This document provides information about withanolides and umbelliferone. It discusses their biological sources, properties, biosynthesis, extraction, isolation, uses, and characterization. Withanolides are steroidal lactones found in ashwagandha that have anti-cancer and anti-inflammatory properties. Umbelliferone is a yellow coumarin found in plants like carrot and coriander that exhibits blue fluorescence and is used as an indicator in chemical tests. The document outlines methods for extracting, isolating, and identifying these compounds using techniques like column chromatography, TLC, UV-Vis, IR, NMR, and mass spectroscopy.

1. SEMINAR ON
BIOSYNTHESIS, ISOLATION,
IDENTIFICATION AND
CHARACTERIZATION OF WITHANOLIDES
& UMBELLIFERONE
Presented By :- Tejaswini C

2. CONTENTS
Biological source
Properties
Biosynthesis
Extraction and isolation
Uses
Chemical test
Characterisation
• WITHANOLIDES
• UMBELLIFERONES
• REFERENCES

3. • Withanolides are a group of naturally occurring
polyoxygenated steroidal lactones assembled on a C28
ergosterone skeleton.
•It includes the various steroidal lactones such as
withaferin A, withanone, withanolide-A, B, E, F, G, H,
I, J, K, L, M, withanoside IV, withanoside V
WITHANOLIDES

4. GENERAL STRUCTURE
5,6-Epoxy-4,20,22-trihydroxy-1-oxoergosta-2,24-dien-26-oic acid alpha-lactone

5. ASHWAGANDHA
• Synonym: Indian Ginseng;
Withania root, ashwagandha, winter
cherry
• Biological source: Ashwagandha
consists of the dried mature roots of
Withania somnifera Dunal (Fam.
Solanaceae).

7. PROPERTIES
color: white
solubility: soluble in methanol.
Molecular Formula: C28H38O6
Molecular Weight: 470.606 g/mol
Melting point: 305℃
Density :1.264 g/ml

8. BIOSYNTHESIS

9. Extraction (Method-1)
10g of ashwagandha
+ 100ml 1% H₂SO₄,
mix & keep overnight
horizontal shaker
for ½ hour
filter and wash with
40ml of 1% H₂SO₄
Filter & repeat the
process twice until the
supernatant liquid is
obtained
Add calcium hydroxide
solution to precipitate the
starch and impurities
Filter & concentrate it
by rotary evaporator
Crystals separate out,
collect and Subject for
further analysis

10. Method-2
Marc is again extracted
with fresh solvent 3 times
Soak 250g of ashwagandha
stem powder in 50%
ethanol for over night.
Concentrate the filtrate
under the pressure at
50 ± 5 ºC to obtain
ethanolic extract
Combined all extract and
concentrate to 1/8th of the
original volume under
reduced pressure at 50±5°C.
Subjected to column
chromatography
Boil it for 24
hours , filter.

11. Isolation by column chromatography
• The chloroform extract was subjected to column chromatography on
silica gel 250 mesh, eluted with gradient of increasing order of ethyl
acetate in hexane (5–10% each step).
 White crystals in ethyl acetate were obtained from ethyl acetate:
hexane (1:3) fraction.
 Recrystallization was done in acetone. The percent of isolated
alkaloids from shoot of Withania somifera is 0.08%.

12. IDENTIFICATION TESTS
By TLC (withanolide)
• Stationary phase: silica gel G
• Mobile phase:chloroform:
methanol (9:1)
• Spraying agent: Vanillin-
Sulphuric acid
• Rf value of withanolide A: 0.65
⁂Leiberman’s test: (for
steroidal moiety)
sample+ acetic
anhydride+ conc. Sulphuric
acid produces Reddish violet
turns to green
⁂Salkowski test:
Sample +
trichloromethane. The color
of the chloroform layer turns
yellow color when conc
H2SO4 is added.

