5. Taxol
06/11/20 5
Taxol is isolated from the bark of the Pacific yew, Taxus
brevifolia
Family : Taxaceae
Molecular formula : C47H51NO14
Molecular mass: 853.918 g·mol−1
Melting point: 216–217° C
Boiling point: 957.1±65.0 °C
6. Structure of Taxol
Figure 5
Source: https://www.researchgate.net/figure/2-The-structure-of-Taxol-R-paclitaxel_fig2_260629519
06/11/20 6
7. Isolation of Taxol
ISOLATION
Tissue Collected
Bleached With HCL
Thin Sections Using Sterile
Scaple
Samples Were Placed On The
Surface Of Sterile Potato Dextrose
Agar Media
Incubated At 300C For 48-72 Hours
PURIFICATION
With Growing Clonies They
Were Then Purified Through
Sub Culturing
PRESERVATION
Preserved In Refrigerator At 4 O
C And Then In A -20 O C Freezer
In The Presence Of Glycerol As
Preservative Agent For Long
Time
06/11/20 7
8. Estimation
06/11/20 8
1. TLC- Purity of the isolated compound was checked on TLC in
different solvent systems such as (A) chloroform: acetonitrile (7:3);
(B) chloroform: methanol (7:1), and (C) ethylacetate:2-propanol
(95:5), and spots were detected using anisaldehyde-sulfuric acid
reagent or vanillin sulfuric acid reagent.
Partially purified Taxol obtained from preparative TLC showed a
single dark blue spot that later turned to grey (with Rf value 0.5)
when sprayed with the anisaldehyde-sulfuric acid
reagent/vanillin-sulfuric acid reagent.
9. Continued…..
06/11/20 9
2.UV Absorption analysis- The absorption maximum of the purified
compound was determined by a Shimadzu PC 101 spectrophotometer.
The UV absorption analysis showed a peak with absorption maxima at
227 nm
2.HPLC The purity and characteristics of Taxol were determined by HPLC
using a C18 Symmetry column (Waters). Sample was taken in 10-µl
chloroform, injected in the HPLC column, and gradient elution was
performed using 25% to 95% acetonitrile at a flow rate of 0.5 ml/min. A
dual-wavelength recorder set at 227 and 254 nm was used to detect the
compounds eluting from the column
The homogeneity of the purified compound was confirmed by HPLC
analysis, which showed a single symmetrical peak with a retention
time of 25.5 min on the C18 symmetry column.
10. Utilization
06/11/20 10
1. Taxol is used to treat breast cancer, ovarian cancer and lung
cancer.
2. It is also used to treat Aids related Kaposi’s sarcoma.
12. Vinca Alkaloids
Vinca alkaloids derived from Catharanthus roseus
(Apocynaceae). They are well-known clinical cytotoxic drugs
inhibiting the ability of cancer cells to divide. It include
vinblastine, vincristine, vindesine, and vinorelbine
06/11/20 12
Vinblastine
• Molecular formula: C46H58N4O9
Vinorelbine
• Molecular formula : C45H54N4O8
Vincristine
• Molecular formula : C46H56N4O10
Vindesine
• Molecular formula : C43H55N5O7
14. Extraction & Isolation
Extract the powder with the mixture solution of
alcohol –water:acetic acid in a ratio of 9:1
Filter and evaporate the filtrate to dryness
Dissolve the residue in 2 % Hcl . Filter and adjust
the pH of filtrate to 7
Extract the neutralized filtrate with benzene and
separate benzene layer
Evaporate the benzene layer to give crude alkaloidal
residue.
06/11/20 14
15. Continued….
Subject the dried residue to column chromatography
containing alumina adsorbent. Moniter the column
fractions with TLC ( silica gel, ethyl acetate: methanol:,
9:1)
Use the gradient elution technique starting from pure
benzene then benzene –chloroform (1: 1) and lastly
chloroform methanol (1:1)mixture
Collect and combine the fractions giving single spot
of vinblastine.
