Rutin is a flavonoid glycoside that can be isolated from various plant sources, including buckwheat. It has antioxidant, anti-inflammatory, and antimicrobial properties. Rutin is typically isolated using solvent extraction and fractional crystallization. It appears as a yellow crystalline powder that is sparingly soluble in water but soluble in alcohols. Rutin has been shown to have potential health benefits for conditions like arthritis, cancer, and cardiovascular issues due to its antioxidant and anti-inflammatory activities.

1. B.PHARM. 5th SEMESTER
PHARMACOGNOSY ANDPHYTOCHEMISTRY-II
BP504T.
UNIT-III
Isolation, Identification and Analysis of Phytoconstituents
Glycosides: Glycyrhetinic acid, Rutin
Wasim Raza Ali
Associate Professor
SOP, ITMBU, Vadodara (GJ)

2. Introduction
Naturally occurring organic compounds
Found in both plants and animals
On hydrolysis (by acids or enzymatic) gives one or more sugar moiety
(glycone) and non-sugar moiety (aglycone).
Non-sugar (aglycon) called genin.
The pharmacological acivity is due to the presence of genin part.
Glycone part facilitates the transportation of genin to the site of
action.

3. Glycosides
Definition
• These are the organic compounds obtained from the
plants or animals sources which on enzymatic or acid
hydrolysis gives one or more sugar moieties along
with non-sugar moiety.
Glycosides
Enzymatic or acid hydrolysis
sugar
moieties(glycone)
non-sugar moiety
(aglycone or genin)

4. The sugar involved in glycosides are of different types, but most commonly, it is β-D
glucose. Other are galactose, mannose, rhamnose, digitoxose, cymarose, etc.
The linkage between alycone & aglycone is called glycosidic linkage.

5. Types of glycoside
1. Based on atoms involved in glycosidic linkage
• O- glycosides
• C- glycosides
• S- glycosides
• N- glycosides
2. According to chemical nature of aglycone part
• Cardiac/sterol glycosides-Digitalis, indian squill, strophanthus,
• Anthracene(Anthraqunone) glycosides-Senna, rhubarb,aloe,
• cascara
• Cyanogenetic glycosides-Bitter almond, white cherry bark,
• Saponin glycosides-Dioscorea, liquorice, Ginseng,sersaparilla
• Isothiocynate glycosides-Black mustard

6. Physical & chemical properties
Colorless, solid, amorphous, non-volatile (flavonoid-yellow, anthra-
quinone-red or orange.)
Give positive reaction with Molisch's and Fehling's solution test
(after hydrolysis).
They are water soluble compounds, insoluble in organic solvents
Most of them have bitter taste (except: populin, glycyrrhizin,
stevioside)
Odorless except saponin (glycyrrhizin).
When a glycosides has a lot of sugars its solubility in water
decrease.
Glycosides hydrolyzed by using mineral acids and temperature or by
using enzymes

7. General method of isolation of Glycosides
Isolation of Glycoside :The method by which glycoside are isolated is called
Stas-Otto method.
Extraction
• Finely powdered (drug containing glycoside) is extracted by successive
extraction in a soxhlet apparatus with alcohol as solvent (Various enzyme
deactivated due to heating).
• The thermo-labile glycoside should be extracted at below 45°C.
• The extract is treated with lead acetate to precipitate tannins (eliminate non
glycosidal impurities) and filtered it.
• To the filtrate H2S gas is passed, the excess of lead acetate is precipitate as
lead sulphide.
• The extract is again filtered and concentrated to get crude glycoside.
Isolation
• The filtrate is subjected to fractional crystallization, distillation or
chromatography to obtained pure component.
Characterization
• Pure component is determined by the spectro-scopy method i.e. UV, IR,
NMR, MS, etc.