13. CHARACTERISATION OF WITHANOLIDES
UV: Withanolides shows
an absorption maxima in
the range 220- 223 nm in
methanol or ethanol
Mass Spectroscopy: The
Mass spectrum showed
 m/z = 470.6028 for
withanolide A- C28H38O6
 m/z = 474.6342 for
withanolide D- C28H42O8

14. Infrared spectroscopy
Sample is prepared with KBr pellets.
Finger print region shows peaks at 1318, 1253,
1083, 1016, 889, 836, 775, 670cm-1.
The IR spectrum bands
Functional groups IR bands
Hydroxyl 3465
Lactone 1710
Ketone 1680
Epoxide 1130

15. NMR
Hydrogen position Delta value
(2-H) 5.87
(1H, m, 3-H) 6.60
(1H each 4-H) 2.85, 2.45
(1H, br s, OH at 5-C) 3.13
(1H, d, 6-H) 3.07
(1H, dd, 7-H) 3.32
(3H, s, 18-H) 0.98
(3H, s, 19-H) 1.21
(3H, d, 21-H) 1.13
(1H, dt, 22-H) 4.73
(2H, m, 23-H) 2.58
(3H, s, 27-H) 1.88
(3H, s, 28-H) 1.93
1H NMR:-(300 MHz, CDCl3)

16. 13C NMR
Compound 1- withanolide
Compound 2- withaferin

17. USES
Anticancer property
Immunosuppressive effect on B & T lymphocytes.
antibacterial , antitumour, antiartritic and hepatoprotective activity.
Used in the treatment of anxiety neurosis
Treatment of nervous disorders, intestinal infections and leprosy.
 Treatment of impotence and infertility

18. Coumarins
Coumarin is present either in a free state or in glycosidal form
naturally occurring in the different families such as
Umbelliferae, Rutaceae, Leguminosae, etc.
Coumarin and others forms like umbelliferone, scopoletin,
scopoline, aseculetin, aesculin etc are derivatives of Benzo
alpha pyrone.

19. These are of 3 types
Hydroxy coumarins - aesculin, umbelliferone
Furanocoumarins- angelicn, psoralen
Pyranocoumarins- xanthyletin, lomatin

20. UMBELLIFERONE
• Umbelliferone also known as 7 Hydroxycoumarin, hydrangine, skimmetine, and
beta-umbelliferone is a natural product of the coumarin family
• Biological Source: Umbelliferone occurs in many familiar plants from the
Umbelliferae family such as mentioned below
Drug name Biological source Family
Ammi Ammi majus umbelliferae
Visnaga Ammi visnaga umbelliferae
Carrot Daucus carota L., Umbelliferae
Coriander Coriandrum
sativum
Umbelliferae.

21. STRUCTURE PROPERTIES
 Colour: yellowish-white crystals
 Taste: bitter
 Solubility- slightly soluble in hot water (1g in
100ml), have good solubility in ethanol,
chloroform, and acetic acid.
 It shows distinct blue fluorescence.
 Melting point: 225C – 228C
 Molecular formula: C9H6O3
 Molecular weight-130g/mol
Umbelliferone or 7-hydroxychromen-2-one

22. BIOSYNTHESIS

23. EXTRACTION AND ISOLATION
Umbelliferone has extracted from the solvents CHCl3, ethyl acetate
and methanol , hexane by keeping them in solvent for 24hr at room
temperature and the hexane soluble fraction of the methanol extract is
opted for column chromatography
Silica gel column chromatography eluted with n-hexane and ethyl
acetate or CHCl3/MeOH solvent mixtures of increasing polarity were
employed for the fractionation and isolations.

24. PREPARATION OF UMBELLIFERONE
 from asafoetida:
umbelliferone is prepared by treating ferulic acid with HCl which gets converted
to umbellic acid and the latter loses a molecule of water to give rise to
umbelliferone
 umbelliferone may also be obtained from the distillation of resin from
Umbelliferae family drugs.
 Umbelliferone can also be prepared synthetically by pechmann condensation as
follows,

