Presented By : Ankit M Mehta

Guided by: Dr Aheda Saber

Dr Timothy Gsell
 Rhubarb is a rhizome,
belong to polygonaceae
family .
 Rhubarb leaves are large
and heart-shaped
 Rhubarb root is thick,
oval shape ,externally
brown color and
internally deep yellow
color
 Most important
constituents are
Anthraquinone
Derivatives .
 Emodine , Rhein,
Anthralin ,chrysophanol
and physcion
 Stilbene , sennoside,
Gallic acid and Cinnamic
acid
Compounds Molecular Polarity Molecular
Weight Formula
Emodine 270 Polar C15 H10 O5
Rhein 284 Polar C15 H8O6
Sennoside 848 Polar C42H40O19

Stilbene 180 Non-Polar C14 H12

Anthralin 226 C14 H10 O3
Chrysophenol 254 Non-polar C15 H10 O4

Phyosterol 681 Non-polar C47H84O2,

Calcium oxalate 146 CaC2O4

 Hepatic Stimulant
 Laxative

 Thermal burn

 Emollients

 Skin inflammation

 Cancer treatment ( skin and renal)

 EXTRACTION OF RHUBARAB
 Soxhlet method :25 gm of
rhubarb powder dissolved in
300ml of Methanol and
attached to a Soxhlet
Apparatus.
 Temperature was 78 ºC –
Extraction was carried out
for 8 hours.
 Final concentration were
stored in refrigerator at 3 ºC
THIN LAYER CHROMATOGRAPHY:
 Stationary phase – Silica Gel

 Mobile Phase-

(1) mixture of ethyl acetate: petroleum ether

(75:25)
(2) mixture of Acetic acid : chloroform (85:15)
(3) mixture of Acetic anhydride, water and
petroleum ether (50:10:40)
 Silica gel plates wear placed inside TLC chamber for
development
 Iodine- spot visualization –Observed under UV light

 The best separation was observed with mixture of Ethyl

Acetate and Petroleum ether.(75:25)
 Under UV light – three bands were observed.
Measuring Rf values :(Retardation Factor)
Retardation factor = Distance traveled by
solute /Distance traveled by solvent .

The marked bands were scratched out-transferred

each into different vials.
Sample Distance Retardatio
traveled n
by solute Factor
Band 1 4.2 cm 0.3000

Band 2 4.7 cm 0.3357

Band 3 9.3 cm 0.6642

 Gas Chromatography
– Best separation
technique
 The components are
separated according
to their partition co-
efficients .
 Compound should be
volatile and
thermally stable
 Experimental Conditions
Gas Chromatograph HP 5890 series 2
Column ZB-1
Method SK3MIX.M
Program Temperature
Oven Initial Temperature 40 ºC
Injection port Temperature 200 ºC
Detector Temperature 300ºC
Rate 7ºC per minute
Hold Up Time 5 minutes
Run Time 45.20 minutes
 GC/MS- determination of
structural elucidation and
molecular weight .
 GC/MS- separates the
mixture into molecular
compounds ,MS- ionized
molecules and separates
molecular ions and
fragments according to
their mass to charge ratio
 Only Volatile compounds and
compounds which have molecular
weight between 50-650 amu.
 Determination of structural elucidation
by using (1) fragmentation patterns, or
(2) spectral identification with library ,
 Experimental conditions
Method Ank solvcut1.5
Mass scan range 50-600 EI
Injection Port Temperature 200ºC
Initial oven temperature 40ºC
Final Oven temperature 300ºC
Rate 7ºC/min
Hold Time 5 min
Total time 42.64 min
226 m/z Molecular Ion Peak/Base
peak
198 m/z Loss of CO (-28)
181 m/z Loss of OH (-17)
151 m/z Loss of CO (-28)
76 m/z m/3e (triply charged ion)
180 m/z Molecular Ion Peak/Base
Peak
181 m/z M+1

166 m/z Loss of –CH2 ( -14)

103 m/z Removal of benzene moiety

77 m/z Removal of benzene moiety

91 m/z Tropilium Ion Formation

 LC/MS – Very sensitive and
accurate chromatography
technique
 Electro spray – generates
ion under atmospheric
pressure
 Mass spectroscopy –
detecting specific mass-to-
charge(M/Z) ratio related to
component .
  HPLC Gradient
Elution Program
Method Ankit
rhubarab2.m
Time Acetonitril Water
Technique Gradient Elution e% %
0 15 85
Flow rate 0.5 ml/min
8 20 80
Wavelength 254, 270 and 360

