Industrial production estimation and utilization of phytoconstituents

March 7, 2022 0 Comments

Industrial production estimation and utilization of phytoconstituents

Here we are covered with industrial production, estimation, and utilization of some phytoconstituents like Forskolin, Sennosides, Artemisinin, Diosgenin, Digoxin, Atropine, Podophyllotoxin, Caffeine, Taxol, Vinblastine, and Vincristine. we have covered drugs in detail with their synonym, biological source, family.



Structure of Forskolin

Industrial production:-

♦ Roots and bark powder extracted with toluene at 60°c for 2 hours.

♦ Filtrate collected and concentrated at a temperature not exceeding 40°c.

♦ Concentrated extract mixed with n-hexane, yields crude FORSKOLIN in the form of brown ppt.

♦ Purified using column chromatography.

Formula:- C22H34O7

Molecular weight :- 410.5 g/mole

Solubility:- Soluble in organic solvents such as ethanol, chloroform, and DMSO.

Melting point:- 230 – 232°c

Appearance:- White crystalline powder


  1. TLC Method

Mobile phase – Toluene: Ethyl acetate ( 8.5: 1.5 w/v )

Stationary phase – Silica gel F254

Visualizing agent – 5% vanillin in glacial acetic acid and 10% sulfuric acid in water.

2. HPLC Method

Column  – ODS

Mobile phase – Acetonitrile : H2o (50 : 60)

Wavelength – 220 nm

Flow rate – 1.6 ml/min

Injection volume – 20µl

3. HPTLC Method

Stationary phase – Absorbent silica gel 60 F254

Mobile phase – Benzene : Ethyl acetate ( 85:15 )

Solvent front – 9cm²

Detection – 550nm after spraying C Vanillin H2so4

Extraction and isolation

Utilization of forskolin

  1. Antidepressant
  2. Vasodilation
  3. Obesity
  4. Glaucoma
  5. Anti asthmatic


♦ Forskolin is a herbal supplement that can be used as a natural remedy for treatment in cancer, obesity, glaucoma, allergies and asthma, heart failure, intestinal spasms, painful cramps during period irritable bowel syndrome, urinary tract infection, high blood pressure ( hypertension ) chest pain (angina) difficulty falling asleep



Structure of sennosides

Industrial production

♦ Senna leaves are powdered to 20-40 mesh and loaded into verticle / continuous extraction. acetone at ambient temperature is circulated through the material to remove adherent impurities of pesticides and others acetone soluble unwanted material of no therapeutic value.

♦ It is then made free of acetone and extracted with 70% v/v alcohol (ethyl or methyl ) adjusted to pH 3.9 with citric acid at temperature 45 – 50°c.

♦ The extraction is continued till washing glycosides (color reaction or TLC) after extraction, the marc is de solvents and discarded. the extraction liquid is filtered and transferred to a tank fitted with a stirrer. the pH is adjusted to 6.0 – 6.2 with lime water. it is then concentrated to a paste of 65 – 70% total solids in a multiple-effect evaporator.

♦ The paste is dried in a rotary vacuum drier at a temperature of 50- 55°c. the flakes obtained are pulverized to a fine powder. it is then shifted to 80 mesh and packed preferably by vacuum sealing.


Sennosides may be estimated by a number of spectrophotometric, spectrofluorimetric, and chromatography. (HPLC), high-performance thin-layer chromatography (HPTLC ) methods.

♦ Column – C 18

♦ Mobile phase – 1% acetic acid in water: acetonitrile (82:18)

♦ Flow rate – 1 ml/ min

♦ Detection – 350 nm


♦ Sennosides are used as effective cathartic agents for a long time they are used to treat constipation.

♦ It is also used as a purgative, cathartic, laxative in acute constipation.

♦ They may also be used to clean out the intestines before a bowel examination/ surgery. they work by keeping water in the intestines, which causes movement of the intestines.

♦ It is used in haemohoridial, after the anorectal operation, anal fissure.



Structure of artemisinin

Physical properties-

Color – A white crystalline powder

Odor – odourless

Melting point – 156-157°C

Solubility – freely soluble in methanol, ethanol, very soluble in dichloromethane, and partially soluble in water.

