In this presentation, i will be talk about Taxol that is a chemotherapeutic drug. how it is isolate, elicititate and their anti-cancerous mechanism.

1. ISOLATION, ELICITATIONAND
MECHANISMOFANTI-CANCEROUS
ACTIVITYOFTAXOL
Presented by-
HINA CHAUDHARY
M.Sc. FINAL BOTANY

2. INTRODUCTION
Cancer is group of disease, characterized by rapid and uncontrolled formation of abnormal cells,
which mass together to form tumor, or may proliferate throughout the body.
Anything that may cause a normal body cell to develop abnormally, potentially can cause cancer.
General categories of cancer-related or causative agents are as follows: chemical or toxic
compoundexposures,ionizingradiation, some pathogens, and humangenetics.
https://www.cancervic.org.au/images/CISS/cancer-types/cancer

3. The drugs that are used for inhibiting the abnormal cell growth or killing the cancer,
called anti-cancerousdrugs. Example of such anti-cancerous drug ,are-
Family Species Part used Active
compound
Target
Apocynaceae Cathranthus
roseus
Dried whole
plant
Vincristine
Vinblastine
Leukaemia, skin
cancer, lung
cancer, brain
tumor
Berberidaceae Podophyllum
peltatum
Dried rhizomes
& roots
Podophyllotoxin Small cell lung
cancer, brain
tumor,
malignant l
Taxaceae Taxus
brevifolia
Stem bark Paclitaxel
Docetaxel
Ovarian cancer,
Lung carcinoma,
Gastric &
Cervical cancers
Zingiberaceae
Curcuma
longa
Rhizoids curcumin Anti cancerous
activity.

4. Taxol, is a chemotherapeutic (antineoplastic agent and antimitotic) drug,
i.e., chemically used to treat and control diseases such as cancer.
Taxol is the commercial trade name used by Bristol-Myers Squibb company
for this compound, while paclitaxel is chemical name.
PHYSICALPROERTIES
• Paclitaxel is a crystalline powder which is white to
off-white in appearance.
• It is highly lipophilic and highly insoluble in water.
Freeze dried powder of
Paclitaxel
Preparation,characterization,andinvitrotargeteddeliveryof folate-decoratedpaclitaxel-loadedbovineserumalbumin
nanoparticles.ZhaoD,ZhaoX, ZuY,LiJ, ZhangY,JiangR,ZhangZ - (2010)

5. Taxol 30mg, 100mg & 300mg ( Paclitaxel ) Injection is used to
treat breast cancer, lung cancer, and ovarian cancer. It is also used
to treatAIDS-related Kaposi's sarcoma.

6. CHEMICALPROERTIES
• Taxol has a novel and complex chemical structure along with a chemically
similar analog, taxotere (Docetaxel).
• Taxol (Paclitaxel) is one of the natural diterpenoid plant alkaloid.
• The paclitaxel molecule consists of a tetracyclic core called baccatin III and an
amide tail. The core rings are conveniently called ring A(a cyclohexene), ring B
(a cyclooctane), ring C (a cyclohexane), and ring D (an oxetane)
Chemical structureof paclitaxel
Abriefhistoryof taxol,JustynaZwawiak,LucjuszZaprutko;JournalofMedicalScience1 (83)2014

8. BIOSYNTHESIS OF TAXOL
Taxol biosynthetic pathway. ggpps: geranylgeranyl diphosphate synthase; ts: taxadiene synthase; h-5: cytochrome
P450 taxadiene 5-hydroxylase; tat: taxa-4(20), 11(12)-dien-5a-ol-O-acetyltransferase; h10: cytochrome P450 taxane
10-hydroxylase; tbt: taxane 2a-O-benzoyltransferase; dbat: 10-deacetyl baccatin III-10-O-acetyltransferase. Multiple
arrows indicate several as yet undefined steps.
B.H.Guo,G.Y.Kai,H.B.JinandK.X.Tang;Taxolsynthesis;AfricanJournalof BiotechnologyVol.5 (1),pp.015-020,2 January,2006

9. It was essential to elaborate some different methods – more costless and
higher yielding to obtain this valuable substance.
• The presence of various microorganisms from the bark of the yew tree
was found to be capable of producing TAXOL.
1. In 1993, Stierle et al. (1993) reported the first Taxol producing
fungus Taxomyces andreanae.
2. Pestalotiopsis microspora, a coelomycetous endophyte in Taxus
brevelifolia under cultural condition was observed to produce
TAXOLin an amount considerably more than that of Taxus sp.
Synthetic production of Taxol

10. CONTINUE..
• Within the confines of searching of optimal solution, it was found that
in a result of extraction of fresh Taxus baccata needles, different
taxane diterpene was gained. It was 10-deacetylbaccatin, which was
structurally closed to Taxol. The process of isolation go through from
more favorable material and with higher yield – from 3 tones of
needles, about 1 kg of substance was obtained. Moreover, the
procedure did not require destroying of yew trees, because needles are
easily renewable.