15 30 70
Column Agilent XBD C18

Solvent Acetonitrile:water
 EMODIN

Molecular formula
:C15H1005
M.W - 270
 RHEIN

Molecular
W.C15 H8O6

M.W-284
 Rhubarb Extract treated with Human
Digestive Enzyme .(pepsin stimulation
study)
 Procedure: 10ml of Rhubarb Extract
treated with 5ml pepsin
 Mixture keep a side for 24 hours-
Measured the PH
 Adjust PH at 5.20 (approx) by adding Acid
(HCL), Base ( NaoH) and Buffer .
 Extracted was injected to LC/ms
Peaks Retention Rhubarb Rhubarb
Time Extract extract
treated
with Pepsin

1 2.6 Present present

2 2.7 Present present
3 3.7 Absent Present
4 4.4 Absent Present
5 6.0 absent Present
6 6.2 absent present
7 6.5 Present Present
8 7.0 Absent Present
9 7.7 Present Present
10 8.7 Present Present
11 12 present present
 GC- FID detection of
unknown compound.
 GC- various information
about retention time
 Major peak were observed
at 11.93, 14.15, 17.77,
18.70 ,30.42and 32.93
 TLC Band – cannot give any
confirmed results
 GC/MS- only volatile and
Polar compound –M.W
between 50-600 AMU
 By comparing literature
survey and NIST library
ANTHRALIN (M.W- 226) and
STILBENE (M.W- 180) are
present in Rhubarb extract.
 More research need to be
done in Unknown Peaks
 LC/MS –unknown higher molecular
weight compound can be found.
 Non-Volatile compounds .

 From literature and Molecular weight

and fragmentation ,we can confirm that
EMODIN(M.W 270 )and RHEIN (M.W-
284) are present in Rhubarb extract
 Measure a microbial growth.
 To study the anti-microbial activity ,the
rhubarb extract and commercially
Rhubarb ,several microbial test were
conducted.
 The microorganism
Micrococcus roseus( MR) used for this
purpose are
Bacillus Subtilis (BS)
Micrococcus Luteus( ML)
Staphylococcus Epidermis (SE)
Eschericia coli b ( EC)
Cornebacterium Psudodiphterine(CP)
Enteroccoocus Faecalis (EF)
 Broth Culture Method:
 Susceptibility of the microorganism was
determined by measuring their Absorbance and
Transmittance .
 Spectrophotometer at 600 nm
 8 gm of Nutrient broth dissolved in 1L distil water.
Microorganism were cultured in tryptic soy broth .
100µl of microorganism transferred in to tryptic
soy broth then transmittance and absorbance
was measured .
 Six set containing 7 tube in each set were
sterilized and label .
SET QUANTITY IN THE TUBE

TSB Medium and Microorganism

First set 100µl of solvent methanol

Second set 100µl of 100% extract of Rhubarb

Third set 100µl of 1% concentration of

commercially Rhubarb in methanol

Forth set 100µl of 10% concentration of

commercially Rhubarb in methanol
Fifth set 100µl of 25% concentration of
commercially Rhubarb in methanol
Sixth set 100µl of 100% concentration of
commercially Rhubarb in methanol
 Microorgani Absorbanc Trans Microorgani Absorbanc Trans
sm e mittan sm e mittan
ce ce
Micrococcus 0.840 18.3 Micrococcus 0.18 65.5
Roseus (MR) Roseus (MR)
Bacillus 0.681 32.8 Bacillus 0.00 100.2
Subtilis (BS) Subtilis (BS)
Micrococcus 0.810 21.3 Micrococcus 0.006 99.9
Luteus(ML) Luteus(ML)
Stephylococas1.00 0.03 Stephylococas0.211 61.9
ermis (SE) ermis (SE)

Escheria Coli 1.00 O.05 Escheria Coli 0.08 93.2

B(EC) B(EC)
Corynebacteri 0.730 23.5 Corynebacteri 0.339 46.5
upsudediphtri upsudediphtri
um(CP) um(CP)
Enteroccus 0.639 37.2 Enteroccus 0.05 99.0
Faecalis(EF) Faecalis(EF)
TSB Medium,
Methanol
SB Medium & Microorganisms
 Microorganis Absorban Transmi
m ce ttance

Micrococcus 0.48 32.1

Roseus (MR)
Bacillus 0.384 41.7
Subtilis (BS)
Micrococcus 0.336 46.4
Luteus(ML)
Stephylococas 0.422 38.1
ermis (SE)
Escheria Coli 0.382 41.6
B(EC)
Corynebacteri 0.459 35.3
upsudediphtri
um(CP)
Enteroccus 0.308 49.2
Faecalis(EF)