Loss of drying- not more than 0.5%

Storage – Tightly closed container.

Isolation or production of artemisinin

Method -1

♦ The leaves are dried, coarsely powdered and extracted with petroleum ether (40-60°)

♦Filter the extract and concentrate, dried and dissolved in chloroform, and then add acetonitrile which precipitates the sugar and waxes present in the extract.

♦ Elute the concentrate (extract) on silica gel by using chloroform: ethyl acetate which yields the fraction of artemisinin.

♦ Crystalized by the fraction containing artemisinin by using cyclohexane or 50% ethanol.

♦ The highest yields are obtained from the levels just before flowering.

Method – 2

♦ 100 mg of fine powder was placed into an extraction thimble and extracted with 170ml of methanol via hot soxhlet extraction method for 6 hours over a water bath. the extract was evaporated in vacuo.

Method -3

♦ 100mg of fine powder was extracted under the influence of microwave energy using n-hexane, benzene, toluene, chloroform, methylene dichloride, ethyl acetate, acetone, acetone, methanol, and acetonitrile.

♦ Extraction parameters (160 watts, 120s,  10ml per extraction cycle, two extraction cycles, and cleanup with 2 ml of the corresponding solvent at the end of the second cycle of extraction) for microwave-assisted extraction (MAE) were the same for every solvent.

♦ The extract thus obtained was evaporated in vacuo.

Estimation of Artemisinin

TLC Densitometer

Dissolve extract of artemisinin in chloroform apply the spots of the sample and standard on the silica gel G plate solvent system petroleum ether: ethyl acetate (1:2) spray with p-dimethyl amino- benzaldehyde and heat at 80°c.

The spot of the sample and standard artemisinin can be quantitated by densitometry at 600m, the use of 2% solution of vanillin- Sulphuric acid produce color which is measured at 560nm.

HPLC Method

Equipment model – waters 501

Column– Waters C18

Detector – UV 260NM

Mobile phase– Phosphate buffer: methanol (6:4) pH -7.9

Flow rate – 1ml/min calibration curve was plotted with diff concentration.

Utilization of artemisinin

  1.  It is effective in treating malaria including cerebral malaria.
  2. Artemisinin and its analogs were shown to possess immunomodulatory and antitumor effects.
  3. The semisynthetic derivatives such as Artesunate, dihydroartemisinin, and arteether, etc have helped in deciphering the mechanism of action as an antimalarial, antitumor, anti-inflammatory, or immunosuppressive agent.
  4. It has remarkably strong activity against helicobacter pylori, the pathogen responsible for peptic ulcer diseases.



Structure of diosgenin

Extraction and isolation

Method 1

Method 2


Method 3


TLC method

Stationary phase – Absorbent precoated silica gel

Mobile phase – Benzyl: Ethyl acetate (85:15)

Sample- Rhizome extract

Detecting agent – 50% H2SO4 or anisaldehyde

RF value – 0.42

HPTLC Method

Standard preparation – Prepare a solution containing a known concentration of Diosgenis in CHCl3 i.e. concentration range of about 2-25mg.

Sample preparation – Dissolve the extracted residue in 5 ml of CHCl3 and make up the volume to 10 ml.

Solvent system – Toluene: Ethyl acetate (70:30)

Spray system – Libermann Burchard heat at 120°c

Detection – Violet or blue color spot.


  1. It is used as a precursor for the synthesis of steroids.
  2. T is first converted to 16 -dehydropregnolone acetate which acts as a precursor for steroidal syntheses like corticosteroids.
  3. It is used in the synthesis of sex hormones and oral contraceptives.
  4. It is used in the treatment of Rheumatism.



Structure of Digoxin

Geographical source –

It is cultivated and collected in England other parts of Europe, the United States, and India.

