This topic covers the notes for unit 1 of phytochemistry in 1st semester in M Pharm of department of pharmacognosy. This includes biosynthesis, characterization, purification and uses of steroids.

1. PHYTOCHEMISTRY
BIOSYNTHETIC PATHWAYS AND
RADIO TRACING TECHNIQUES
SEMINAR-I
Topic: STEROIDS - HECOGENIN
Presented
By
P M MOHAMED INSAAF
FIRST YEAR M.PHARM
1
DEPARTMENT OF PHARMACOGNOSY

2. STEROIDS
2

3. STEROIDS
• Steroids constitute a natural
product class of compounds
that is widely distributed
throughout the nature.
• A steroid is any compound
that contain a
cyclopentanoperhydrophen
anthrene nucleus.
• Plant steroids are a diverse
group of natural products.
3

4. STEROIDS
• They are biosynthetically derived from S -
squalene-2,3-epoxide through acetate
mevalonate pathway.
• Phytosterols are universal steroids in the
plant kingdom, and have been reported to
show hypocholesterolemic activity.
• Withanolides are large steroidal lactones
group having many biological activities.
4

5. STEROIDS
• A small group of plant steroids, i.e.,
brassinosteroids, exhibit plant growth
hormonal activity.
• Phytoecdysteroids (polyhydroxylated plant
steroids) show anabolic effects with no un
wanted side effects.
• Steroidal alkaloids are nitrogen-containing
plant steroids possessing various biological
activities. In some plants, minute amounts
of cholesterol and mammalian steroidal
hormones including progesterone have
been detected.
5

6. STEROIDS
• Plant steroid analysis is done by
chromatographic methods like thin layer
chromatography (TLC), high performance liquid
chromatography (HPLC), ultra high performance
liquid chromatography (UHPLC), and gas liquid
chromatography (GLC); while photodiode array
(PDA), evaporative light scattering (ELS), an
fluorescence (FL) detectors, and mass
spectrometry (MS) are used for the detection,
quantification, and identification of plant steroids.
6

7. STEROIDS
• Steroidal alkaloids found in the plants of
Solanaceae and Melanthiaceae family
(specially the genus Veratrum), cardiac
glycosides, phytosterols, and
brassinosteroids (which include many plant
hormones) are the sources of plant steroids.
• Steroids contribute to a wide range of
therapeutic applications, such as cardiotonics
(digitoxin), vitamin D precursors (ergosterol),
oral contraceptive agents (semisynthetic
estrogens and progestins), anti-inflammatory
agents (corticosteroids), and anabolic agents
(androgens).
7

8. HECOGENIN
8

9. INTRODUCTION
• Hecogenin is a sapogenin found in Agave
genus in high quantities and is responsible
for the many therapeutic effects of these
medicinal plants.
• In addition, this compound is also widely
used in the pharmaceutical industry as a
precursor for the synthesis of steroidal
hormones and anti-inflammatory drugs.
9

10. HECOGENIN
SYNONYMS:
Sisal, sisal fiber.
SOURCE:
The steroidal saponin obtained from the dried leaves of Agave sisalana.
FAMILY:
Agavaceae.
GEOGRAPHICAL SOURCE:
Sub tropical America and Kenya.
CHEMICAL CONSTITUENTS:
Juice and pulp from the leaves:
• Hecogenin
• Smilagenin
• Tigogenin.
10

11. HECOGENIN
CHEMICAL TESTS:
Libermann burchard test
Alcoholic extract of drug was evaporated to dryness and
extracted with CHCl3, add few drops of acetic anhydride
followed by conc. H2SO4 from side wall of test tube to the
CHCl3 extract. Formation of violet to blue coloured ring at
the junction of two liquid, indicate the presence of steroid
moiety.
Salkowaski test
Alcoholic extract of drug was evaporated to dryness and
extracted with CHCl3, add conc. H2 SO4 from sidewall of
test tube to the CHCl3 extract. Formation of yellow
coloured ring at the junction of two liquid, which turns red
after 2 min, indicate the presence of steroid moiety.
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12. BIOSYNTHESIS
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13. BIOSYNTHESIS (CONTINUED)
13

