Analytical Profile of Coleus Forskohlii | Forskolin .pdf

SWAPNIL THERKAR
ANALYTICAL PROFILE OF COLEUS FORSKOHLII 24-04-2024
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Government College of Pharmacy, Amravati
1) Introduction: Plant Profile
Synonyms: Plectranthus barbatus, Coleus Barbatus, Forskohlii.
1. Sanskrit: Mayani, Makandi24-Apr-24
2. Hindi: Patharchur, Pashanabhedi
3. Marathi: Mainmul, Garamar
Biological Source:
It consists of roots of Coleus forskohlii belonging to family the Labiatae
(Lamiaceae) and should contain not less than 0.4 per cent of forskolin on dried basis.
Chemical Constituents:
It contains the diterpene Forskolin about 0.6 per cent (coleonol) which was
discovered at the Hoechst Research Centre, Mumbai, India and Central Drug Research
Institute (CDRI), Lucknow, India. It also contains various diterpenoid derivatives. The
leaves give coleon E (methylenequinone), barbatusin and coleon F. The roots contain
coleonol B, coleonol C, deoxycoleonol and labdane diterpenoids (I, II and III) (Kokate et
al.,2008, P. 14.98-14.99).
Facts:
▪ Coleus forskohlii is the only source of forskolin & one of the most potential medicinal
crops of the future, as its pharmacological properties have been discovered recently.
▪ Forskohlii almost have traces of forskolin, the roots are the main source possessing 0.1
to 0.5 per cent and preferred for its extraction.
▪ The pharmaceutical industries are mainly dependent upon the wild population of the
plant for the supply of tuberous roots for forskolin extraction.
▪ In India, the major medicinal species of Coleus are the tuberous C. forskohlii., C.
amboinicus, C. blumei, C. malabaricus and C. scutellaroides are other species
▪ The forskolin is biosynthesized from acetate- mevalonate pathway [slideshare, n.d.].

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2) Forskolin
A Forskolin is a labdane diterpenoid and considered as the active secondary metabolite of
Coleus forskohlii.
Labdane diterpenoids are bicyclic structures, with decalin as the parent nucleus,
including its demethylation, cleavage and rearrangement derivatives, which could be
considered as precursor of higher diterpenoids. Diterpenes or diterpenoid are C20
compounds, derived from four isoprene units, and in contrast to mono or sesquiterpenes,
are non-volatile in nature. These Co-natural compounds are biosynthetically prepared from
geranyl pyrophosphate. The forskolin is biosynthesized from acetate- mevalonate pathway
(Kokate et al.,2008, P.14.98-14.99).

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3) Uses
1. Heart disorder
Forskolin has a positive inotropic action on cardiac tissue via increased cAMP levels.
Detailed pharmacological studies established that forskolin lowered normal or elevated
blood pressure in different animal species through a vasodilatory effect and it had a positive
inotropic action on the heart muscle.
2. Glaucoma
The effect of forskolin on aqueous humor dynamics and intraocular pressure was first
described by Capriole and Sears (1983). The topical application of forskolin lowered the
intraocular pressure in rabbits, monkeys and healthy human volunteers and it was
associated with a reduction in aqueous inflow and no change in outflow facility indicating
the potential of forskolin as a therapeutic agent in the treatment of glaucoma. However,
Lee et al. reported that forskolin had no lasting effect on intraocular pressure in monkeys
with glaucoma. It also showed no effect on humans in reducing aqueous flow when applied
topically to the eye.
3. Asthma
Forskolin was studied as bronchodilator for its potential use in the treatment of asthma. It
blocked bronchospasm, the chief characteristic of asthma and bronchitis in guinea pigs
caused by histamine and leukotriene C-4 (Kreutner et al., 1985). In human basophils and
mast cells, forskolin blocked the release of histamine and leukotriene C-4 (Marone et al.,
1987). A study involving human revealed that inhaled forskolin powder formulations were
capable of causing bronchodilation in asthma patients (Bauer et al., 1993). Forskolin seems
to be a promising drug if used in an appropriate dosage for treatment of patients with
congestive heart failure, glaucoma and asthma

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4. Antithrombotic effect
Forskolin inhibits platelet aggregation through adenylate cyclase stimulation, augmenting
the effects of prostaglandins (Siegl et al., 1982; Adnot et al., 1982). Its antithrombotic
properties may be enhanced by cerebral vasodilation and it was observed in rabbits. This
vasodilation was not potentiated by adenosine (Wysham et al., 1986). The use of crude C.
forskohlii extract as a rational phototherapeutics antithrombotic has been proposed.
5. Anti-obesity
Henderson et al. (2005) suggested that C. forskohlii does not appear to promote weight loss
but may help mitigate weight gain in overweight females with apparently no clinically
significant side effects. The antiobesity effects of C. forskohlii were investigated in
ovariectomized rats (Han et al., 2005) and the administration of C. forskohlii extracts
reduced body weight, food intake and fat accumulation in those rats suggesting that C.
forskohlii may be useful in the treatment of obesity.
6. Dryness of eyes
Early research shows that taking a supplement containing coleus extract and other
ingredients for 30 days moderately decreases dry eye symptoms. It’s unclear if this benefit
is due to coleus, other ingredients, or the combination (Kavitha et al., 2010 P. 278-285).