Industrial production

Method 1

Method 2


Method 3

Production media

  1. Production medium 1 (PM1) i.e. an 8% glucose solution with the pH adjusted to 5.5 was used as the production medium for all experiments run in the batch mode.
  2. For the production of digoxin under semi-continuous culture conditions a medium termed PM3 (16% GLUCOSE ) solution, pH 5.5 was used to replace part the GM2 at the beginning of the pre-incubation phase.
  3. Growth of cell suspension culture was added to 28 1 Gm2 in a 40-1 airlift bioreactor, which was used to produce the inoculum for a 300-1 bioreactor.


Calorimetric estimation

Digoxin extract is treated with 3.5 – nitro benzoic acid and benzyl trimethyl ammonium hydroxide

Bluish Red color is formed

Calorimetrically measured at 550nm

Assay of Digoxin

Test and standard solution

Weight about 40mg of test and standard digoxin, dissolve it in 95% of ethanol, and wake up the volume to 50ml pipette out +5 ml from the above stock solution and make up the volume to 100 ml with 95% ethanol.


Pipette out 5 ml from the above solution and add 3 ml of alkaline picric acid. allow to stand for 30 min and measure the absorbance at 495nm using as the blank a mixture of 5ml of ethanol (95%) and 3 ml of alkaline picric acid solution. calculate the content of digoxin from the absorbance obtained by repeating the operation using digoxin in the place of substance under examination.

% Assay = (sample Abs/ stand Abs) X (stand conc /sample conc) X 100

TLC method

Stationary phase – Absorbent pre-coated silica gel

Mobile phase – Benzene: Ethanol

Sample – plant extract (leaf)

Detecting agent – p- Anisaldehyde, perchloric acid

Observation – UV at 350nm blue spot was produced.


  1. Cardio-tonic
  2. Used in various forms like tablets or capsules in the treatment of congestive cardio failure, atrial failure, trial fibrillation, and paroxysmal atrial tachycardia
  3. Employed in the treatment of CHF.
  4. Duration in cardiac edema.


Structure of atropine


Industrial production-

♦ The regional research laboratory Jammu has developed a technique for the production of total phytoconstituents.

♦  Total alkaloid being extracted from belladonna leave at drug factory of C1MAP, Jammu.

Method 1

Method 2

♦ Atropine isolated from juice of powdered drug.

♦ The powdered drug material is thoroughly moistened with aqueous solution of sodium carbonate and extracted with ether or benzene and acidified solution of acetic acid.

♦ Acid solution is taken with solvent ether to remove coloring water.

♦ Alkaloids are precipitated with sodium carbonate filtrated off washed and dried.

♦ Dried mass is dissolved in solvent or acetone and dehydrated with anhydrous sodium sulfate before filtration.

♦ Filtrate after concentration and cooling yield & crystals of hyoscyamine and atropine from solution. this mass is dissolved in alcohol and mixed with hydrochloric solution and the mixture is allowed to stand until hyoscyamine is completely racemized to atropine.

♦ The crude atropine purified by crystallization from acetone.

♦ The crude atropine purified by crystallization from acetone.

♦ Atropine sulfate is the most important salt of atropine.

Method 3



Stationary phase – silica gel

Mobile phase – chloroform: methanol : water ( 30:15:12)

Detection– spray with 2 naphthol reagent and heat at 110°c (for 5 min )

Detecting agent – Dragendorff’s reagent


Stationary phase – C18

Mobile phase – A- 0.1% formic acid in water B- Acetonitrile

Gradient – 18% B to 22% B in 8 min: 22% B tp 45% B form 8-11 min : 045 B to 80% B from 17-23 min

Detection – (AO detector charged ) 205-215nm.





Marketed preparation

Atropine eye drops/ ointments

Assay of atropine

Weight accurately 0.5mg dissolved in 5 ml of alcohol previously neutralized to the solution of methyl red. add 20 ml of 0.1N HCl and titrate with excess acid (0.1N NaOH using methyl red indicator )

Each ml of 0.1N HCl = 0.02894 gm of atropine

Non-aqueous titration

Weigh accurately 0.5 gm dissolve in 30ml of anhydrous glacial acetic acid (CH3COOH) & titrate 0.1N HCl or determine the endpoint potentiometrically.