TSB Medium , Rhubarb Extract & Microorganisms

Microorganis Absorbanc Transmi
m e ttance

Micrococcus 0.488 32.6

Roseus (MR)
Bacillus 0.433 37.0
Subtilis (BS)
Micrococcus 0.333 46.6
Luteus(ML)
Stephylococas 0.357 44.1
ermis (SE)
Escheria Coli 0.411 38.9
B(EC)
Corynebacteriu 0.430 37.3
psudediphtriu
m(CP)
Enteroccus 0.347 44.9
Faecalis(EF)

TSB Medium , Commercially 100% Rhubarb

Microorganis Absorbanc Transmi
m e ttance

Micrococcus 0.228 59.4

Roseus (MR)
Bacillus 0.200 63.3
Subtilis (BS)
Micrococcus 0.170 67.8
Luteus(ML)
Stephylococas 0.046 90.8
ermis (SE)
Escheria Coli 0.021 95.3
B(EC)
Corynebacteriu 0.235 58.1
psudediphtriu
m(CP)
Enteroccus 0.025 94.8
Faecalis(EF)

TSB Medium , Commercially 1% Rhubarb

Microorganis Absorbanc Transmi
m e ttance

Micrococcus 0.152 70.7

Roseus (MR)
Bacillus Subtilis 0.124 75.3
(BS)
Micrococcus 0.135 73.5
Luteus(ML)
Stephylococas 0.124 75.4
ermis (SE)
Escheria Coli 0.162 69.2
B(EC)
Corynebacteriu 0.122 75.7
psudediphtrium
(CP)
Enteroccus 0.170 67.8
Faecalis(EF)

TSB Medium , Commercially 10% Rhubarb

&MO
Microorganis Absorbanc Transmi
m e ttance

Micrococcus 0.232 74.1

Roseus (MR)
Bacillus Subtilis 0.139 74.8
(BS)
Micrococcus 0.187 71.4
Luteus(ML)
Stephylococas 0.144 71.7
ermis (SE)
Escheria Coli 0.197 74.9
B(EC)
Corynebacteriu 0.143 72.3
psudediphtrium
(CP)
Enteroccus 0.185 65.5
Faecalis(EF)

TSB Medium , Commercially 10% Rhubarb

&MO
TSB, MICROORGANISM& RHUBARAB EXTRACT
VS TSB, MICROORGANISM & 100 %
Commercially Rhubarb
 Rhubarb shown a very pronoun anti-
microbial activity
 10% concentration of commercial
rhubarb showed maximum anti-
microbial activity
 Slightly difference between Rhubarb
Extract and Rhubarb commercial
Extract
 ANTHRALIN and Stilbene were identified by GC/MS
 EMODIN and RHEIN were identified by LC/MS
 10% concentration of commercial rhubarb
showed maximum anti-microbial activity
 Further studies are required for Unknown Peaks
from GC/MS and LC/MS (specially Rhubarb Extract
treated with Pepsin)
 Further studies are required to show the Anti-
Cancer Activity of Rhubarb
 www.rhubarbinfo.com
 www.drugs.com/npp/rhubarb.htm, Text book of
Pharamcognosy , second edition by Dr kokate
 http://www.jstage.jst.go.jp/article/cpb/54/11/54_149
 Determination of five major anthraquinoids in
Chinese herbal preparations by micellar electro
kinetic capillary electrophoresis – huenn-jyi-sheu
and Hong-Ren
 Analysis of nine rhubarb anthraquinones and
bianthrones by micellar electrokinetic
chromatography using experimental design -
Ching Hua Kua and Shao-Wen Sun
 - C-liu , L-liu, L-zhu , Computer-aided development of a high-
performance liquid chromatographic method for the determination of
hydroxyanthraquinone derivatives in Chinese herb medicine rhubarb-
Department of Chemistry ,Lanzhou University , Lanzhou -China
 min-ye , jain –ha ,hubio chen ,junhua zheng and dean geo , Analysis
of Phenolic Compounds in Rhubarbs Using Liquid Chromatography
Coupled with Electro spray Ionization Mass Spectrometry, The State
Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing, People’s
Republic of China.
 Hai-Xia Zhang and Man-Cang Liu, Separation procedures for the
pharmacologically active components of rhubarb, Department of
Chemistry, Lanzhou University, Lanzhou 730000, China
 Ming s fuh , hung –jian lin , Analysis of rhubarb by LC/MS ,
department of chemistry, soochow university , Taiwan
 Dr Aheda Saber
 Dr Timothy Gsell

 Dr Karen D’Arcy

 Professor Kent

 Dr Joseph Edition

 Mr. Rahul Khanke

 Krishna