8. Glycyrrhetinic acid
Pentacyclic triterpenoid aglycone

9. Glycyrrhetinic acid
• Biological source : It consist dried, unpeeled roots and stolons of
Glycyrrhiza glabra linn
• Family : Leguminosae
• Geographical source: Commercially cultivated in Spain, Sicily and
England.
• Varieties of glycyrrhiza glabra: G. glabra var. typica (spanish
liquorice) G. glabra var. glandulifera (russian liquorice) G. glabra
var. violacea (persian liquorice)
Main constituent is glycyrrhizin (K & Ca salt of Glycyrrhizinic acid,
a glucoside) glycoside on hydrolysis it yields glycyrrhetinic acid (tri-
terpenoide). It should contain not less than 3% of glycyrrhinic acid.
Other constituents are sucrose, glycyramarin (bitter principle), resins,
fat, asparagin. It contain flavonoids like liquiritin and isoliquiritin.

10. Glycyrrhetinic acid
The isolation of glycyrrhetinic acid based on its solubility, it is
slightly soluble in hot water and alcohol but Insoluble in cold water
and insoluble in ether.
Basically three method for the isolation of glycyrrhetinic acid
•Acid precipitation method
•Ammonia extraction method
•Chloroform extraction method

11. Acid precipitation method
Required quantity of coarse powder of Glycyrrhiza roots is extracted
with boiling water.
Filtered and concentrate the extract to obtain a crude liquorice extract.
Then this extract is again extracted in water then,
Acidified with HCl to maintain pH 3-3.4 to precipitate Glycyrrhetinic acid
and filter.
The residue is washed with cold water to yield Glycyrrhetinic acid.
Dry the precipitate in china dish until trces of water get evaporated.

12. Ammonia extraction method
Transfer 20g powedered liquorice +50ml acetone +2 ml
di. HNO3 , mix, macerate for 2hrs with stirring.
Filter, transfer the marc to stoppered conical flask & add
20 ml acetone & filter
Combine both filtrate, conc. under vacuum,
Add dil NH3 sol. For ppt of NH4
+ glycyrrhizinate
Separate ppt by filtration and wash it with 5ml acetone
(2x) followed by drying & weigh the product

13. Chloroform extraction method
Weigh accurate qty of liqurice powder + CHCl3 in soxhlet apparatus for
3hrs.
Filter the content of flask & discard the filtrate.
The residue left on filter paper is then extracted with 0.5M H2SO4 for few
hrs.
Filter the content of flask. Transfer the filtrate in and extract with CHCl3
Separate & combine the CHCl3 layer.
The combined CHCl3 layer is evaporated to dryness to get glycyrhetinic
acid

14. Glycyrrhetinic acid
Properties
• Appearance :White crystalline powder
• Odor :Characteristic
• Taste :Characteristic
• Solubility :In soluble in water but freely
soluble in Soluble in ethanol,
chloroform, dioxone, pyridine or
acetic acidetc.

15. Glycyrrhetinic acid
Identification by chemical test
• Libermann test: 3ml of extract and 3ml of
acetic anhydride is heated and cooled. To this a
drops of conc. H2SO4 is added. Blue colour is
observed which indicate the presence of
triterpenoid.
• Libermann-Burchard test: In 3ml of extract,
2ml of chloroform, 1ml of acetic anhydride and
one drop of conc. H2SO4 is added. Blue-green to
red orange color is observed which indicate the
presence of triterpenoid or steroids

16. Glycyrrhetinic acid
Estimation
• Reflux about 25 g of liqurice root powder + 250ml water
for 6 hrs + NH3 to adjust pH 6.5-7, keep aside for few
hrs.
• Centrifuge extract & evaporate to 75% of original vol.
Add H2SO4 with stirring
• Decant supernatant liq. & suspend the ppt in 25ml H2O
& adjust the pH 4 by Na2CO3 & stir about 2-3 hr & then
centrifuge
• Again suspend the residue in H2O, pH 6, followed by
centrifugation, drying in oven.
• Calculate % on basis of air dried drug calculated as
glycyrrhetinic acid.