Industrial production

♦ Required quantity of rhizomes or roots of P. emodils taken with methanol, filtered and evaporated to a semisolid mass.

♦ Semisolid mass is dissolved into acidic water.

♦ Precipitate is formed which should be allowed for at least 2 hours.

♦ Filtered it and the filtrate is washed with cold water.

♦ The residue is collected, washed with acidified water, and dried to obtain a dark brown amorphous powder.

♦ Then the residue is extracted with hot alcohol, filtered, and evaporated to dryness.

♦ Finally residue is re-crystallize in benzene to yield podophyllotoxin

Identification by chemical test

♦ Sample drug is treated with 50% sulphuric acid it will show violet-blue color.

Analysis by TLC

Sample preparation -1 mg of podophyllotoxin is dissolved in 1ml of methanol.

standard sample – podophyllotoxin

stationary phase – silica gel- G

Mobile phase – Chloroform: methanol (90:10) for about 6cm (only glycosides are separated but aglycone-like podophyllotoxin resins in the region of the front.

The same plate is again eluted with more weakly polar solvent chloroform: acetone (65:35) up to 12 cm.

Detecting agent – spray with methanol sulphuric acid and heat 10 minutes at 110°c.

RF value– 0.65

Colour spot – Yellow spot

Analysis by HPLC – Isocratic

Stationary phase – C18 column

Mobile phase – methanol : water (6:4) at flow rate 0.8 ml/min

Detection– photodiode detector at 283 nm


Podophyllotoxin and its derivatives are used as a cathartic, purgative, antiviral agent



Industrial production

♦The powder tea leaves are extracted with boiling water and the aqueous extract is filtered while hot.

♦ The warm extract is treated with lead acetate to precipitate tannins and filter.

♦ The filtrate is treated with excess dilute sulphuric acid to precipitate lead in the form of lead sulfate.

♦ The filtrate is boiled with activated charcoal to remove coloring matter it any and filtered to remove the charcoal.

♦ The filtered decolorized solution is extracted with chloroform successively.

♦ Combined with chloroform extracts and evaporated on a water bath to yield caffeine (white powder)

♦ It is recrystallized with alcohol

Identification by chemical test

♦ Murexide test- sample is taken in a Petri dish to which hydrochloric acid and potassium chlorate are added and heated to dryness.

-A purple color is obtained by exposing the residue to the vapor of dilute ammonia, the purple color is lost on the addition of fixed alkali.

Analysis by TLC

Sample preparation – 1 mg of caffeine is dissolved in 1 ml of methanol or chloroform

standard sample– caffeine

stationary phase – silica gel G

mobile phase– Ethyl acetate : methanol: acetic acid (80:10:10)

Detecting agent– expose to vapors of iodine

RF value – 0.41

color spot – Brown spot

Analysis by HPLC

Method– Isocratic

Stationary phase– C18 column

Mobile phase – Methanol: water (25:75)

Detection– UV- residue detection 254nm.


♦ Caffeine is a CNS stimulant and Diuretic

♦ It is used in beverages.

Storage condition

♦ It should be stored in well-closed and airtight containers protected from light and in a cool place.



Yew, Himalaya yew

Biological source

Taxol is a natural diterpenoid. it is isolated from the bark of plant Taxus bravifolia, Taxus cuspidata, and Taxus Canadensis. these are the main important species.

♦ Taxus Bravifolia (pacific yew) mainly from stem bark. bark contains 0.01 – 0.02 % of taxol.

♦ Taxus cuspidata (Japanese yew ) mainly from leaves.

♦ Taxus Canadensis ( Canadian or American yew) from leaves & roots

Family – Taxaceae

Chemical constituents – All the part of Taxus brevifolia contains a wide range of diterpenoid derivatives termed taxanes.

♦More than 100 taxanes have been characterized from various species of Taxus of which taxol is a member of a small group of compounds possessing a four-membered oxetane ring. a complex ester side chain in the structures both are essential for anti-tumor activity & cytotoxic activity of taxol.

Industrial preparation

♦ The dried ground bark is extracted with methanol or ethanol & alcohol is removed by concentrating the combined extract.