25. IDENTIFICATION TEST
 When 0.5g of sample is triturated
with pure sand (SiO3) and add 5 ml
of HCl, added 5ml of water, filtered
and to the filtrate added an equal
amount of ammonia solution, it
gives a distinct blue fluorescence. –
umbelliferone test (asoefaetida)
 TLC of umbelliferone shows a
blue emission band at kmax =
460–480 nm
Mobile phase- chloroform:
methanol:: 97:3 or 9:1
Rf value- 0.74 or 0.35
respectively
 Umbelliferone gives a
positive test with FeCl3
indicated by the deep blue
color

26. CHARACTERISATION OF UMBELLIFERONE
UV :The UV spectra of methanolic solution of umbelliferone shows absorption
maxima at knm (log e) are
• 339(0.50), 294 (0.36), 242 (0.77)
The absorbance maxima in
• Acid solution is 325 nm
• alkaline solutions it shift to 365 nm

27. Fluorescencece:
Umbelliferone shows distinct blue
fluorescence.
The fluorescence excitation maxima in
Acid solution- 330nm
alkali solutions-370 nm
emission maxima-460 nm

28. Stretching IR BANDS IN cm-1
Ar-OH 3165
lactone 1715-1690
1628-1603
C=C 1575, 1109
CH 835
Infrared spectra
Mass Spectroscopy
The EI-MS (rel. intensity) spectra
showed peaks at m/z
162 , 134 , 106 , 105 and 78 .

29. Position H (400 MHz) dH C (100 MHz) dC
2 160.5 [162.6]
3 6.16 (1H) [6.19 (1H)] 112.0 [112.8]
4 7.87 (1H) [7.86 (1H)] 144.2 [144.5]
4a 111.9 [111.9]
5 7.50 (1H) [7.46 (1H)] 129.7 [129.3]
6
7 6.83 (1H,dd) [6.87 (1H, dd)] 113.2 [113.7]
161.6 [161.4]
8 6.74 (1H) [6.78 (1H)] 102.5 [103.0]
8a 156.2 [155.9]
NMR data for umbelliferone in CDCl3 and [CD3OD].
NMR

30. REFERENCE
1. Anjaneyulu AS, Rao DS, Lequesne PW. Withanolides, biologically active natural steroidal
lactones: a review. Studies in natural products chemistry. 1997 Jan 1;20:135-261.
2. Kokate CK, Purohit AP, Gokhale SB. Text book of Pharmacognosy. Pune: Nirali Prakashan.
2003;8(66):1-624.
3. https://pubchem.ncbi.nlm.nih.gov/compound/Withanolide
4. Rangari VD. Pharmacognosy & phytochemistry.2nd edition, Career publications; 2009
5. Mishra S, Bansal S, Mishra B, Sangwan RS, Jadaun JS, Sangwan NS. RNAi and
homologous over-expression based functional approaches reveal triterpenoid synthase
gene-cycloartenol synthase is involved in downstream withanolide biosynthesis in Withania
somnifera. PLoS One. 2016 Feb 26;11(2):e0149691.
6. Ha JW, Yu JS, Lee BS, Kang DM, Ahn MJ, Kim JK, Kim KH. Structural Characterization of
Withanolide Glycosides from the Roots of Withania somnifera and Their Potential Biological
Activities. Plants. 2022 Mar 13;11(6):767.

31. 7. Mali PC. Isolation, characterization and evaluation of antimicrobial activity of
Withanolide-A of Withania somnifera. IJPR. 2013;3(3):48.
8. Kar A. Pharmacognosy and pharmacobiotechnology 2nd edition, New Age
International; 2003.
9. Mazimba O. Umbelliferone: Sources, chemistry and bioactivities review. Bulletin of
Faculty of Pharmacy, Cairo University. 2017 Dec 1;55(2):223-32.
10. https://pubchem.ncbi.nlm.nih.gov/compound/Umbelliferone
11. Venugopala KN, Rashmi V, Odhav B. Review on natural coumarin lead compounds
for their pharmacological activity. BioMed research international. 2013 Oct;2013.
12. Matos MJ, Santana L, Uriarte E, Abreu OA, Molina E, Yordi EG. Coumarins—an
important class of phytochemicals. Phytochemicals-isolation, characterisation and role
in human health. 2015 Sep 30;25:533-8.

32. Thank you