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Methods of Isolation and Analysis of
Forskolin from Coleus forskohlii 109
Mohamed Saleem A.
Contents
1 Introduction ............................................................................... 3326
1.1 Physicochemical Properties of Forskolin .. . ....................................... 3328
2 Isolation by Column Chromatography ................................................... 3328
3 Isolation by Vacuum Liquid Chromatography .. ........................................ 3329
4 Isolation by Charcoal Column Chromatography .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 3329
5 Isolation by Immunoaffinity Column Chromatography .. . . . . . . ......................... 3330
6 Isolation by Hydrotropic Extraction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 3330
7 Isolation by Microwave-Assisted Extraction .. . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . 3331
8 Isolation by Using Selective Adsorbent Designed by Molecular Simulation .......... 3332
9 Extraction by Three-Phase Partitioning ................................................. 3333
10 TLC Analysis of Forskolin ............................................................... 3334
11 HPTLC Analysis of Forskolin .. . . . . . . . . . . . . . . . . . ........................................ 3336
12 HPLC Analysis of Forskolin .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . 3336
13 GC Analysis of Forskolin .. . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . 3337
14 Analysis of Forskolin by ELISA ......................................................... 3338
15 Hyphenated Techniques .................................................................. 3339
16 Conclusion ................................................................................ 3339
References ...................................................................................... 3340
Abstract
Forskolin is a well-known activator of adenylyl cyclase, obtained from the
roots of Coleus forskohlii. Several methods were reported for the isolation of
pure forskolin from the roots of C. forskohlii. The various methods of
isolation, the purity, and yield of isolated forskolin are described. The methods
included column chromatography, vacuum liquid chromatography, charcoal
M. Saleem A.
Department of Pharmaceutical Biology, Faculty of Pharmaceutical Sciences, UCSI University,
Cheras, Kuala Lumpur, Malaysia
e-mail: saleemskma@yahoo.com
K.G. Ramawat, J.M. Me
´rillon (eds.), Natural Products,
DOI 10.1007/978-3-642-22144-6_177, #Springer-Verlag Berlin Heidelberg 2013
3325
column chromatography, immunoaffinity column chromatography, hydrotropic
extraction, and adsorption on a selective adsorptive ligand designed by molecular
simulation. Forskolin with a maximum purity of 98 % w/w was obtained through
adsorption on a selective adsorptive ligand designed by molecular simulation.
Similarly, several analytical techniques like, TLC, HPTLC, HPLC, GLC, and
ELISA are described for the quantitative determination of forskolin in standard-
ized crude extracts and pharmaceutical formulations. Among the analytical
methods, HPLC and HPTLC appear to be more popular and widely used in the
industry and academia. Analysis of forskolin by ELISA appears to be highly
sensitive than other techniques.
Keywords
Analytical techniques • Charcoal column chromatography • Chromatography •
Coleus forskohlii • Forskolin • ELISA • GLC • HPLC • HPTLC • Hydrotropic
extraction • Immunoaffinity chromatography • Microwave-assisted extraction •
Molecular simulation • TLC
Abbreviations
BSA Bovine serum albumin
cAMP Cyclic adenosine monophosphate
CNBr Activated Sepharose 4B – Cyanogen bromide-activated-Sepharose 4B
ELISA Enzyme-linked immunosorbent assay
GC Gas chromatography
GLC Gas liquid chromatography
HAS Human serum albumin
HPLC High performance liquid chromatography
HPTLC High performance thin layer chromatography
IgG Immunoglobulin G
LC-MS Liquid chromatography mass spectrometry
MAb Monoclonal antibody
NaCl Sodium chloride
NaHCO
3
Sodium bicarbonate
PBS Phosphate buffered saline
TLC Thin layer chromatography
UV Ultraviolet
1 Introduction
Coleus forskohlii Briq. (Lamiaceae) is a perennial herb, occurs naturally in Indian
subcontinent and is also distributed in Egypt, Arabia, Ethiopia, tropical East Africa,
and Brazil [1,2]. In traditional medicine, C. forskohlii is commonly used in
different countries for various disorders. In Egypt and Africa, the leaf is used as
an expectorant, emmenagogue, and diuretic [1]. In Brazil, it is used as a stomach
3326 M. Saleem A.
aid, for treating intestinal disorders and to interrupt pregnancy [1,3]. In India, it is
used as a condiment and the root tubers are prepared as pickle and eaten.