Further elution of the column results in separation
vincristine.
06/11/20 15
16. Chemical test
06/11/20 16
Tests for identification
1-Vanillin /Hcl reagent gives with:
Vinblastine a pink colour.
Vincristine an orange-yellow colour.
2-Van-Urk's reagent: → Reddish-brown colour.
17. Estimation
06/11/20 17
TLC
• Stationary phase : Silica Gel G
• Mobile phase : Acetonitrile: Benzene (30:70)
• Sample: 1 mg in 0.1 ml of 25% water in methanol
• Detecting agent : 1% solution of cerric ammonia sulfate in
85% phosphoric acid.
• Rf value: 0.39
18. Utilization
06/11/20 18
1. Vinblastine is an anti tumour alkaloid used in the treatment
of Hodgkin’s disease.
2. Vincristine is a cytotoxic compound and used to treat
leukemia in children.
3. It is also used in the treatment of diabetes.
4. Vincristine sulphate is recommended for the treatment of
acute haemocytic leukemia and in other types of sarcomas.
20. Atropine
• Atropine belongs to the tropane group of alkaloids.
• Atropine is a mixture of two isomers of hyoscyamine. That is
atropine is a racemic mixture of the alkaloids D-hyoscyamine and L-
hyoscyamine, with most of its physiological effects due to L-
hyoscyamine.
• Commercially, it is manufactured largely by using L-hyoscyamine,
taken from Hyoscyamus niger and partially converting this
enantiomer into the D form (D-hyoscyamine).
Molecular Formula: C17H23NO3
Molecular Weight: 676.8 g/mol
Melting Point: 190 to 1940C
06/11/20 20
21. Production of atropine
06/11/20 21
Method 1
1. Atropine is extracted either from belladonna roots or from the
juice of datura plant.
2. The powdered drug or the juice is treated with aqueous solution
of potassium carbonate. The juice contains hyoscyamine.
3. It is then extracted with chloroform.
4. The chloroform is recovered by evaporation process and the
residue is then extracted with dilute sulphuric acid. This solution
is made alkaline with potassium carbonate where atropine
precipitated out.
5. The precipitated atropine is extracted with ether and purified by
converting it into corresponding oxalate or sulphate salt.
22. Continued…..
06/11/20 22
Method 2
1. Take the powdered drug and carry out extraction using 95%
alcohol.
2. Collect the ethanolic extract and then concentrate it.
3. Add dil. Hydrochloric acid and then filter it.
4. Again carry out extraction of the filtrate using petroleum ether in
order to remove the impurities.
5. The aqueous solution is made alkaline by using ammonia.
6. Carry out extraction using chloroform ( 3 times). Combine all the
extracts and then evaporate the chloroform under vaccum.
7. Again extracted with dilute oxalic acid solution.
8. Finally get the crystals of atropine.
23. IDENTIFICATION AND ANALYSIS
06/11/20 23
Chemical test –
• Vitalin–morin test - Take small quantity of the solid atropine
and add 2 drops of Conc. nitric acid in an evaporating dish and
evaporated to dryness on water bath. Then dissolve the
residue in 1ml of acetone. Add few drops of freshly prepared
alcoholic potassium hydroxide solution. Violet colouration
takes place due to tropane nucleus.
24. Estimation of atropine
06/11/20 24
• Thin layer chromatographic method
1. 1% solution of atropine is dissolved in 2N acetic acid.
2. This mixture is then spotted over silica gel G plate and eluted
in the solvent system of strong ammonia solution and
methanol (5:100).
3. TLC plate is spread with acidified iodoplatinate solution.
4. Rf value of 0.18 is obtained.
25. Utilization
06/11/20 25
1. Atropine eye drops have been shown to be effective in slow
progression of myopia in children.
2. Atropine injections are used in treatment of bradycardia.
3. Atropine has also been used in an effort to prevent a low
heart rate during intubation of children.
4. Atropine can be useful in treating hyperhidrosis.
5. Atropine used in treatment of poising by organophosphate
insecticide.