14. BIOSYNTHESIS (CONTINUED)
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15. ISOLATION
15

16. CHARACTERIZATION
1. TLC Densitometric
method
2. GC-MS
Characterization
Method

17. 1. TLC DENSITOMETRIC METHOD
• In this method, sample and standard
solutions are prepared and applied in
triplicate on a precoated silica gel TLC plate
using automatic sample spotter.
• The plate is developed in a suitable solvent
system in the development chamber and
after development the plate is dried in air,
derivatised and scanned at suitable
wavelength in UV spectrophotometer.
• The peak areas can be recorded and the
calibration curve can be obtained by plotting
peak areas vs concentration of hecogenin
applied.
17

18. 1. TLC DENSITOMETRIC METHOD
(CONTINUED)
• The amount of hecogenin in different
samples can be calculated from calibration
curve.
• This method of characterization is found to
be simple, precise, specific, sensitive,
accurate and can be used in routine
quality control.
18

19. 2. GC-MS CHARACTERISATION
METHOD
• It can be performed on a GC-MS series
apparatus with electron impact ionization
detector.
• Helium is used as carrier gas and general
analysis conditions such as capillary column,
injector temperature, injection volume and oven
temperature is been programmed.
• The peaks are recorded and hecogenin could be
characterised by comparing with standard.
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20. PURIFICATION
STEP 1: PREPARATION OF CRUDE EXTRACT:
• Reflux in distilled water at 1:1 for 3hours.
• Filtrate the extract. This is done through forced filtration
machines to obtain a dilute extract.
• The concentration by forced circulation at 50̊ to yield
8.7% sisal crude extract.
STEP 2: SEPARATION OF POLYSACCHARIDES:
• Re-suspension in an ethanol : water solution is 80:20
and maintain at room temperature for 18h to remove
polysaccharides.
• Separation of precipitated polysaccharides done by
filtration.
20

21. • The concentration of hydroalcoholic extracts
(without polysaccharides) done by rotary
evaporator.
STEP 3: PARTITIONING:
• Hydroalcoholic extracts is partitioned with ethyl
acetate(1:2 v/v) and organic phase was
concentrated under reduced pressure in rotary
evaporator.
STEP 4: HYDROLYSIS REACTION:
• Ethyl acetate extract is subjected to hydrolysis
reaction.
21

22. HYDROLYSIS REACTION:
Ethyl acetate extract (1g) is refluxed for 3h in
hydroethanol solution 80% (30ml) containing 5ml
of concentrated HCl.
NEUTRALISATION REACTION:
The reaction product is neutralized with NaOH and
extracted with ethyl acetate to yield sapogenins.
Then concentrated by rotary evaporator.
Reserved product is dissolved in ethyl acetate
0.5 mg/ml.
22

23. IMPORTANCE (OR) USES
• Hecogenin has been indicated as the
responsible agent for sisal therapeutic
effect due to its beneficial properties
involving anti-inflammatory, antioxidant,
antifungal, hypotensive, anti-hyperalgesic
and anti-nociceptive effects.
• Hecogenin is used in the pharmaceutical
industry as a precursor of steroidal anti-
inflammatory and steroidal hormone
drugs.
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24. RECENT ARTICLES OF HECOGENIN
• Saponins and sapogenins of Agave with respect
to diverse Pharmacological role of hecogenin,
Deepa K. Ingawale (Mandlik), International
Journal of Pharmacy and Pharmaceutical
Sciences, Vol-12, 2020.
• Effect of Hecogenin on DNA instability, M.S.
Cruz et al, Toxicological reports, Elsevier, 2016.
• Effects of Hecogenin and its possible
mechanism of action on experimental models of
gastric ulcer in mice, G. Santos Cerqueira et al,
European journal of Pharmacology, Elsevier,
2012. 24

25. REFERENCES
• Tyler, Varro E.; Brady, Lynn R.; Robbers, James E;
Pharmacognosy, Lee & Febiger, 9th edition, Pg:156
• https://www.researchgate.net › 2705...GC-MS
Characterisation of Sapogenins from ... -
ResearchGate
• https://www.researchgate.net › 2256...Densitometric
Determination of Hecogenin from Agave americana ...
• C.K.Kokate, S.B.Gokhale, A.P.Purohit; A textbook of
Pharmacognosy, Nirali Prakashan, 54th edition,
Pg:9.44.
• www.slideshare/ Peter Kakema/ Isolation of hecogenin
from sisal.
• N.Biren Shah, A.K Seth; Textbook of Pharmacognosy
and Phytochemistry, Elsevier, 1st edition, Pg:253
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