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A Report onPhytochemical and analytical study of Calotropis procara(Aiton)
in partial fulfillments of the course GE -511
Submitted by :-Gaurav
M.S.(Pharm) Semester -1Registration No. 16TMM2363
Department of natural product National Institute of Pharmaceutical Education and Research
Flow of presentation
1 : Introduction
3 : Taxonomical Classification
5 : Phytoconstituent Including Marker Compounds
6 : Biological Potential
8 : Analytical Study
a) HPLC Analysis b)1H NMR Analysis
c) LC-MS Analysis
Calotropis procara(Aiton)Common names - Sodom apple, Usher, dead sea apple, swallow wort, and milk weed. English name - Calotropis, Mudar plantHindi 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.
Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order :Gentianales
Family :Asclepiadaceae
Genus :Calotropis
Species :Calotropis procera(Aiton)
Taxonomical classification
https://plants.usda.gov
Phytoconstituents including marker compounds:Glycoside- Asclepin,calotropin,calotropagenin,calotoxin,calactin,uscharin,proceroside,proceragenin. Flavonoids- Quercetin,Quercetin-3-O-rutinoside, Kaempferol,Kaempferol-3-O-rutinosideSteroids -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, 1989Lbrahim.S.R.M,a.b* , Phytochemistry Lett.5 (2012) 490–495.
Figure 1. (1) Uscharin (2) Calotropin (3) calatoxin (4) asclepine (5) celetropagenin (6) 2-(3,4-Dimethyl-phenyl)-3,5,7-trihydroxy-chromen-4-one
O O
O
O O
H3C H H
H
HOH H
H
H
CH3
OH
OSN
O O
O
O O
OH
H3C H H
H
HOH H
H
H
CH3
OH
O
O O
O
O O
OH
H3C H H
H
HOH H
H
H
CH3
OH
O
HO
O O
O
O O
O
H3C H H
H
HOH H
H
H
CH3
OH
O
O
H
OH H
O
HO
HO
O
O
12
3 4
5
O
OHOH
HO
O6
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.
Extracts of Calotropis
procara
Anti- inflammat
oryAntimicro
bial
antimalarial
Anthelmintic
Anti-hyperglyce
mic
antipyreticHepatoprotective
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.
Analytical Analysis
1-HPLC Analysis 2- 1H NMR Analysis
3- LC-MS Analysis
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.
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.
NMR Analysis Calotropin:1H NMR (CDCl3): ppm 0.86 (3H, s, CH3 ) 1.30 (3H, d, J = 6 MHz, -O-CH-CH3), 3.40-4.20 (4H, m, broad, C-2H, C-3H, C-3’H and C-5’H), 4.59 (1H, s, C-1’H), 4.93 (2H, broad, C-21 H-2,), 5.90 (1H, s, olefinic), 9.98 (1H, s, CHO).
O O
O
O O
OH
H3C H H
H
HOH H
H
H
CH3
OH
O9.98
0.869.93
5.90
4.591.30
SINGH.B, RASTOGI.R.P, Phytochemistry,1972, Vol.11,pp. 757-762Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.
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. 757-762Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.
O O
O
O O
OH
H3C H H
H
HOH H
H
H
CH3
OH
O
Mass Spectroscopy Analysism.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. 757-762Moustafa.A.M.Y, Ahmed.S.H, Pharmaceutical Biology, 2010; 48(10): 1080–1190.
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 : AcetonitrileSolvent B : MethanolColumn & 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/minPlant part and extract: : Leaves and hydro-alcoholic (20:80)Injection volume : : 10 uLGradient(Time in min/solvent B%): : 80–30% B in 0–30 min; 5% B in 40–60 minWavelength range : 200 - 400 nmMarker 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
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.
ConclusionPhytochemical 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
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