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4) Physico-chemical Properties
Forskolin is relatively a nonpolar compound, thus sparingly soluble in water. However, it
is also insoluble in petroleum ether and xylene. Thus, it is soluble organic in solvents like
toluene, chloroform, ethanol, benzene and methanol.
Forskolin is supplied as a crystalline solid. Forskolin is soluble in organic solvents such as
ethanol, DMSO, and dimethyl formamide (DMF). The solubility of forskolin in ethanol is
approximately 15 mg/ml and approximately 30 mg/ml in DMSO and DMF. Forskolin is
sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, forskolin
should first be dissolved in DMSO and then diluted with the aqueous buffer of choice.
Forskolin has a solubility of approximately 0.1 mg/ml in a 1:10 solution of DMSO: PBS
(pH 7.2) using this method.
▪ Color: White Crystalline powder
▪ Molecular formula: C22H34O7
▪ Molecular weight: 410.5 g/ mol
▪ Solubility: It is non-polar in nature. Soluble in acetone, chloroform, benzene and
sparingly soluble in Pet. ether and water
▪ Melting Point: 232-235 C
▪ Boiling Point: approximately 519.9°C at a pressure of 760 mmHg. (Cayman
Chemical, n.d.)

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5) Analytical Profile
1. TLC (Thin Layer Chromatography)
TLC method is used to monitor the presence of
forskolin during the isolation process. However,
it can also be used to determine the quantity of
forskolin present in the crude extract as well as
in formulations. Inamdar et al. described a TLC
method for the quantification of forskolin in
pharmaceutical preparations. In this method,
forskolin was detected by using vanillin in
acetic acid and perchloric acid as the detection
agent. Forskolin gave violet color spot and the
intensity of violet color was quantitatively
estimated.
The method was developed on TLC aluminium plates pre-coated with SILICA GEL 60F-
254 using solvent system benzene: methanol (9:1 v/v).
The detection wavelength was 210 nm, and the injection volume was 5 μL. (Rf value
0.25±0.02). Densitometric analysis of forskolin was carried out in the absorbance mode at
545 nm after spraying with anisaldehyde-sulfuric acid visualization reagent. After spraying
with anisaldehyde-sulfuric acid regent, the TLC plate was dried and heated to 110–120 C.
Forskolin was detected as a dark violet or purple color spot.
Another TLC method, which was mainly used and performed with precoated plates of
silica gel 60F-254 in toluene: ethyl acetate (80:20, v/v) as mobile phase. Rf value of 0.27
was obtained. Anisaldehyde-sulfuric acid was used as the spraying reagent.
Fig 1: TLC of Forskolin with Rf Value
0.25 ± 0.02

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2. HPTLC (High Pressure Thin Layer Chromatography)
It is an automated multiple development HPTLC
method for the separation of several forskolin
derivatives. The HPTLC development used a 25-
step gradient with a polarity range of methylene
chloride–methanol to hexane and detection is done
by chloro-sulphonic acid reagent.
Standard preparation: Weigh accurately 12.5 mg
of Forskolin Ref. Standard to a 25 mL volumetric
flask. Dissolve with 50 mL of chloroform and
make up to 100 mL with chloroform, from this
pipette 5 mL into a 100 mL volumetric flask and
make up the volume with chloroform.
Sample preparation: Weigh the extract accurately
equivalent to 50 mg of Forskolin into a 100 mL volumetric flask. Dissolve with the aid of
chloroform and make up to 100 mL with chloroform, from this pipette 5 mL into a 100 mL
volumetric flask and make up the volume with chloroform.
HPTLC METHOD: Extract of C. forskohlii roots was applied to HPTLC plate. Benzene:
ethyl acetate (80:20 v/v) was used as the mobile phase and anisaldehyde-sulfuric acid was
used as the spray reagent. After developing and spraying, the plate was dried at 100–105°C.
Forskolin gave violet colored spot with Rf value of 0.45.
Table 1; Overview of HPTLC conditions for the analysis of forskolin
Mobile Phase St. Phase Detection Rf Value
Benzene: Ethyl Acetate
(80:20)
HPTLC Plate Anisaldehyde-sulfuric acid
/560nm
0.45
Toluene: Methanol
(18:1.5)
Silica Gel 60
F254
Anisaldehyde-sulfuric
acid/545 nm
0.27±
0.02
Benzene: Ethyl Acetate
(75:25)
Perforated Plate 200 nm 0.49±
0.01
Fig 2: HPTLC of Forskolin a) 366nm
b) Visible Light