27. Podophyllotoxin
• Podophyllotoxin is a non- alkaloid toxin lignan extracted from the
roots and rhizomes of Podophyllum hexandrum or Podophyllum
emodi. Family Berberideceae.
• It is a potent spindle poison, toxic if taken internally and has been
used as a cathartic.
Molecular Formula: C22H22O8
Molecular Weight: 414.4 g/mol
Melting Point: 183.3 to 184 °C
06/11/20 27
28. Production of Podophyllotoxin
06/11/20 28
Method 1
1. Collect the rhizomes of Podophyllum hexandrum and dried.
2. Take the dried rhizome powder.
3. Carry out Soxhlet extraction using ethanol followed by
distillation.
4. Collect the ethanolic extract and concentrate it to obtain a syrupy
like mass.
5. Add dilute hydrochloric acid to this syrupy mass ad then cool it at
around 50C.
29. Continued…
06/11/20 29
6. Allow to stand for 2 hours and then filter it under vaccum.
7.Wash the residue or filtrate with acidified water and cool it below
50C.
8. Collect the residue and then mix it with 90% hot ethanol.
9.Filter it and evaporate the solvent to obtain a dry residue to
constant weight at 800C.
30. Method 2
1. Extract powdered Podophyllum emodi root (4g) by stirring and
heating with ethanol ( 40ml) for about 10 min cool and filter the
mixture.
2. The re-extract the residue with further 100 ml of ethanol combine
the extract and evaporate to about 20 ml.
3. Pour the ethanolic solution into water (100 ml) and extract with
ethyl acetate combine the extract and evaporate to dryness.
4. Isolate the podophyllotoxin by preparative TLC (4 plates).
5. Crystallize the product from ethanol allowing to crystallize at 00C for
several days.
06/11/20 30
31. IDENTIFICATION AND ANALYSIS
06/11/20 31
Chemical test –
1.Macerate 0.5gm drug with 10 ml alcohol and filter. Then in filtrate
add 0.5 ml copper acetate then brown precipitate formed.
2.Treat podophyllotoxin with 50% Sulphuric acid it will show violet –
blue colour.
3.In Alcoholic extract of sample add 5 ml 1NKOH then stiff jelly
produced.
32. T.L.C Method
06/11/20 32
• Sample preparation – Dissolved about 1mg of
podophyllotoxin in 1ml of methanol
• Stationary phase - Silica gel –G
• Mobile phase – Toluene : ethyl acetate(5:7)
• Standard drug – Podophyllotoxin
• Detection – Spray with methanol Sulphuric acid and heat 10
minutes at 1100 C
• RF Value – 0.89 (Violet colour)
33. Utilization
06/11/20 33
1. It is best known lignan among hundreds of natural plant
lignans.
2. It has been used as a poison, an antihelmintic and a local
antifungal.
3. It is used for cathartic herbal preparation.
4. Used in treatment of gential infection.
5. Starting compound for th chemical synthesis of etoposide
and teniposide.
35. Artemisinin
06/11/20 35
• Artemisinin is obtained from traditional Chinese medicinal herb
Artemisia annua, family Asteraceace.
• Use of Artemisia annua to treat malaria has been known for atleast
1600 years.
• Artemisinin, a sesquiterpene lactone, is a secondary metabolite
from this plant that is widely dispersed throughout the temperate
region.
Molecular Formula: C15H22O5
Molecular Weight: 282.332 g/mol
Melting Point: 156–157°C
37. Production of artemisinin
06/11/20 37
Method 1
1.The leaves are dried, coarsely powdered and extracted with
petroleum ether (40-600C).
2.Filter the extract and concentrate, dried and dissolved in
chloroform and then add acetonitrile which precipitate the sugar
and waxes present in the ethanol.
3. Elute the concentrate ( extract) on silica gel by using chloroform
: Ethyl acetate which yields the fraction of Artemisinin.