♦ The obtained concentrate is re-extracted with dichloromethane & the solvent extract is concentrated to a powder.

♦ this powder is stirred with a mixture of acetone and ligroin (1:1) & filtered to remove the insoluble matter.

♦ The filtrate containing taxol is concentrated dissolved in 30% acetone is ligroin and applied to a column of the florist.

♦ The taxol fraction from the column is twice purified by crystallization.

♦ The crystalline taxol is subjected to chromatography on silica column the closely related analog, cephalomannine separate out from taxol.

♦The purified taxol obtained from the column is twice crystallized.

♦ The unseparated mixtures & mother liquors are recycled through the silica column to obtain move pure taxol.

Estimation- The presence of taxol in plant extract can be detected by two dimensional TLC method/ HPTLC using diphenyl modified silica gel plates. the eluting systems are hexane isopropanol acetone (15:2:3)0

♦ The other system uses a cyan modified silica gel plate with dichloromethane hexane acetic acid (3:10:1) and water methyl cyanide methanol THF (8:5:7:0:1) as an eluting system.

HPLC METHOD – Isocratic stationary phase-C18

Mobile phase – Methanol: Acetonitrile: water (25:35:40 by volume)

Detection by UV detector at 232nm


♦ Taxol is used clinically in the treatment f metastatic carcinoma of the ovary after failure of the first-line treatment or subsequent chemotherapy breast cancer & non-small cell using lung cancer.

♦ Taxon / Taxotere is a side chain analog of Taxol which is also been produced by semi-synthesis from 10- deacetyl baccatin III used for the treatment of breast cancer.

♦ Kaposi’s sarcoma forms patches of abnormal tissue growth under the skin in the nose in the mouth lining in the throat or other organs.




vinblastine & vincristine

Biological source –

Vinblastine & vincristine are two important indole alkaloids obtained from the plant Catharanthus roseus

Family – Apocynaceae

Chemical constituents – About 90 alkaloids have been isolated from Catharanthus alkaloids are present in the entire shrub but leave fruits to contain more alkaloids. the important alkaloids in Catharanthus are the dimer indole indoline alkaloids vinblastine & vincristine possess anti-cancer activity.

Extraction & isolation

Vinca leaves & herbs are minced & extracted with aqueous alcoholic acetic acid (CH3COOH) solution. after the concentration of the aqueous extract, the residue is further extracted with 2% HCl. NaOH is used to adjust the pH of the extract to 0.4. This is again extracted with benzene. the pH of the solution is then raised to 7 and again extracted with benzene. the pH 7 benzene extracted are mixed together concentrated dried and dissolved in benzene methylene chloride (65:35) solution. this is separated by chromatography on a neutral alumina column with benzene methylene chloride solution as an eluent using gradient elution technique. evaporation of the vinblastine-rich fractions gives vinblastine sulfate which is recrystallized from alcohol. further elution of the column gives vincristine which is dried & recrystallized from alcohol.

Estimation:- Vinblastine & vincristine sulfate are estimated with the help of the ‘HPLC’ method. the following solution is prepared for the estimation.

Solution 1 :- 0.1% w/v of substance being examine.

solution 2 ;- contain 0.2% w/v each vinblastin sulphate RS & vincristine Rs sulphate solution.

Solution 3,4 & 5 :- 0.1 % w/v 0.002 % w/v, 0.0001 w/v respectively of vinblastine sulphate. column packed with stationary phase.

Flow rate – 1.0 ml/min

Mobile phase ( for vinblastine ) mixture of 70 volume of methanol 30 volume of 15% w/v of diethylamine.

( pH adjusted 7.5 min phosphoric acid ) 12 volume of acetonitrile detection on wavelength ( for vincristine sulfate) 262nm.

Utilization :

♦ In the vinblastine sulfate is highly used in the treatment of neoplasm, lymphocytic lymphoma, Hodgkin’s disease, testicular carcinoma.

♦ Vincristine sulfate is given in intravenously in the treatment of leukoma of children.

♦ These alkaloids show antimitotic activity which inhibits cell growth.

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