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3. HPLC (High Pressure Liquid Chromatography)
HPLC analysis of forskolin has gained wider
acceptance among academic and industrial
researchers due to its high accuracy and
repeatability. It is used for the quantitative
estimation of forskolin in C. forskohlii root crude
extracts and in formulations. Inamdar et al.
described a HPLC method for the determination
of forskolin in crude extracts and in
pharmaceutical formulations.
A rapid method was developed for the evaluation
of forskolin in the root and stem of dried C.
forskohlii and in 17 market products by reversed-
phase high performance liquid chromatography (RP-HPLC) with a photodiode array
detector at 210 nm. The retention time of forskolin was approximately 6.8 min (Ciotonea
and Cernătescu, 2010; Saleem A., 2013; Thimmegowda, 2020; Kumar and Spandana,
2013).
HPLC Method- The samples were extracted with acetonitrile by sonication.
• HPLC Column- Reversed-phase high performance liquid chromatography (RP-HPLC)
and C18 Column.
• Solvent System- The analysis was performed by water-acetonitrile gradient elution
(50:50 v/v)
• Standard preparation- Weigh accurately 10 mg of Forskolin in a 25 mL volumetric
flask. Dissolve in 15 mL of Acetonitrile and make up to 25 mL with Acetonitrile.
• Sample preparation- Weigh 250 mg of. Dissolve in 25 mL of Acetonitrile with the aid
of heat, filter and make up to 100 mL with Acetonitrile in a volumetric flask.
• Flow Rate- at a flow rate of 1.0 ml /min
• Injection volume- 20uL
• Detector and Wavelength- photodiode detector at 210 nm
• Retention Time- approximately 6.8 min (RT was 9.5 at Solvent System 60:40 V/V)

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4. UV Spectroscopy
Forskolin is not very much UV sensitive. Its UV-absorption maximum is
around 200 nm. Benzene extract of C. forskohlii is used followed by concentration
Process.
5. IR Spectroscopy
The FTIR spectra have been recorded in KBr pellet, using a: Fourier Transmission Infrared
Spectra by: SHIMADZU (Ciotonea and Cernătescu, 2010).
Wavenumber Functional Group
3394 cm-1
Alcoholic -OH
2957 cm-1
1735 cm-1
C=O
1268 cm-1
C-O-C
1162 cm-1
C-O
Table 2: IR Functional Groups
Fig 5: FTIR of Forskolin

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6. MASS Spectrometry
The LC/MS experiment was performed to
confirm the identity of the peak of interest.
The flow rate was modified to 0.5 mL/min to
allow better detection of the compound.
Although the retention time increased to
about 12.3 min, the UV spectra were a match
for the peak of interest in the LC and LC/MS
methods. In positive ESI (Electron Spray
Ionization) mode, the spectrum of forskolin showed signals at m/z 428.2 [M+NH4]⁺ and
411.1 [M+H]⁺. The efficiency of the extraction procedure was verified by repeatedly
extracting sample with 3 mL acetonitrile. Each extract was analyzed individually. Forskolin
was no longer detectable after the fourth repetition. Forskolin was already 99.2% extracted
after the third repetition, which confirmed that the extraction procedure was exhaustive.
Process Requirements:
• Stationary phase- RP C18 column
• Sample- Benzene extract of C. forskohlii is used followed by concentration Process.
Mobile Phase- acetonitrile–water (65:35 v/v).
• The flow rate was modified to 0.5 mL/min
• Retention time increased to about 12.3 min in the LC
• Mass-Spectrometer (Wang et al., 2016; Schaneberg and Khan, 2003).

