Calotropis procera is a shrub with various medicinal properties. It contains several bioactive compounds including calotropin and calotoxin. HPLC and NMR analysis identified and characterized calotropin, with a retention time of 12.3 minutes by HPLC and characteristic proton signals. LC-MS analysis of calotoxin showed a molecular ion of m/z 405 that shifted during metal cationization, confirming its molecular weight of 404 Da. Thus, several analytical techniques were used to study the marker compounds in Calotropis procera.

1. Submitted by :-Gaurav
M.S.(Pharm) Semester -1
Registration No. 16TMM2363
Department of natural product
National Institute of Pharmaceutical Education and Research

3. Calotropis procara(Aiton)
Common names - Sodom apple,
Usher, dead sea apple, swallow wort,
and milk weed.
English name - Calotropis, Mudar
plant
Hindi names - Aaka, Aanka, Ak
• small, erect and compact shrub
• Family- Asclepiadaceae
• Have analgesic, antitumor,
antihelmintic, antidiarrhoeal,
antimalarial , antiviral activity
• Native to North Africa, Tropical
Africa, Western Asia, South Asia,
and Indochina
Introduction
Mohamed, N.H.a,b*, Bioorg. Med. Chem.Lett.,2015, 25 , 4615-4620.

4. Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order :Gentianales
Family :Asclepiadaceae
Genus :Calotropis
Species :Calotropis procera(Aiton)
Taxonomical classification
https://plants.usda.gov

5. Phytoconstituents including marker compounds:
Glycoside-
Asclepin,calotropin,calotropagenin,calotoxin,calactin,uscharin,proceroside
,proceragenin.
Flavonoids- Quercetin,Quercetin-3-O-rutinoside, Kaempferol,Kaempferol-3-O-rutinoside
Steroids -procesterol
Enzyme- Cysteine protease,Plant endopeptidase.Procerain,Anti-procerain.
Terpinoids- :Amyrin, amyrin acetate, calotroproceryl acetate A ,
calotroprocerone A,calotroprocerol A, and calotroproceryl acetate B
KHAN.A.Q, and MALIK.A ,Phytochemtstry, Vol 28, No 10. pp 2859-2861, 1989
Lbrahim.S.R.M,a.b* , Phytochemistry Lett.5 (2012) 490–495.

6. O O
O
O O
H 3 C
H H
H
H
O H H
H
H
C H 3
O H
OS
N
O O
O
O O
O H
H 3 C
H H
H
H
O H H
H
H
C H 3
O H
O
O O
O
O O
O H
H 3 C
H H
H
H
O H H
H
H
C H 3
O H
O
H O
O O
O
O O
O
H 3 C
H H
H
H
O H H
H
H
C H 3
O H
O
O
H
O H H
O
H O
H O
O
O
1
2
3 4
5
O
O H
O H
H O
O
6
KHAN.A.Q, and MALIK.A ,Phytochemtstry, Vol 28, No 10. pp 2859-2861, 1989.
Lbrahim.S.R.M,a.b* , Phytochemistry Lett.5 (2012) 490–495.

7. Extracts of
Calotropis
procara
Anti-
inflammat
ory
Antimicro
bial
antimalari
al
Anthelmin
tic
Anti-
hyperglyce
mic
antipyretic
Hepatopro
tective
Antifertile
Analgesic
Larvicidal
Anti ulcer
Biological Potential
Mossa.J.S.a.b*,American Journel of Chinese Medicine , Vol ,XIX,Nos.3-4, pp 223-231.

9. HPLC analysis
Marker compounds : Calotropin
Sample preparation : Plant material (dried latex) was extracted with ethanol (5
kg) by maeceration at room temperature for two weeks
Extract concentration 5mg/ml in methanol. Filtered through
0.22 µm filter, injection volume 10 µl.
Standard solution and calibration detail : 1mg/ml in acetonitrile (ACN)
Chromatography system : : HPLC
Column details : ChromSpher C18 reverse-phase column, 5 µm particle size,
20 x 0.3c (Chrompack, Middelburg, The Netherlands)
Mobile phase detail : ACN : Water
Elution mode : Gradient (27.5-70 % ACN), flow rate 0.35 ml/min.
Detection wavelength : 220 nm with a Hewlett Packard model 1040A diode array
detector
Peaks identification : Calotropin, Rt = 12.3 min
GROENEVELD.H.W a,b*, J.Chem. Ecol. Vol. 16, No. 12, 1990.

10. Figure 2. HPLC chromatogram of methanolic extract of Calotropis procera(Aiton)
latex (Calotropin Rt = 12.3 min)
GROENEVELD.H.W a,b*, J.Chem. Ecol. Vol. 16, No. 12, 1990.

11. O O
O
O O
OH
H3C
H H
H
H
OH H
H
H
CH3
OH
O
9.98
0.86
9.93
5.90
4.591.30
SINGH.B, RASTOGI.R.P, Phytochemistry,1972, Vol.11,pp. 757-762
Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.

12. Marker Proton signal (characteristic peak)
Calotropin 5.90 (olefinic proton)
9.98 (aldehydic proton)
Figure 4. Proton NMR of Calotropin (ChemDraw)
SINGH.B, RASTOGI.R.P, Phytochemistry,1972, Vol.11,pp. 757-762
Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.

13. O O
O
O O
OH
H3C
H H
H
H
OH H
H
H
CH3
OH
O
Mass Spectroscopy Analysis
m.p. 207-209ᵒC, mass spectrum m/e 532 (M+), 404, 386, 357, 233
(base peak), 215, 192, 187, 128, 113, 91, 85, 79
calotropin
SINGH.B, RASTOGI.R.P, Phytochemistry,1972, Vol.11,pp. 757-762
Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.

14. LC-MS ANALYSIS
Alkali metal cationisation in ESI‐MS , capillary voltage 3.5 kV; cone potential at 40 V, drying gas at flow
rates of 10 and 350 L/h, respectively
Solvent A : Acetonitrile
Solvent B : Methanol
Column & flow rate : JASCO 980 LC (equipped with JASCO AS‐
205plus auto‐sampler and JASCO MD‐
2010plus PDA multi‐wavelength detector)
using a Thermo‐ODS‐2 analytical column (250
× 4.6 mm, 5 μm) and 1.0 mL/min
Plant part and extract: : Leaves and hydro-alcoholic (20:80)
Injection volume : : 10 uL
Gradient(Time in min/solvent B%): : 80–30% B in 0–30 min; 5% B in 40–60 min
Wavelength range : 200 - 400 nm
Marker Identification : Calotoxin at Rt 10.29 min showed an [M + H]+
ion at m/z 405 that was shifted to m/z 411 [M +
Li]+ and 427 [M + Na]+ during corresponding
metal cationisation, thus confirming the
molecular weight as 404 Da.
Kanojiya.S ,Madhusudanan.K.P, Volume 23, Issue 2,March/April 2012 Pages 117–125

15. Figure 4. Metal cationised electrospray ionisation mass spectra of calotoxin: (A)
ammonium cationised, (B) lithium cationised, (C) sodium cationised
Kanojiya.S ,Madhusudanan.K.P, Volume 23, Issue 2,March/April 2012 Pages 117–125.

16. Conclusion
Phytochemical and analytical study of calotropis procera (Aiton)was reviewed
1-marker compound of calotropic procera (calotropin ) studied by
HPLC and 1 H NMR
3-Another marker compound Calotoxin was studied by LC-MS