17. Glycyrrhetinic acid
Analysis by TLC
• Standard sample : Glycyrrhetinic acid
• Sample preparation : 1mg of Glycyrrhetinic acid is
dissolved in 1ml of methanol :
Chloroform (1:1)
• Stationary phase : Silica gel-G
• Mobile phase : Toluene: Ethyl acetate: Glacial
acetic acid (12.5:7.5:0.5)
• Detecting agent : 1% vanillin-sulphuric acid reagent
or Anisaldehyde-sulphuric acid and
heated for 10minutes at 110C
• RF Value : 0.41
• Colour spot : Purplish spot UV Blue fluorescence
under at 365 nm.

18. Glycyrrhetinic acid
Analysis by colorimetric method
• In the sample anisaldehyde and sulphuric acid is
added, which shows purple color.
• The intensity of color is measured in colorimeter at
556nm.
Utilization:
• It is used in Rheumatoid Arthritis, Inflammation and
Addisions disease, ulcer, expectorants
Storage condition:
• It should be store in well closed and air-tight
containers protected from light and in cool place.

19. Rutin

20. Introduction
Discovered in 1842 and it has been used in medicine to treat vascular disorders
related to capillary permeability and fragility.
The concentrations of rutin reported in grapes and buckwheat are the highest
among the other plant species.
Among the flavonoids, rutin belongs to the flavonols and it is the glycoside form
of quercetin.
Rutin has significant antioxidant, chelating, and antimicrobial properties, and
therefore many beneficial health effects.
Therefore, can be incorporated into functional foods or used in nutraceuticals
and medicinal products.
Antioxidant and anti-inflammatory effects of rutin have been extensively studied
in neurodegenerative disorders and brain pathologies in which rutin has
demonstrated a potential neuroprotective role.

21. Introduction
Rutin is also called vitamin P, which originally described the
group of flavonoids.
P stands for “permeability” (penetrability), because flavonoids
are able to contribute to reducing the permeability of blood
vessels.
There is not enough scientific data to provide a recommended
dose of rutin. The appropriate dose for you may depend on
factors including age, gender, and medical history.
The usual recommendation is 250 mg two times a day. Adults
using rutin to treat osteoarthritis may be advised to take 250
mg three times per day, or every 12 hours.

22. Rutin
Biological source
Rutin is a flavonoids glycosides obtained from the powered of dried food grains
of Fagopyrum esculentum (Buck wheat) .
Family: Polygonaceae. It is also obtained from various citrus fruit
It was first reported in Ruta graveolens L. (Common Rue)
Family : Rutaceae
Rutin has been found in the extracts obtained from the skin of
different grape (Vitis vinifera L.) varieties.
Family: Vitaceae

23. Rutin is also called rutoside, quercetin-3-O-rutinoside
and sophorin.It is a citrus flavonoid glycoside which is a
low molecular weight polyphenolic compound.
OCCURRENCE
Rutin is one of the phenolic compounds found in the
invasive plant species Carpobrotus edulis and
contribute to the anti bacterial and antioxidant
properties of the plant.
Its name comes from the name Ruta graveolens, a
plant that also contain rutin.
Rutin

24. Isolation: Rutin
Sufficient quantity of solid NaCl is added with stirring.
Sufficient quantity of 5% aqueous solution of borax is added (until resulting pH
is 7.5) with continuous stirring.
Residue is dissolved in sufficient quantity of 30% acetone, filtered and
evaporated the solvent to ¼ th of its original volume.
Then the marc is extracted with 78% of alcohol for 1 hour, filtered it and
evaporated to obtain dried residue.
Required quantity of Fagopyrum powder is defatted with n-hexane and filtered.

25. Isolation: Rutin
HCl is added to the mixture in hot condition to bring pH to 1.5, cooled
and kept in a refrigerator overnight. Crystal of rutin is separate out
Again mixture is filtered and evaporated the filtrate to 500°C to ¼ th of
its original volume.
Then the mixture is filtered and residue is washed with 20% NaCl
solution.
Mixture is filtered and acidify with phosphoric acid to bring pH to 5.5
and stirred for 15 minutes.

