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ISOLATION AND ESTIMATION OF PHYTOCONSTITUENTS:
GLYCERRHIZIN AND PHYLLANTHIN
By: A.M. Naseefa
Glycerrhizin
By: A.M. Naseefa
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
3
INTRODUCTION TO
GLYCYRRHIZA
Common name: Liquorice, Sweet Wood, Regolizia
(Italian), Jeshtamadh, Yastimadhu.
Biological ...
4
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
5
CHEMICAL CONSTITUENTS
 Chief constituent of Liquorice- Glycyrrhizin.
 Glycyrrhizin:
 A triterpenoidal saponin glycosi...
6
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
7
INTRODUCTION TOINTRODUCTION TO
GLYCYRRIZINGLYCYRRIZINGlycyrrhizin is the main sweet tasting compound from liquorice
root...
8
ISOLATION OF GLYCYRRHIZINISOLATION OF GLYCYRRHIZIN
The isolation of glycerrhizin from glycerrhiza is based on
its solubi...
9
1. Acid precipitating method:
Procedure:
1. wiegh 50g liquorice powder add 300ml water in the beaker and
boil with stirr...
10
2. Alcohol extraction method
Procedure:
1. wiegh 50g of powdered drug into 500ml beaker & add 100ml
methanol mix it .
2...
11
3. Ammonia extraction method
Procedure:
1. Glycerrhiza is extracted with hot water and filtered.
2. Filtrate is acidifi...
12
1. TLC- densitometry
2. Colorimetric method
3. UV- spectroscopic method
4. HPTLC method
5. HPLC method
Estimation:
AZEE...
13
1.TLC- densitometry:
TLC plates- kieselgel 60HF 254
Solvent system: n Butanol: acetic acid:water 7:1:2
Detection:1% cer...
14
2. Colorimetric method:
Glycerrhizin gives purple color with anisaldehyde in the
presence of H2SO4 . this forms the bas...
15
3.Spectrophotometric method
1g extract+50ml 3%
solution of
trichloroacetic
acid in acetone
stand for few
minutes &
deca...
16
4. HPTLC METHOD:
Sample Preparation:
Sample Application: Bandwise with linomat 2, 3, 4 μl of standard & sample
solution...
17
USES:
• Demulcent
• Anti-inflammatory
• Flavoring agent and sweetner
• Anti-tussive and expectorant
• Anti-bacterial
• ...
18
Biological source : It consists of the aerial parts of the plant Phyllanthus
amarus belong to the Family – Euphorbiacea...
19
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
20
PROCEDURE :
1.Mix the powder drug with lime water , It is allowed to stand for
overnight .
2.The mass is then transferr...
21
IDENTIFICATION
BY T.LC :
Adsorbent – Silica gel GF254
Solvent system – n- hexane: ethyl acetate (2:1)
Detection – Vanil...
22
By HPLC :
Column: - µ- Bondapak-18 ( 3.9mm X 30cm)
Mobile phase :– Methanol – Water (66: 34) Flow rate – 1.8ml/Minute
D...
23
REFERENCE:
Textbook of Pharmacognosy pulse publications.
en.wikipedia.org
www.authorstream.com
www.webmd.com
www.slides...