11. CASE STUDY

12. Synthetic production of Taxol
Semi-synthetic method of obtaining Taxol from 10-deacetylbaccatine
First step concerns triethylsilylation of C-7 OH;
additionally C-10 hydroxyl group has to be
acetylated.
This semi product may be coupled in reaction
with enantiomeric O-protected N-benzoyl-α-
hydroxy-β-amino acid.
After deprotection of hydroxyl groups with
HCl, Taxol is obtained with yield up to 90%

13. Isolation of Taxol
Taxol, a mitotic inhibitor first isolate from the bark of
the pacific yew tree (Taxus brevilifolia) by Monroe E.
Wall and Mansukh C.Waniin 1967.
Isolation of 1 kg of raw natural product required the
use of 10 tons of yew tree bark. It means that 3000 of
trees ought to be cut out. Also, it should be point out,
that 1 kg of Taxol is sufficient for 500 patients.
Moreover, Taxus is a slow growing evergreen shrub
or small tree (11 species all over the world)
uncertainly distributed throughout Northern
hemisphere temperate zones.
Wanietal 1971; https://landscapeplants.oregonstate.edu/plants/taxus-brevifolia;Abriefhistoryof taxol,JustynaZwawiak,LucjuszZaprutko;
Journalof MedicalScience1 (83)2014

14. ALTERNATIVE METHOD
• Due to low taxol content from bark of Taxus sp., different callus
culture methods have been examined to increase quantitatively the
taxol level. Although some success has been achieved with these
protocols, Taxus sp. is a slow-growing rare plant.
• In addition, Taxus cells are very sensitive to shear stresses and
dissolved oxygen level.
• Thus, the search for alternative microbial source of taxol production
was expanded during the past two decades

15. CASE STUDY

16. Endophytic fungi
• Plant endophytic fungi are defined as the fungi which pass the
whole or part of their lifecycle inside the healthy tissues of the
host plants, typically causing no distinct symptoms of disease.
• Some endophytic fungi have the ability to produce bioactive
compounds as those originated from their host plants.
• One of the most important “gold” bioactive compound is taxol
and there is a defined gene cluster for biosynthesis of taxol in
fungi such as Cladosporium sp., Fusarium sp., Rhizophora sp.,
and Taxomyces sp.

17. Isolation,
Tissue collected
Bleached with HCl
Thin sections using
sterile scaple
Samples were placed on the surface of sterile
potatodextroseagar(PDA, Merck) media
Incubated at 30 °C for 48–72 h
With growing colonies, they
were then purified through
sub-culturing.
The purified fungi were
transferred to PDA slants,
Purification,
preserved shortly in
refrigerator at 4 °C and
then in a −20 °C freezer
in the presence of
glycerol as preservative
agent for long time
Preservation

18. Taxol KeyGeneAmplificationandFungi Colony-PCR
One of the key genes in taxol biosynthesis is tdc-1 (taxadine synthase)
FungalTaxol ProductionandAssay
The synthetic medium containing 1% (w/v) yeast extract, 2% (w/v)
peptone and 2% (w/v) glucose was used to taxol production.
ForquantificationofTaxanes
They used an HPLC system based on anAgilent 1100
Viability,MTTAssay, andGenesExpressionEvaluation
Cell viability analysis was conducted in the presence of different concentration of
commercial (Sigma–Aldrich) and fungal taxols (F1, F2, and F6) in the range of 10–1000
μg/ ml using Trypan blue and Neobar Lama..

19. OBSERVATION
Two purified native endophytic fungi belong to
Cladosporium sp. that grow on solid PDA plates at
30 °C for 48–72 h. It shows colony appearance (a,
c) and morphology (b, d) of F1 and F3 strains,
respectively
Colony-PCR products on 1% agarose gel.
F1 to F8 are isolated and purified
endophytic fungi that harbor tdc1 key
gene and have positive PCR results. M is
DNA size marker (100 bp) and C(−) is
negative control

20. RESULT and DISCUSSION

21. CONCLUSION
As a result, one of the best advantages of using endophytic taxol-
producingfungi is-
• that the culture medium for the fungal cells is simple and inexpensive.
• In addition, endophytic fungi can produce taxol with antitumor activity in a
short period of time and high efficiency.
• It appears that optimization of taxol-producing endophytic fungi from Iranian
yew could be a safe and confident procedure to overcome challenges of using
other methods.