4.Crystallized the fraction containing artemisinin by using
cyclohexane or 50% ethanol.
5.The highest yield is obtained from the leaves just before
flowering.
38. ANALYSIS
06/11/20 38
T.L.C Method
Sample preparation – Dissolved 1mg of Artemisinin in 1ml of
Chloroform
Stationary phase - Silica gel –G
Standard sample -Artemisinin
Detecting agent – p- dimethylamino benzaldehyde and heat at
800C to produce color
Mobile phase – Petroleum ether - Ethyl acetate (1:2)
RF Value – Compare with standardArtemisinin
39. Utilization
06/11/20 39
1. It is effective in treating malaria including cerebral malaria.
2. Artemisinin and its analogues were shown to possess
immunomodulatory and antitumor effects.
3. The semisynthetic derivatives such as artesunate,
dihydroartemisnin and artemether etc. has helped in deciphering
the mechanism of action as an antimalarial, antitumor,
antinflammatory or immunosuppressive agent.
4. It has remarkably strong activity against helicobacter pylori the
pathogen responsible for peptic ulcer diseases.
41. Digoxin
06/11/20 ISF College of Pharmacy, MOGA 41
• Digoxin is dried leaves of Digitalis purpurea and Digitalis lanata.
Family: Scrophulariaceae
• Digoxin is the most potent cardiac glycoside.
• It is a secondary glycoside produced from Primary glycoside
Lanatoside C. On hydrolysis gives three molecules of
digitoxosesugar and digoxigenin.
• Digitalis lanata is more potent than Digitalis purpurea
• Physical properties
• Colour : Colourless crystal or white powder
• Odour : Odourless
• Taste : Bitter
• Solubility : Soluble in pyridine, 80% ethanol and a mixture of equal
volume of chloroform and methanol.
42. Structure of Digoxin
• Formula: C41H64O14
• Molar mass: 780.938 g/mol
• Boiling point: 931.6 °C
• Melting point : 249.3 °C
06/11/20 ISF College of Pharmacy, MOGA 42
43. Isolation or production of Digoxin
06/11/20 ISF College of Pharmacy, MOGA 43
Method 1
1.The drug is pulverized and extracted with 50% ethanol at low
temperature.
2.Filter the extract and add lead acetate to the filtrate for the
removal of impurities which is removed by centrifugation.
3. The supernatant extracted with chloroform.
4.The chloroform extract is evaporated and residue left behind is
further purified by chromatography.
44. Continued….
Method II
1.Fresh leaves are ground with neutral salt to inactivate the enzymes
and the pulp is further extracted with ethyl acetate.
2.The leaves are defatted with benzene prior to extraction , for better
yield of glycoside.
3. The ethyl acetate extract is concentrated, dried and further
subjected to chromatographic purification to yield lanatoside A (46%),
lanatoside B (17%), lanatoside C (37%).
4. The fractions obtained are crystallized from alcohol.
5. Lanatoside C after subsequent hydrolysis affords digoxin.
6. Digoxin on acid hydrolysis yield digoxigenin as an aglycone and
th0r6e/1e1/20molesof digitoxose. ISF College of Pharmacy, MOGA 44
45. Identification test
06/11/20 ISF College of Pharmacy, MOGA 45
• Digoxin is dissolved and diluted in the hot methanol. The aliquot
of the solution is evaporated to dryness. Acid ferric chloride is
added to the residue. A green colour develops that slowly
changes to a deep green blue colour.
46. Analysis
06/11/20 ISF College of Pharmacy, MOGA 46
T.L.C Method
Sample preparation – Dissolved 1mg of glycoside in 1ml of
alcohol.
Stationary phase - Silica gel –G
Standard sample - Digoxin
Detecting agent- 50% aqueous sulphuric acid
Mobile phase – Cyclohexane :Acetone: Acetic acid (49:49:2)
RF Value – Compare with standard Digoxin. Digoxin appears as
a blue spot under 385 nm UV light.