24
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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ISOLATION AND ESTIMATION OF GLYCERRHIZIN AND PHYLLANTHIN

ISOLATION AND ESTIMATION OF GLYCERRHIZIN AND PHYLLANTHIN

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ISOLATION AND ESTIMATION OF GLYCERRHIZIN AND PHYLLANTHIN

  1. 1. ISOLATION AND ESTIMATION OF PHYTOCONSTITUENTS: GLYCERRHIZIN AND PHYLLANTHIN By: A.M. Naseefa
  2. 2. Glycerrhizin By: A.M. Naseefa AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  3. 3. 3 INTRODUCTION TO GLYCYRRHIZA Common name: Liquorice, Sweet Wood, Regolizia (Italian), Jeshtamadh, Yastimadhu. Biological sources: It is obtained from the dried, unpeeled, roots and stolons of Glycyrrhiza glabra. Family: Leguminosae Part used: Roots & Stolons Geographical Source: commercially cultivated in Spain, and England. Allied species: Glycyrrhiza uralensis AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  4. 4. 4 AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  5. 5. 5 CHEMICAL CONSTITUENTS  Chief constituent of Liquorice- Glycyrrhizin.  Glycyrrhizin:  A triterpenoidal saponin glycoside.  Also called Glycyrrhizic acid or Glycyrrhizinic acid.  Present as potassium and calcium salt of Glycyrrhizinic acid.  Being a glycoside, Glycyrrhizic acid, on hydrolysis gives an aglycone and a glycone portions: Glycyrrhizinic acid Glycrrhetinic acid (Glycyrrhetic acid), which is an aglycone of triterpenoidal structure + 2 moles of Glucuronic acid. Upon hydrolysis, the glycoside loses its sweet taste.AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  6. 6. 6 AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  7. 7. 7 INTRODUCTION TOINTRODUCTION TO GLYCYRRIZINGLYCYRRIZINGlycyrrhizin is the main sweet tasting compound from liquorice root. It is 30–50 times as sweet as sucrose. Pure glycyrrhizin is odorless. Glycyrrhizin and other products with this component have a long history of medicinal value and are used in the treatment of peptic ulcer, especially glycyrrhetinic acid (triterpene derivative). IUPAC Name: (3β,18α)-30-hydroxy-11,30-dioxoolean-12-en-3-yl 2-O-β-D- glucopyranuronosyl-β-D-glucopyranosiduronic acid Molecular formula: C42H62O16 Molecular weight: 822.94gm/mol. NASEEFA. JSSCP ,MYSORE
  8. 8. 8 ISOLATION OF GLYCYRRHIZINISOLATION OF GLYCYRRHIZIN The isolation of glycerrhizin from glycerrhiza is based on its solubility. Glycerrhizin is soluble in hot water, alcohol, but slightly soluble in cold water and insoluble in ether. The three methods of isolation are: 1. Acid precipitating method 2. Alcohol extraction method 3. Ammonia extraction method AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  9. 9. 9 1. Acid precipitating method: Procedure: 1. wiegh 50g liquorice powder add 300ml water in the beaker and boil with stirring 2. decant the supernant liquid 3. filter the remaining residue and collect the filterate 4. Adjust pH 2.8 by the addition of acid, Glycerrhizin precipitates out 5. Filter and collect the ppt 6. Wash the ppt with cold water to make it free from acid 7. Transfer the ppt to china dish and heat gently to remove the water content, shiny brown mass of glycerrhizin is seen. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  10. 10. 10 2. Alcohol extraction method Procedure: 1. wiegh 50g of powdered drug into 500ml beaker & add 100ml methanol mix it . 2. Add another 100ml methnol & left for 24hrs 3. Filter and collect the filterate 4. Extract this methnolic extract with 3 portions of petroleum ether, subsequently with benzene, ethylacetate chloroium and solvent ether. 5. Transfer methnolic layer into china dish & evaporate on water bath to get glycerrhizin. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  11. 11. 11 3. Ammonia extraction method Procedure: 1. Glycerrhiza is extracted with hot water and filtered. 2. Filtrate is acidified with con H2SO4 to pH2.8 3. Ppt is dissolved in dil.NH4OH and poured into acetone to ppt ammonium glycerrhizinate. 4. The ppt is dissolved in hot water and evaporated to get ammonium glycerrhizinate. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  12. 12. 12 1. TLC- densitometry 2. Colorimetric method 3. UV- spectroscopic method 4. HPTLC method 5. HPLC method Estimation: AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  13. 13. 13 1.TLC- densitometry: TLC plates- kieselgel 60HF 254 Solvent system: n Butanol: acetic acid:water 7:1:2 Detection:1% cerric sulfate in 10% H2SO4 heating at 105oC for 5min Std solution: A known conc. Of glycerrhizin (0.5-2.0mg/ml) Sample solution: Reflux 1gm of samplewith 50ml of 50%ethanol cool and filter. Reflux the marc with 50ml of 50% ethanol. Evaporate and dissolve the residues in 50% ethanol(10ml). Development and chromatograms: 13-14cm.distance application and 10µl std and sample Visualisation of spots: visualise and scan in densitometer. Integrate the area of spots corresponding to glycerrhetic acid. Calculate the % of it in the sample. