5. Contents
• Synonym: Forskohlii, Placranthus barbalus
• B.S.: It is a labdane diterpene obtained from the roots
of Coleus forskohlii
• Family: Labiatae
• G.S.: tropical and subtropical regions of India,
Pakistan, Srilanka, Brazil
• In India, at high altitudes in Himalayas in the region
Garhwal, Kumaon and Nepal
• Commercially cultivated in Gujarat
Dr Payal Rahul Dande 6
6. Forskolin
• Was first discovered by Hoechst Research Centre, Mumbai & CDRI
Lucknow
• Other diterpene dervatives in plants are
• Coleon E & F
• Coleonol B, C,
• Deoxycoleonol
• barbatusin
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7. Forskolin: physical & chemical properties
• Molecular formula: C22H34O7
• Mol. Wt.: 410.51
• Melting point: 232- 235oC
• Appearance: white crystalline powder
• Solubility: Soluble in chloroform, benzene and DCM and Sparingly
soluble in petroleum ether
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8. Extraction of Forskolin
• Dried roots powder of Coleus forskohlii
• Extract with chloroform & ethyl acetate (1:10) using soxhlet apparatus
• Collect organic solvent, evaporate to dryness using rotary evaporator
• This yields a crude extract of foskolin
• Separation was done by column chromatography and by using
activated charcoal
• The residue obtained from the elute is purified and crystallized using
organic solvent
• Pure form of forskolin is obtained
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9. Identification test: TLC
• Sample : Dissolve 1mg forskolin in 1ml chloroform
• Stationary phase: Silica Gel G pre coated plates
• Mobile phase: Benzene: Ethyl acetate (85:15)
• Solvent front – 8 cm2
• Detecting agent: Anisaldehyde – H2SO4
• Rf: 0.25
• Color: Appearance of violet or purple spot
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10. Estimation by HPLC
• HPLC equipped with LC 8A pump and
• Photo diode Array detector with Lc10A software
• Column: ODS (Octadicylsilane) C18, (250X4.6 mm)
• Mobile phase : acetonitrile: water (50:50)
• Wavelength: 220 nm
• Flow rate: 1.6ml/min
• Injecting volume : 10 microlitre
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11. Estimation by HPLC
• Standard preparation: dissolve 10 mg forskolin in 15 ml acetonitrile
and make up volume up to 25 ml
• Sample preparation: dissolve 250 mg of sample extract in 25 ml
acetonitrile and make up volume up to 100 ml
• Procedure:
• Inject the 10 μl of standard and sample &
• record the chromatogram.
• Calculate the percentage of forskolin content from the peak areas
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12. Estimation: HPTLC
• HPTLC equipped with densitometer, applicator anddeveloping chamber
• Sample : Dissolve1mg forskolin in 1ml chloroform
• Stationary phase: Silica Gel 60 F254 pre coated plates
• Mobile phase: Benzene: Ethyl acetate(85:15)
• Solvent front – 8 cm2
• Detection – 550 nm after spraying with
• Detecting agent: Anisaldehyde– H2SO4 or vanillinH2SO4
• Rf: 0.25
• Color: Appearance of violet or purple spot
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13. Estimation by HPTLC
• Standard preparation: dissolve12.5 mg forskolin in 50 ml chloroformand make up volume
up to 100 ml
• Take 5 ml and dilute up to 100 ml again
• Sample preparation: dissolve50 mg of sample extract in 50 ml chloroformand make up
volumeup to 100 ml
• Take 5 ml and dilute up to 100 ml again
• Procedure:
• By means of syringeapply 5, 10, 15 and 20 μl of standard and sample of 4mm bandwidth
• Add mobile phase in the developingchamber and allow to saturate
• Allow to develop thechromatogram upto 8 cm in saturated chamber
• Remove,Dry and spray the plate with spraying agent
• Heat at 105oC-110oC
• Record the chromatogram
• Calculate the contentby extrapolation method with conc.On x axis and AUC on Y axis
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14. Uses
• Used in congestivecardiomyopathy,glaucoma and asthma
• Forskolin has vasodilatorand cardiac stimulant action
• Used as hypotensive and spasmolytic agent
• Forskolin works on muscles in the heart and in the walls of the blood vessels.
• It produces a more powerful heartbeat and widening of the blood vessels,which
lowers blood pressure.
• Forskolin works by boosting the levels of a compoundcalled cyclicAMP. This helps
relax the muscles around the bronchial tubes to make breathing easier.