26. Isolation: Method-II
The filtrate was evaporated to dryness to give yellow powder rutin.
The un-dissolved part of the precipitate was dissolved in hot methanol and filtered.
The precipitate was filtered and washed with a mixture of chloroform : ethylacetate : ethanol
(50:25:25).
After extraction, the aqueous layer was collected and left to stand in a cold place for 72 hours;
A yellow precipitate separated out of the solution.
The extract was filtered and concentrated by evaporation under vacuum to about 10ml+ 25 ml
distilled water.
Extracted with petroleum ether (50ml x 3), then with chloroform (50 ml x 3).
Rutin extracted from 5 g of the powder in Soxhlet apparatus with 250 ml of 80% ethanol till
exhaustion.

27. Rutin
Properties: Appearance : Greenish yellow powder.
: It occurs as yellow crystalline powder.
Odor : Odorless
Taste :Tasteless
Solubility :Sparingly soluble in water but its solubility
increase in boiling water.
Soluble in methyl alcohol, isopropyl alcohol, pyridine and
solution of alkali hydroxides
On hydrolysis yield quervetin, rhamnose and glucose.
It decrease capillary fragility and are therefore employed in
cases of hypertension and radiation injuries.
Rutin is dextrotatory in neutral and acidic solvent and
levorotatory in alkaline solvents.

28. Rutin
Identification
by
chemical
test
Shinoda test: 3ml of ethanol and few drops of
sulphuric acid are added to the sample. To this 0.5g of
magnesium turnings is added. Pink colour is developed
which indicate the presence of flavonoids.
Lead acetate is added to the sample. Yellow coloured
precipitate is observed which indicate the presence of
flavonoids.
Ferric chloride is added to the sample. Dark green
colour is observed which indicate the presence of
flavonoids

29. Rutin
Analysis
by
TLC Standard sample :Rutin
Sample preparation :1mg of Rutin is dissolved 1ml
of methanol
Stationary phase :Silica gel –G
Mobile phase :Ethyl acetate: butanone: formic acid: water (50:30:10:10),
:Ethyl acetate: butanone: formic acid: water (100:10:11:27)
Detecting agent :Anisaldehyde sulphuric acid reagent
RF Value :0.43 (10% aqueous sodium chloride
solution)
Color spot :Yellow spot

30. Benefits & Uses

31. Role of Rutin Mechanism of action Main findings in studies
Antimicrobial
effects
• Inhibits DNA gyrase
• Prevents cytoplasmic
membrane function
• Inhibits energy
metabolism
• In combination of rutin with morin,
quercetin, kaempherol, myricetin and
fisetin against S. enteritidis, the
antibacterial action gets enhance,
significantly
Anti-arthritic
activity
• Inhibits nitric oxide
production from
macrophages and T-
cells proliferation
• The dual effects of rutin (e.g., anti-
arthritic and anti- candidal) is useful as
an all-in-one treatment for septic
arthritis.
Antioxidant
activity
• Neutralizes free
radicals
• Chelates transition
metals
• The antioxidant capacity of rutin is
depended on its concentration.
• The inhibitory effect of rutin on the
lipid peroxidation increases with an
increase in its concentration.
Anticancer
activity
• Inhibits cell
proliferation
• Removes reactive
oxygen species
• The tumor size and number of
papillomas significantly decreases by
rutin treatment.
• Level of lipid peroxidase reduces
significantly by rutin treatment

32. Role of
Rutin
Mechanism of action Main findings in studies
Anti-
inflammatory
activity
• Inhibits eicosanoid
biosynthesis, COX,
lipoxygenase and PLA2
activities
• The significant dose dependent
inhibitory effect of rutin is
there
Anti-diabetic
activity
• Enhances the release of
insulin from islets of
langerhans, decreases in the
expression of resistin,
increases in the expression
of PPARgene
• Decrease in the fasting plasma
glucose, as well as increase in
insulin levels in diabetic group
after a period of 45 days' oral
administration of rutin is there
Anti-allergic
activity
• Inhibits mast cell activation
through the inhibition of
Ca2+ influx, histamine,
leukotrienes and
prostaglandins release
• By rutin treatment, a
significant increase in mast
cells occurs.
• The reduction of ROS levels in
antigen ± IgE activated mast
cells occurs.
• Histamine released from mast
cells is also inhibited by rutin