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  14. 14. 14 2. Colorimetric method: Glycerrhizin gives purple color with anisaldehyde in the presence of H2SO4 . this forms the basis of estimation. The intensity of color developed is proportional to the amount of glycerrhizin, which is measured at 556nm. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  15. 15. 15 3.Spectrophotometric method 1g extract+50ml 3% solution of trichloroacetic acid in acetone stand for few minutes & decant the liquid repeat the extraction 2 more times by adding 25ml of trichloroacetic acid in acetone filter after the third extraction wash the residue with 25ml of acetone . combine all the extracts Add drop by drop con.ammonia until abandunt cheesy ppt is formed. fliter in buchner funnel wash residue with 50ml of acetone 3- 4times dissolve the residue in 50ml of water make up the volume upto 250ml (solutionA) take 10ml from above solution and dilute upto 500ml this is solution B measure optical density of solution B at 258nm using water as blank AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  16. 16. 16 4. HPTLC METHOD: Sample Preparation: Sample Application: Bandwise with linomat 2, 3, 4 μl of standard & sample solution. Band length remains 6mm; distance between bands 4mm. Chromatography: 2 step chromatogram development: 1st run: chloroform- acetone (9:1). 2nd run: Chloroform- diethyl ether- formaldehyde (80:15:1) Quantitative evaluation: Done at 254nm using Mercury lamp as monochromator. AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  17. 17. 17 USES: • Demulcent • Anti-inflammatory • Flavoring agent and sweetner • Anti-tussive and expectorant • Anti-bacterial • Useful in gastric and duodenal ulcers • Anti-spasmolytic activity Phyllanthin AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  18. 18. 18 Biological source : It consists of the aerial parts of the plant Phyllanthus amarus belong to the Family – Euphorbiaceae common names: Bhuiamla, Bhumyamalaki Chemical Constituents: Phyllanthin and Hypophyllanthin are the two major constituents of the drug phyllanthus. Chemically , Phyllanthin is a diaryl butane, where as hypophyllanthin is an aryl tetra-hydronaphthalein. other constituents are : phyllanirurin, niranthin, nirtetralin, tannins like amariniic acid , geraniic acid. Phyllanthin Hypophyllanthin PHYLLANTHIN
  19. 19. 19 AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  20. 20. 20 PROCEDURE : 1.Mix the powder drug with lime water , It is allowed to stand for overnight . 2.The mass is then transferred to a Soxhlet apparatus and extraction is carried out with petroleum ether 3.Collect the petroleum ether extract and concentrate under reduced pressure. 4. Mix the extract with methanol and boil , collect the dewaxed methanol extract , evaporate to dryness, collect the residue. 5. Dissolve the residue in petroleum ether , concentrate and allow to stand (Yellow oil gets separated) 6. The residue is subjected to column chromatography on Alumina and elute with n-hexane: ethyl acetate (99:1) 7. 99- 108 fractions corresponds to hypophyllanthin and 109- 137 fractions corresponds to phyllanthin Subject them to chromatography further to yield pure phyllanthin and hypophyllanthin ISOLATION: AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  21. 21. 21 IDENTIFICATION BY T.LC : Adsorbent – Silica gel GF254 Solvent system – n- hexane: ethyl acetate (2:1) Detection – Vanillin in sulphuric acid reagent Rf value – Phyllanthin – 0.20 ( Blue spot) Hypophyllanthin – 0.25 ( Brown spot) AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  22. 22. 22 By HPLC : Column: - µ- Bondapak-18 ( 3.9mm X 30cm) Mobile phase :– Methanol – Water (66: 34) Flow rate – 1.8ml/Minute Detection: – UV at 230nm Standard – Known concentration (0.05- 2µg) Sample: – Macerate 1gm of the drug powder with lime water at room temperature for 18hrs .Reflux with 30ml methanol containing 3% KOH for 1hr .cool and filter .collect the filtrate Reflux mark with methanol containing 3% KOH . Filter and collect the filtrate . Combine the extracts and concentrate under reduced pressure. Make up to 50ml. Sampling: – Apply 10µl of both standard and sample solutions Determination: – Note the peak areas corresponding to phyllanthin and hypophyllanthin in both standard and samples and calculate their percentages accordingly. ESTIMATION : AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  23. 23. 23 REFERENCE: Textbook of Pharmacognosy pulse publications. en.wikipedia.org www.authorstream.com www.webmd.com www.slideshare.com AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
  24. 24. 24 AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE

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