• Forskolin stimulates adenylate cyclase activity lowers the intraocular pressure by
reducing the aqueous humor inflow and thus used in glaucoma
• In Ayurveda, used in treatment of heart disease and abdominal colic pain
• Also used for its platelet aggregation inhibitoractivity
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16. Sennosides
• Sennosides are the anthraquinone glycosides obtained from
dried leaflets and pods of
• Indian species c/as Tinnevelley senna (Cassia angustifolia) and
• European species c/as Alexandrian senna (Cassia acutifolia)
• Contain NLT 1-2% of anthraquinone glycosides calculated as
sennosides a & b.
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17. Indian Senna
• Common name: Tinevelley senna, Cassia senna, Sennae
folium
• BS: dried leaflets and pods of Cassia angustifolia or
Cassia senna Vahl
• Family: Leguminosae
• NLT 1-2% anthracene derivatives calculated as
sennosides A & B
• Generally 2.5% of anthraquinone glycosidescalculated
as sennosides B
• G.S.: native to Tamilnadu (Tinevelley,Ramnathpuram
and Madurai) and grown in AP, Gujarat & Rajasthan
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18. Alexandrian Senna
• Synonyms: Folia sennae alexandrinae, Cassia senna, Egyptian senna
• B.S.: it consist of dried leaflet of Cassia acutifolia Delile
• Family: Leguminosae
• G.S.: Middle & Upper Nile territories, Tropical Africa & specially
Sudan.
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19. Extraction of sennosides
• Take dried leaf powder of senna and extract with 70% methanol using
soxhlet apparatus
• Filter and concentrate the filtrate to 1/8th volume using rotary
evaporator
• Adjust pH to 3 with HCl, filter
• Neutralize it with ammonia
• Extract the filtrate with chloroform
• Centrifuge and get crude extract of sennosides
• Purify using column chromatography
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22. Chemistry
• Anthraquinone glycosides (o- glycosides)
• Dimeric sennosides A & B
• On hydrolysis produce aglycone sennidin A & B
• Sennidin A is dextrorotatory and B is mesoform
• Molecular formula – C42H38O20
• Melting point- 220-240oC
• Molecular weight 862.7g/mol
• Solubility: sennedin is soluble in chloroform, benzene, DCM and pet. Ether
• Positive test for borntrager’s reagent
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24. Identification test: TLC
• Sample : Dissolve 1mg sennosides in 1ml chloroform
• Stationary phase: Silica Gel G pre coated plates
• Mobile phase: n-propanol: ethyl acetate: water: GAA(3:3:2:1)
n-propanol: ethyl acetate: water (5:3:2)
• Detecting agent: nitric acid and KOH reagent
• Rf: A-0.4, B-0.2, C-0.7 & D-0.55
• Color: Appearance of brown spot in visible light
• Lemon Yellow or Light blue in UV 365 nm
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25. Estimation of sennosides A & B by HPLC
• A conventional RP C18 column (4.6 mm × 150 mm), was used as
stationary phase and
• Mobile phase: Acetonitrile, water, phosphoric acid (200:800:1 V/V/V)
• Flow rate was 1.2 mL/min,
• the column temperature 40 °C,
• the detection wavelength 380 nm, and
• the injection volume 20 μL.
• the chromatogram shows 2 peaks due to sennoside A/B at Rt-14 min.
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26. uses
• Purgative in habitual constipation
• Causes reduction in water reabsorption by body
• Results in Soft & bulky faeces
• 40-60% activity is due to sennoside A & B
• The glycoside part helps in transport up to large intestine and protect
it to get oxidized in lesser inactive anthraquinone form
• I.V. is given in emergency to directly act in colon
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28. Diosgenin
Is a steroidal sapogenin obtained from saponin
glycosides c/as Dioscin from dried tubers of
various species of Dioscorea which is
commonly known as
Common name: yam, rheumatism root
B.S: dried tuber of the plant Dioscorea
deltoidea, D.composita, D.belophylla
Family: Dioscoreaceae
G.S.: USA & Mexico, North western Himalaya,
Kashmir, Punjab, Nepal & China at an altitude
of 1000-3000m Dr Payal Rahul Dande 29
29. Chemical Class & Chemical constituents
– 75 % of starch (non edible because of bitter taste)
– Steroidal Saponin Glycosides: dioscin
– Dioscin on hydrolysis produce Diosgenin: steroidal
sapogenin 4 to 6%
– Others sapogenins are Smilagenine &
epismilagenine, beta yamogenin
– Enzyme sapogenease
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30. Extraction & Isolation of Diosgenin
• Tubers are washed, sliced and dried
• Extracted with hot water or 95% ethanol for several hours
• Extract is filtered, concentrated under vacuum
• Glycosides are precipitated with solvent ether or by lead acetate
• hydrolyse with 2N HCL &
• extract with petroleum ether (40-60˚C)
• to get Diosgenin
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31. Chemistry of diosgenin
• Is a steroidal sapogenin
• Molecular formula: C27H42O3
• Mol. Wt.: 414.62 g/mol
• Melting point: 204oC – 207oC
• Appearance: crystalline
• Soluble in organic solvent and acetic acid
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33. Estimation of Diosgenin by Colorimetric method:
• Diosgenin is mixed with a solution of antimony trichloride in a
mixture of nitrobenzene and methanol.
