Froskolin a labdane diterpene, is the principal bioactive

compound found in roots of Coleus froskohlii.

It contains a range of pharmacological activities, including

anti-inflammatory, antioxidant, antimicrobial, and antihypertensive

effects.

Extraction method
Plant Material Preparation

Drying: Air-dry the root part of coleus froskohlii in the shade to

remove moisture.

Pulverization: Grind the dried plant material into a fine powder using
a mechanical grinder.

Sieving: Sieve the powder to a uniform particle size (e.g., 60-80

mesh) to ensure consistent extraction efficiency.

Extraction Protocols

This method serves as a baseline for comparison with modern

techniques.

Place 10 g of powdered plant material into a cellulose thimble.

Insert the thimble into the main chamber of the Soxhlet extractor.

Fill a round-bottom flask with 500 mL of ethanol.

Soxhlet method
Assemble the Soxhlet apparatus and heat the solvent to its boiling
point (approximately 80°C for ethanol).

Allow the extraction to proceed for a sufficient duration (e.g., until

the solvent in the siphon tube runs clear), typically several hours.

Concentrate the resulting extract using a rotary evaporator under

reduced pressure to obtain the crude extract.

A green and efficient alternative to conventional methods.

Place a specified amount of powdered plant material (e.g., 10 g) into

the extraction vessel. (10 g of crude plant part in 100 ml of solvent)
in the ratio of 1: 10 to 1:15 w/v.

Add the solvent, ethanol:water (90:10 v/v), at an optimized solvent-

to-drug ratio of 32 mL/g for 6-8 hours at 80*c temperature.

After extraction, filter the mixture to separate the extract from the
plant residue.

Concentrate the filtrate using a rotary evaporator.

This method utilizes ultrasonic waves to enhance extraction

efficiency.

Combine the powdered plant material with the extraction solvent

(e.g., ethanol:water).

Place the vessel in an ultrasonic bath or use an ultrasonic probe.

Apply ultrasound power of 467 W for a duration of 47 minutes.

Maintain a constant temperature if required by the specific protocol.

Filter the extract and concentrate it using a rotary evaporator.

After collecting the extract. it is stored in a well closed container and
covered with an aluminium foil and stored in the refrigerator. The
extract is diluted with the solvent and administered to animals or
further isolation process by using various separation techniques like
chromatography.
Screening methods
Forskolin belongs to the chemical class of labdane diterpenoids
(specifically, it is classified as a bicyclic labdane diterpene).

A) Detection of diterpenoids:
[Link] Copper Acetate Test

This is the most direct qualitative test specifically used to indicate the
presence of diterpenes/diterpenoids in a plant or organic extract.

 Procedure: Dissolve the crude extract in distilled water or an

appropriate solvent. Add 3–4 drops of a copper acetate
solution.

 Observation: The formation of an emerald green color

indicates a positive result.

2. Salkowski's Test

While widely used for the general screening of all terpenoids and
phytosterols, Salkowski's test is standard in phytochemical screening
to detect the presence of the basic terpene skeleton.

 Procedure: Mix 5 mL of the extract with 2 mL of chloroform.

Carefully add 3 mL of concentrated sulfuric acid along the side
of the test tube to form a distinct layer.

 Observation: A reddish-brown or golden-yellow color interface

develops at the boundary of the two liquids, confirming the
presence of terpenoids.
B) Detection of Alkaloids:
A small quantity of extract is treated with few drops of dilute HCl
and filtered. The filtrate was used for the following tests.

A) Mayer’s test

B) Dragendroff’s test

C) Wagner’s test

D) Hager’s test

A) Mayer’s test: Filtrate was treated with Mayer’s reagent. Cream

colour precipitate was produced.

B) Dragendroff’s test: The filtrate was treated with Dragendroff’s

reagent. Orange brown precipitate was obtained.

C) Wagner’s test: The filtrate was treated with Wagner’s reagent.

Reddish brown precipitate was produced.