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7. NMR (Nuclear Magnetic Resonance)
I. Carbon NMR
Spectrum recorded on a Bruker AM 400 spectrophotometer operating at a basic frequency
of 100.62 MHz for carbons.
Starting with the 13C NMR spectrum of forskolin, and all carbon resonances were
numbered according to their chemical shifts. The spectrum can be separated into the five
different classes of carbon atoms by using the editing 32 technique DEPT. All 13C NMR
signals are well resolved and reflect the expected classes of carbons according to their
chemical shift and multiplicity; two carbonyl resonances (a, b), two olefinic carbons (c, d),
six hetero-substituted alkyl carbons (e, f, g, h, i, j), six alkyl carbons (k, 1, m, n, 0, r) and
six methyl groups (p, q, s, t, u, v) (Kegler and Fehlhaber,1991).
Table 3: Chemical shifts i.e. δ (ppm) and assignments of carbon NMR resonance
Carbon δ value in CDCl₃
(ppm)
Multiplicity Type of Carbon
C11 a 205.60 s C=O
C21 b 169.77 s Acetyl C-O
C14 c 146.30 d olefinic carbons
C15 d 110.69 t olefinic carbons
C9 e 82.65 s Hetero-substituted alkyl carbons
C8 f 81.41 s Hetero-substituted alkyl carbons
C7 g 76.58 d Hetero-substituted alkyl carbons
C13 h 75.01 s Hetero-substituted alkyl carbons
C1 i 74.35 d Hetero-substituted alkyl carbons
C6 j 69.89 d Hetero-substituted alkyl carbons
C12 k 48.75 t alkyl carbons
C10 l 42.95 s alkyl carbons
C5 m 42.79 d alkyl carbons
C3 n 36.09 t alkyl carbons
C4 o 34.42 s alkyl carbons
4α
-CH₃ (18) p 32.97 q methyl groups
13-CH₃ (16) q 31.52 q methyl groups
C2 r 26.55 t methyl groups
4β-CH₃ (19) s 24.33 q methyl groups
8-CH3 (17) t 23.59 q methyl groups
Acetyl-CH₃ (22) u 21.17 q methyl groups
10-CH3(20) v 19.82 q methyl groups

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II. Proton NMR
Table 4: Chemical shifts i.e. δ (ppm) and assignments of Proton NMR resonance
Hydrogen δ value In CDCl₃ (ppm) Multiplicity
9α
-OH 6.2 s
H-2β 2.16 d
H-7α 5.46 d
H-12α 3.1 7 d
H-12β 2.38 d
Fig 8: NMR Spectrum of Forskolin recorded on a Bruker AM 400 spectrophotometer.

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6) References
• Ciotonea c. and Cernătescu c. (2010) “forskolin morphology extraction
characterization” Buletinul Institutului Politehnic, Universitatea Tehnică „Gheorghe
Asachi
• Kavitha C., Rajamani K. and Vadivel E. (2010) “Review Coleus forskohlii: A
comprehensive review on morphology, phytochemistry and pharmacological aspects”
Journal of Medicinal Plants Research, Vol. 4(4); Page No- 278-285
• Kegler H. and Fehlhaber H.W. (1991) “NMR Investigations of Forskolin Complete
Assignment of Proton and Carbon NMR Spectra and Conformational Analysis”
MAGNETIC RESONANCE IN CHEMISTRY, VOL. 29, 993-998
• Kokate C.K., Purohit AP, Gokhale SB. (2008) “Textbook of Pharmacognosy” Pune:
Nirali Prakashan, Page No: - 14.98-14.99
• Kumar H., Spandana M. (2013) “Method of Extraction and Determination of Forskolin
from Coleus forskohlii of Nepal; Journal of Pharmacognosy and Phytochemistry; 37-
39
• Mohamed Saleem A. (2013) “Methods of Isolation and Analysis of Forskolin from
Coleus forskohlii” Springer-Verlag Berlin Heidelberg
• Schaneberg B. and Khan I. (2003) “Quantitative Analysis of Forskolin in Coleus
forskohlii (Lamiaceae) by Reversed-Phase Liquid Chromatography” Journal of AOAC
International Vol. 86.
• Wang Y. et al. (2016) “Kinetic study of the degradation of forskolin in aqueous systems
by stability-indicating HPLC method and identification of its degradation products”
Journal of Liquid Chromatography & Related Technologies.
• Thimmegowda S., Hussain F.H.M., Patil R. Et.al. (October 2020) “Antioxidant,
antimicrobial and cytotoxic activity of Curkolin (Curcuma longa and Coleus forskohlii
formulation)”; Article in Indian Journal of Traditional Knowledge.
• Wikipedia (April 2024) Forskolin – Wikipedia
• ChemSpider (April 2024) Forskolin | C22H34O7 | ChemSpider
• Sigmaaldrich.com (April 2024) Forskolin Coleus forskohlii, = 98 HPLC, powder 66575-
29-9 (sigmaaldrich.com)
• SlideShare (April 2024) https://www.slideshare.net/PrasanthBalakrishnap/forskolinpptx

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