Analysis of Chemical Constituents of Rhubarb and Study

Presented By : Ankit M MehtaGuided 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 Weight

Polarity Molecular Formula

Emodine 270 Polar C15H10O5

Rhein 284 Polar C15H8O6

Sennoside 848 Polar C42H40O19

Stilbene 180 Non-Polar C14H12

Anthralin 226 C14H10O3

Chrysophenol 254 Non-polar C15H10O4

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 traveled by solute

Retardation 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 ConditionsGas 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 .

Method Ankit rhubarab2.m

Technique Gradient Elution

Flow rate 0.5 ml/min

Wavelength 254, 270 and 360

Column Agilent XBD C18

Solvent Acetonitrile:water

HPLC Gradient Elution Program

Time Acetonitrile %

Water %

0 15 85

8 20 80

15 30 70

Molecular formula :C15H1005

M.W - 270

EMODIN

Molecular W.C15H8O6

M.W-284

RHEIN

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 Time

Rhubarb Extract

Rhubarb extract treated with Pepsin

1 2.6 Present present

2 2.7 Present present

3 3.7 Absent Present


5 6.0 absent Present

6 6.2 absent present

7 6.5 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 used for this purpose are

Micrococcus roseus( MR)

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

Microorganism

Absorbance

Transmittance

Micrococcus Roseus (MR)

0.840 18.3


0.681 32.8

Micrococcus Luteus(ML)

0.810 21.3

Stephylococas ermis (SE)

1.00 0.03

Escheria Coli B(EC)

1.00 O.05

Corynebacteriupsudediphtrium(CP)

0.730 23.5

Enteroccus Faecalis(EF)

0.639 37.2

Microorganism

Absorbance

Transmittance


0.18 65.5


0.00 100.2


0.006 99.9


0.211 61.9

Escheria Coli B(EC)

0.08 93.2


0.339 46.5


0.05 99.0

TSB Medium & Microorganisms

TSB Medium, Methanol &Microorganisms

Microorganism

Absorbance

Transmittance


0.48 32.1


0.384 41.7


0.336 46.4


0.422 38.1

Escheria Coli B(EC)

0.382 41.6


0.459 35.3


0.308 49.2

TSB Medium , Rhubarb Extract & Microorganisms

Microorganism

Absorbance

Transmittance


0.488 32.6


0.433 37.0


0.333 46.6


0.357 44.1

Escheria Coli B(EC)

0.411 38.9


0.430 37.3


0.347 44.9

TSB Medium , Commercially 100% Rhubarb &MO

Microorganism

Absorbance

Transmittance


0.228 59.4


0.200 63.3


0.170 67.8


0.046 90.8

Escheria Coli B(EC)

0.021 95.3


0.235 58.1


0.025 94.8


Microorganism

Absorbance

Transmittance


0.152 70.7


0.124 75.3


0.135 73.5


0.124 75.4

Escheria Coli B(EC)

0.162 69.2


0.122 75.7


0.170 67.8


Microorganism

Absorbance

Transmittance


0.232 74.1


0.139 74.8


0.187 71.4


0.144 71.7

Escheria Coli B(EC)

0.197 74.9


0.143 72.3


0.185 65.5


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_

1491/_article 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

http://www.rhubarbinfo.com/

http://www.drugs.com/npp/rhubarb.htm

http://www.jstage.jst.go.jp/article/cpb/54/11/54_1491/_article

http://www.jstage.jst.go.jp/article/cpb/54/11/54_1491/_article

- 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

Documents

Analysis of Chemical Constituents of Rhubarb and Study