D) Hager’s test: The filtrate was treated with Hager’s reagent.

Yellow colour precipitate was obtained.

Shinoda test:

The extract was treated with one gram Magnesium turnings and few
drops of concentrated HCl and boiled for 5 minutes. Formation of
orange colour shows presence of flavonoids. Small portion of each is
dissolved in alkali, yellow colour was produced. White precipitate
was produced.
C)Test for Cardiac Glycosides:
A) Keller Killani test

B) Legal’s test

C) Baljet test

A) Keller Killani test:  The Extract was dissolved in 3ml of glacial

acetic acid.  To this about 2 drops of ferric chloride were added. 
The contents are then transferred to a test tube containing 2 ml of
concentrated H2SO4. Reddish colour was formed at the junction of
two layers.

B) Legal’s test: To the extract add 1 ml of sodium nitroprusside and

sodium hydroxide. Colour changes from pink to red colour.

C) Baljet test: To the extract add 1 ml of sodium picrate. A colour

change from yellow to orange was observed.

E) Test for Anthraquinone Glycosides:

A) Borntrager’s test: About 1 ml extract was boiled with dilute
sulphuric acid and filtered. The filtrate was extracted with
chloroform. The chloroform layer was separated and equal quantity
of dilute ammonia was added. Formation of pink or red colour in
organic layer indicates presence of glycosides.

B) A little amount of solution was treated with 5N Sodium

hyposulphite. Appearance of red colour indicates the presence of
glycosides.
F) Detection of Carbohydrates:
A minimum of various extracts Detection of Carbohydrates: A
minimum of various extracts was taken and dissolved in respective
solvents and treated with HCl to detect the presence of
Carbohydrates by following tests.

A) Molisch’s test

B) Fehling’s test

C) Benedict’s test

D) Barfoed’s test

E) Seliwanoff’s test

A) Molisch’s test: The extraction solution was treated with 2-3 drops
of alcoholic α -naphthol. Along the sides of test tubes, the
concentrated H2SO4 (2 ml) was added. If the violet colour appears,
thisindicates the presence of Carbohydrates.

B) Fehling’s test: The 2 ml of extraction solution was treated with

Fehling’s [Link] it was heat on a water [Link] the reddish
orange precipitate produced, this indicates the presence of
carbohydrates.

D) Barfoed’s test: 2 ml of solution was treated with Barfoed’s reagent

and boiled on water bath. Formation of red colour indicates the
presence of carbohydrates.
E) Seliwanoff’s test: 2ml solution was treated with few ml of
seliwanoff’s reagent and boiled on water bath. Red colour was
formed.

G) Detection of Proteins and Amino acids:

A) Biuret test

B) Millon’s test

C) Ninhydrin’s test

A) Biuret test: 1ml solution was treated with equal volume of 5%

sodium hydroxide and 1% copper sulphate solution. A violet colour
was produced.

B) Millon’s test: 1ml solution was treated with Millon’s reagent and
heated. A white colour changes into red colour upon heating
indicates presence of amino acids.

C) Ninhydrin’s test: 1 ml of extraction solution was treated with

ninhydrine reagent and then heated. The Purple colour was
produced.

Detection of Phytosterols: A small quantity of extract was

dissolved in 5ml chloroform. The solution is subjected to tests like

A) Salkowski’s test

B) Libermann-buchards test

C) Zark’s test

A) Salkowski’s test: To 1ml solution few drops of concentrated

sulphuric acid is added. Brown colour was produced.
B) Libermann-Buchards test: The prepared chloroform solution was
treated with 2 drops of concentrated sulphuric acid followed by 3
drops of acetic anhydride. Emerald green colour formed.

C) Zark’s test: To 1 ml solution mixture of glacial acetic acid, ferric

chloride and concentrated sulphuric acid was added. Purple colour is
produced.