47. Utilization
06/11/20 ISF College of Pharmacy, MOGA 47
1. Digoxin is used as a cardiotonic.
2. Digoxin is used to treat heart failure.
3. Digoxin is also used to treat atrial fibrillation.
49. Caffeine
06/11/20 49
• Caffeine is a purine alkaloid obtained from Tea leaves, Coffee
seeds, cocoa, and other species
• Biological source -It consists of dried leaves of plant known as
Thea sinensis, Family – Theaceae
• It is chemically 1,3,7, trimethyl xanthine. Tea leaves contains
1-4% of caffeine and coffee contains 1- 2% of caffeine
• It is white powder or white ,glistering needles, odour less,
bitter in taste, Soluble in hot water.
• Caffeine is a CNS stimulant and Diuretic.
Molecular Formula: C8H10N4O2
Molecular Weight: 194.19 g/mol
Melting Point: 235-237 °C
51. Production of caffeine
06/11/20 51
1. Dissolve 10g of calcium carbonate in 350 ml hot water taken in a
500 ml beaker and then add 10 tea bags into it.
2. Heat the mixture over a hot plate for about 20 minutes.
3. Filter the dark, still warm mixture through Gooch type filter and
transfer into a 500 ml Erlenmeyer flask.
4. Use a spatula to press as much of the liquid as possible out of the
tea bags.
5. Cool the flask containing the filtrate by keeping it in cold water.
6. Add 25 ml of methylene chloride to the dark solution and then
pour the mixture to 500 ml separatory funnel.
7. Gently swirl the contents of the funnel for about 30 sec to extract
the caffeine in to the organic phase.
52. Continued….
06/11/20 52
8. Allow the layers to separate over a 5 min period. Draw off the
methylene chloride layer into a beaker.
9.Add 25 ml of methylene chloride to the separatory funnel and
repeat the extraction process. Combine the methylene chloride
extracts. Repeat the extraction with a 3rd 25 ml portion of methylene
chloride and combine the methylene chloride extracts.
10.Add 2-5 gm of anhydrous sodium sulfate to the methylene
chloride solution and swirl the beaker. This will remove any residual
water from the organic phase.
11.Take a conical flask and plug its hole with a wad of cotton or glass
wool. Alternatively, use some filter paper. Filter the methylene
chloride solution through the funnel under the influence of gravity
and collect the filtrate into a previously tared 100 ml beaker or
Erlenmeyer Flask.
53. Continued….
06/11/20 53
12. Evaporate the solvent in the fume hood over a warm hot plate.
13.Once dried, determine the weight of crude caffeine ( slight
greenish solid).
14. Recrystallization process is carried out to obtain pure caffeine.
15.Dissolve the crude solid in minimum amount of hot acetone.
Cool the solution in an ice bath to induce crystallization. Collect the
product by suction filtration.
54. IDENTIFICATION AND ANALYSIS
06/11/20 54
Chemical test –
• Murexide test – To the caffeine add hydrochloric acid and
potassium chlorate, heated to dryness. A purple colour is
obtained by exposing the residue to vapours of dilute
ammonia.
55. • T.L.C Method
• Sample preparation – Dissolved 1mg of caffeine in 1ml of
methanol or chloroform
• Stationary phase - Silica gel –G
• Standard sample - Caffeine
• Mobile phase – Ethyl acetate: methanol : acetic acid
(80:10:10)
• Detecting agent – Expose to vapours of iodine
• Rf Value – 0.41
06/11/20 55
56. References
06/11/20 56
Trease & Evans Pharmacognosy, by William Evans, Elsevier Publisher
16th edition,2009.
Textbook of Pharmacognosy by C.K Kokate , A.P Purohit,S.B.Gokhale
Nirali Parkashan, 47th edition,2012.
Text Book of Pharmacognosy and Phytochemistry, by Biren N. Shah
and A.K. Seth Elsevier Publisher, First edition,2010.
https://en.wikipedia.org/wiki/Vinca_alkaloid