22. CASE STUDY

23.  In this paper, fungi isolated from Taxus chinensis var. mairei were tested for
their ability to produce taxol.
 High performance liquid chromatography combined with mass spectrometry is
used.
 It confirmed that Aspergillus aculeatinus Tax-6, one of the endophytic fungi,
could produce taxol in potato dextrose agar liquid medium.
 NaOAc, Cu2þ, and salicylic acid were introduced into the medium to enhance
taxol production of strain Tax-6 .
 Because NaOAc is an important precursor of taxol,
 Cu2þ may enhance the activity of oxidase and catalyze the formation of taxol.
 salicylic acid could be an elicitor signal.
W.Qiaoet al; Enhancing taxol production in a novel endophytic fungus, Aspergillus aculeatinus Tax-6, isolated fromTaxus chinensis var.
mairei; 2017.

24. HPLC chromatograms
HPLC chromatogram
of standard taxol
HPLC chromatogram
of fungal taxol
The taxol specific peak was at 9.963 ± 0.2 min
W.Qiaoet al;Enhancing taxol production in a novel endophytic fungus, Aspergillus aculeatinus Tax-6, isolated fromTaxus chinensis var. mairei; 2017.

25. HPLC-MS chromatogram
(A) standard taxol
(B) fungal taxol.
Arrow indicates the
molecular ion of taxol at
m/z 854.3 [M D HD] and
m/z 876.3 [M D NaD]

26. The original yield of ax-6, 334.92 mg is higher than other taxol-producing fungi
(Table 1).
When compared with other taxol producing strains, the relatively high yield and ability
to rapidly accumulate taxol of strain Tax-6 indicates that the commercial production of
taxol will be feasible.
Tax-6, 334.92 mg L1

27. .
• Choi et al. reported that Paclitaxel production reached its
highest level value of 67 mg/Lover a period of 42 days,
• Based on the sugar-feeding strategy of suspension culture .
• In addition, application of elicitors such as methyl jasmonate
(MJ) has been shown to increase the production of taxol up to
36 mg/l
As the productivity of taxol from plant cell cultures is not
routine, there are three potential ways for increasing taxol
production

28. ANTI CANCEROUSACTIVITYOF TAXOL
• Taxol is an inhibitor that is effective against solid tumors cancers such
as lung, ovarian etc. it interferes with in the cell division of the cancer
cells in the tumor and can therefore stop its growth.
• Taxol does this by bonding covalently with a protein called tubulin
that’s makes up the structure called microtubule.
• Taxol are categorized as microtubule-stabilizing anticancer agents.
Paclitaxel stabilizes and protects microtubule against disassembly.
Thus, it interferes with the formation of mitotic spindle which cause
chromosome not to segregate, and consequently mitotic arrest.

29. Microtubule assembly Microtubule disassembly
Taxol , prevents disassembly of microtubule
MECHANISM OFACTION

30. Mode of actionof Paclitaxel (Taxol), during Mitosis

31. CONCLUSION
With the discovery of the cancer drug paclitaxel
(Taxol), patients with lung, cervical, and ovarian
cancers have hope for better treatment outcomes,
increased life expectancy, and improved quality
of life.

32. REFERENCES
• Justyna Zwawiak, Lucjusz Zaprutko; 2014, A brief history of taxol,; Journal of Medical
Science 1 (83)
• Zhao D, Zhao X, Zu Y, Li J, Zhang Y, Jiang R, Zhang Z, 2010 Preparation,
characterization, and in vitro targeted delivery of folate-decorated paclitaxel-loaded
bovine serum albumin nanoparticles.
• B.H. Guo, G.Y. Kai, H.B. Jin and K.X. Tang; , 2006 Taxol synthesis; African Journal of
BiotechnologyVol. 5 (1), pp. 015-020
• Wani et al 1971
• https://landscapeplants.oregonstate.edu/plants/taxus-brevifolia
• https://www.cancervic.org.au/images/CISS/cancer-types/cancer
• Weichuan QIAO, Fei LING, Lei YU, Yifang HUANG, Ting WANG; Enhancing taxol
production in a novel endophytic fungus, Aspergillus aculeatinus Tax-6, isolated from
Taxus chinensis var. mairei

33. Thank you!