Quantitative characterization through High Performance

Liquid Chromatography (HPLC)
HPLC method found to be more rapid and less sensitive than GLC
(Gas liquid chromatography). It is used to monitor variation in
forskolin content. Reversed-phase liquid chromatography with a
photo diode array detector at 210 nm was successful for the
qualitative and quantitative evaluation of forskolin in plant material.
HPLC-ELSD finger print method was used for quality control of Coleus
forskohlii was suggested. For quantitative analysis, in HPLC,
temperature was held constant at 300C or room temperature and
flow rate employed for analysis was 1.0 ml/min. The analysis of
sample completed within 30 min. The HPLC chromatogram of
ethanol in vitro and in vivo root extract of Coleus forskohlii showed
12.498 ± 0.3 min. A stock solution of forskolin with a concentration
of 1 mg/ml was prepared in benzene and further diluted with the
solvent and final concentration was made up to 100 mg/ml. The
calibration curve was linear over the concentration range between 5
- 25 μg/ml. The standard curve plotted between concentrations
against area. In the ethanol in vitro root extract maximum forskolin a
major chemical compound was identified with respect to in vivo
ethanol root extract. Identification of chemical constituents was
based on the peak areas which represent the percentage of forskolin
compound. The HPLC chromatogram exhibited 0.54% quantity of
forskolin in in vitro ethanol root extract and 0.48% of forskolin in in
vivo ethanol root extract. The present data showed that percentage
of estimation of in vitro ethanol root extract is higher as compare in
vivo ethanol root extract. Similar report of forskolin quantification by
reverse phase liquid chromatography was showed. Differentiation
capacity of in vitro ethanol root extract was found to be greater
when compared to in vivo ethanol root extracts.

In Vitro Culture of Entamoeba histolytica

The axenic cultivation of E. histolytica, meaning growth in the
absence of any other metabolizing cells, is fundamental for studying
its biology and for drug testing. The most widely used medium for
this purpose is TYI-S-33.

Protocol 1: Preparation of TYI-S-33 Medium

This protocol details the preparation of the complete TYI-S-33
medium for the axenic culture of E. histolytica trophozoites.

Materials:
Casein Digest Peptone

Yeast Extract

Glucose

Sodium Chloride (NaCl)

Potassium Phosphate, dibasic (K₂HPO₄)

Potassium Phosphate, monobasic (KH₂PO₄)

L-Cysteine Hydrochloride

Ascorbic Acid

Ferric Ammonium Citrate

Adult Bovine Serum (heat-inactivated)

Vitamin-Tween 80 Solution

Deionized or glass-distilled water

1N Sodium Hydroxide (NaOH)

Autoclave

Sterile screw-capped borosilicate glass culture tubes (16 x 125 mm)

Sterile filter (0.22 µm)

Procedure
Preparation of TYI Broth Base:

In 870 ml of deionized or glass-distilled water, dissolve the following

components in order:

Casein Digest Peptone: 20.0 g

Yeast Extract: 10.0 g

Glucose: 10.0 g

Sodium Chloride: 2.0 g

Potassium Phosphate, dibasic: 1.0 g

Potassium Phosphate, monobasic: 0.6 g

L-Cysteine Hydrochloride: 1.0 g

Ascorbic Acid: 0.2 g

Ferric Ammonium Citrate: 22.8 mg

Adjust the pH of the solution to 6.8 with 1N NaOH.

Bring the final volume to 1000 ml with deionized water.

Dispense the broth into glass bottles and autoclave at 121°C for 15
minutes.

The sterile TYI broth base can be stored at -20°C for several months.

Preparation of Complete TYI-S-33 Medium:

To prepare 100 ml of complete medium, aseptically combine:

87 ml of sterile TYI broth base10 ml of heat-inactivated adult bovine

serum. (Note: Fetal bovine serum is not suitable

as fetuin can be toxic to the amoebae).

3 ml of sterile Vitamin-Tween 80 solution.

The complete medium should be used within 7-10 days and stored at
4°C in the dark.