• Heating is necessary to develop color for about 20 minutes at 60˚C.
• Absorbance is measured between 400-600nm against blank
• Maximum absorbance is at 486nm
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34. Estimation of Diosgenin by HPTLCmethod:
• Standard Preparation: Prepare a solution containing known
concentration of diosgenin in CHCl3.i.e., concentration range of about
2-25mg.
• Sample preparation:
• Dissolve the extracted residue in 5ml of CHCl3 & make up the volume
up to 10ml.
• Solvent system: Toluene : Ethyl acetate (70:30)
• Spray system: Liebermann Burchard reagent and heat at 120˚C.
• Detection: Violet or blue color spot.
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35. Diosgenin Utilization
• Diosgenin is used as Precursor for synthesis of several
corticosteroids, sex-hormones & oral contraceptives
• Treatment of rheumatic arthritis and natural hormone
replacement therapy
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37. Digoxin
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• Synonym: Foxglove Leaves, digitalis.
• Biological source: Digoxin is a cardiac
glycoside obtained from the dried leaves
of Digitalis purpurea and Digitalis lanata.
• Family: Scrophulariaceae
• Geographical source: Plants are cultivated
and collected in England, other parts of
Europe, US and India.
• In India it is cultivated in Kashmir and
Nilgiris.
38. Digitalis : Known as “foxglove”
Allied drugs
• Digitalis purpurea
• Digitalis lanata
• Digitalis lutea
• Digitalis thapsi
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39. Chemical constituents
• 0.2 -0.45% of primary & secondary cardiac glycoside
• Purpurea glycoside A & B and glucogitaloxin (primary)
• Digitoxin, gitoxin, gitaloxin (secondary)
• Lanatoside A,B,C & digoxin (D.lanata)
• Odoroside H, gitaloxin, verodoxin & glucoverodoxin
• Primary glycosides are less absorbed & less stable than secondary glycosides
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43. Identification tests:
Keller-killani test is employed for digitoxose
• Boil 1 gm drug powder with 10 ml of 70% alcohol for 2-3 min
• Filter extract, add 5 ml water & 0.5 ml lead acetate solution to
filtrate
• Separate filtrate, treat with chloroform & evaporate to get extract
• Take 2ml extract, Dissolve it in GAA, cool & add 2 drops of
ferric chloride
• Add it to 2 ml of conc. H2SO4 (sulphuric acid)
• Reddish brown layer is observed at junction which get bluish
green on standing
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44. Identification test
• Legal test: Aqueous or alcoholic extract is dissolved in 1 ml of
pyridine and sodium nitroprusside & made alkaline, red or pink
color is observed
• Baljet test: to section of leaf, add sodium picrate, shows yellow to
orange color
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45. Identification test TLC method
• Stationary Phase: Absorbent-Pre-coated Silica gel G
• Mobile Phase: Benzene: Ethanol (7:3)
• Sample: Plant extract
• Detecting agent: P-Anisaldehyde, perchloric acid
• Observation: UV 350nm, Blue spot was produced
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46. Estimation of digoxin by colorimeter
• Digoxin extract is treated with 3,5 Dinitro benzoic acid & Benzyl
Trimethyl Ammonium Hydroxide
• Bluish Red color is formed
• Calorimetrically measured at 550nm
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47. Estimation of digoxin by Assay
• Weigh about 40mg of Digoxin, dissolved it in 95% of Ethanol and
make up volume to 50ml
• Pipette out 5ml from the above stock solution and make up the
volume to 100ml with 95% ethanol.
• Again pipette out 5ml from above solution and add 3ml of Alkaline
picric acid
• Allow to stand for 30 minutes.
• Measure the absorbance at 495nm.
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48. Digoxin - Uses
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• Uses:
• Treatment of congestive cardiac failure
• Slows ventricular rate in atrial fibrillation, atrial flutter,
supraventricular tachycardia
• MOA: Blocks sodium potassiumATP ase pump of cardiac muscle
• Helps in forceful contraction of myocardium
• Greater output per beat
• Complete emptying of heart
• Negative chronotropiceffect: Slowing ventricular rate in atrial fibrillationatrial flutter,
superaventricular tachycardia
• Side effect: Has cumulative effect
• Slow elimination from body
• Given by oral & iv route
• Has to be prepared from natural drug only