therapy. Meanwhile, the root and basal stems are rich in
monomeric alkaloids such as ajmalicine and serpentine, 0. 03 to 0.1%, which
are employed to reduce high blood pressure.

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  Conclusion
•In order to produce these useful anticancer drugs much more efficiently,
many scientists have tried to apply plant tissue culture technology. However,
production of vinca alkaloid using the callus or the suspension cultured cell of
C. roseus is so far not promising because the productivity of the cultured cells
reported so far was very low.

•Therefore, the search for active raw materials containing highly bioactive
compounds, suitable extraction and purification methods to serve human
needs is a matter of interest to scientists.

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 Result

A  0.1 mg/l NAA + 0.1 mg/l KIN

B 5 mg/l NAA + 0.1 mg/l KIN

Callus was subcultured to

C
hormone-free medium

Roots was subcultured to

 D 
hormone-free medium.

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THANKS!

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