Dispensing the Medium:

Aseptically dispense approximately 13 ml of the complete TYI-S-33

medium into 16 x 125

mm sterile screw-capped borosilicate glass culture tubes.

Protocol 2: Culturing and Maintenance of E. histolytica
Trophozoites
Procedure:

Initiation of Culture:

Thaw cryopreserved E. histolytica trophozoites rapidly in a 37°C

water bath.

Aseptically transfer the thawed amoebae to a tube containing pre-

warmed complete TYIS-33 medium.

Incubate the culture tubes horizontally at a slight angle at 37°C.

Subculturing:

Examine the cultures microscopically for trophozoite growth and

confluence. Cultures are typically ready for subculture every 48-72
hours.

To detach the adherent trophozoites, chill the culture tubes in an ice

bath for 5-10 minutes.

Invert the tube several times to dislodge the amoebae.

Aseptically transfer an appropriate volume of the cell suspension

(e.g., 1 ml) to a new tube containing fresh, pre-warmed medium. The
transfer volume should be adjusted to achieve a confluent culture in
48-72 hours.
In Vitro Amoebicidal Susceptibility Testing: Nitroblue
Tetrazolium (NBT) Reduction Assay
The NBT reduction assay is a colorimetric method used to determine
the viability of E.

histolytica trophozoites after exposure to a drug. Viable amoebae

reduce the yellow NBT dye to

a blue formazan product, which can be quantified

spectrophotometrically.

a. Preparation of Trophozoites

Harvest trophozoites from a 48 - 72hour culture by chilling the

culture tubes.

Centrifuge the cell suspension to pellet the amoebae.

Wash the pellet with pre-warmed, incomplete culture medium.

Resuspend the trophozoites in fresh medium and determine the cell

concentration using a hemo-cytometer. Adjust the concentration to
the desired level (e.g., 1 x 10^5 trophozoites/mL).

b. Assay Procedure:

In a 96-well microtiter plate, add the test compound in serial

dilutions to the appropriate wells. Include a drug-free control
(negative control) and a positive control (e.g., forskolin crude
extract).

Add the prepared trophozoite suspension to each well. Incubate the

plate at 37°C for the desired time period (e.g., 48 or 72 hours).
Following incubation, add the NBT solution to each well and incubate
for a further 2-3 hours at 37°C.

Stop the reaction by adding a stopping agent (e.g., DMSO or HCl).

Read the absorbance of each well using a microplate reader at a

specific wavelength (e.g., 570 nm).

c. Data Analysis:

The percentage of inhibition is calculated for each drug

concentration relative to the drugfree control.

The IC50 value (the concentration of the drug that inhibits 50% of
the amoebic growth) is determined by plotting the percentage of
inhibition against the drug concentration and fitting the data to a
dose-response curve.

[3H] Thymidine Incorporation Assay

This method measures the proliferation of trophozoites by
quantifying the incorporation of radio labelled thymidine into newly
synthesized DNA.

Protocol:

Assay Setup: Prepare the 96-well microtiter plate with trophozoites

and serial dilutions of the test drug as described in the quantitative
parasite counting method.

Incubation: Incubate the plate at 37°C for 24-48 hours in a

microaerophilic environment.

Radiolabelling:

Add a specific amount of [3H] thymidine (e.g., 0.25 µCi) to each well.
 Further Incubation: Incubate the plate for an additional 18-24 hours
to allow for the incorporation of the radiolabel.

Harvesting: Harvest the cells onto glass fibre filters using a cell
harvester. Wash the filters with phosphate-buffered saline (PBS) to
remove unincorporated [3H] thymidine.

Scintillation Counting: Place the filters in scintillation vials with a

suitable scintillation cocktail. Measure the radioactivity (counts per
minute, CPM) using a liquid scintillation counter.

Data Analysis: The reduction in CPM in drug-treated wells compared

to the control wells indicates inhibition of proliferation. Calculate the
IC₅₀ value as described for the quantitative parasite counting
method.