In traditional Ayurvedic systems of medicine, C. forskohlii has been used
for treating heart diseases, abdominal colic, respiratory disorder, insomnia,
convulsions, asthma, bronchitis, intestinal disorders, burning sensation, constipa-
tion, epilepsy, and angina [4]. The root tubers are employed in the treatment of
worms and to reduce burning sensation in festering boils. The root paste is applied
to treat eczema and other skin infections after mixing with mustard oil. The plant is
also used for veterinary disorders [5]. The root tubers of C. forskohlii are included
in various Ayurvedic formulations and listed in Ayurvedic Pharmacopoeia of India
as Gandira [6].
Forskolin is a bioactive compound obtained mainly from the tuberous roots of
C. forskohlii. Recently, stem of C. forskohlii was also found to contain appreciable
quantities of forskolin [7]. C. forskohlii roots have long been used in Ayurvedic
medicine for treating heart and lung disease, intestinal spasms, insomnia, and con-
vulsions [4]. Forskolin (Fig. 109.1), a labdane diterpene, has exhibited positive effects
in asthma, glaucoma, hypertension, cancer, heart disease, diabetes, and obesity [4].
Forskolin was isolated during 1970s by Indian researchers and initially referred to as
coleonol and later changed to forskolin [8–10]. Its structure and stereochemistry were
established by several studies [8,10–13]. Forskolin occurs exclusively in C. forskohlii
and could not be detected in other Coleus species [14]. Forskolin’s major pharmaco-
logical mechanism of action is linked to its action on adenylyl cyclase enzyme.
Forskolin activates various isoforms of adenylyl cyclase, which results in the increase
in intracellular cAMP. Forskolin is used in many biochemical and pharmacological
experiments as an activator of adenylyl cyclase [15].
The roots contain approximately 0.15–1.5 % w/w of forskolin. The content of
forskolin in stems was found to be around 0.02 % w/w [7]. Apart from the
Fig. 109.1 Structure of
forskolin
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3327
Ayurvedic formulations which contain C. forskohlii as one of the ingredients,
standardized extracts of C. forskohlii have been marketed in the recent days as
nutraceutical supplements mainly for weight loss. Several analytical methods were
developed for the quantitation of forskolin in crude extracts as well as in formula-
tions. There is one clinical study in obese men indicating the efficacy of forskolin in
inducing weight loss, promoting lean body mass, bone mass, and increasing serum
free testosterone levels [16].
1.1 Physicochemical Properties of Forskolin
Forskolin is relatively a nonpolar compound, thus sparingly soluble in water. How-
ever, it is also insoluble in petroleum ether and xylene. Thus, it is soluble in solvents
like toluene, chloroform, ethanol, and methanol. This property is exploited in the
fractionation and isolation of forskolin. Also, forskolin is not very much UV sensi-
tive. Its UV absorption maximum is around 200 nm (Fig. 109.2). Hence, during
isolation process, monitoring of the fractions is done usually by TLC. However, TLC
is cumbersome, as it involves spraying with either anisaldehyde-sulfuric acid reagent
or vanillin-sulfuric acid reagent and heating [7,17].
2 Isolation by Column Chromatography
Column chromatography was used extensively for the isolation of forskolin during
1970s–2000s. Forskolin was isolated by using column chromatography on silica gel
from methanolic extract of root powder by Bhat et al. and other researchers [8,9,
18,19]. The crude extract was chromatographed several times before pure forskolin
was obtained. The mobile phase system consisted of eluting with nonpolar
solvents first to remove impurities, followed by gradually increasing the polarity
of solvents [7].
200nm
ABS
−0.100
1.000
300 400
Fig. 109.2 UV spectrum of
pure forskolin isolated from
C. forskohlii roots [17]
3328 M. Saleem A.
3 Isolation by Vacuum Liquid Chromatography
Vacuum liquid chromatography was employed to separate the forskolin-rich fractions
from the crude extract [Unpublished reports]. This vacuum liquid chromatographic
method was a modification of the published method for the flash chromatography
[20]. The crude extract was obtained by extracting the root powder with toluene or
chloroform. Here, the polar solvents like ethanol and methanol were avoided to
reduce the extractable impurities. The crude extract, after the solvent removed, was
subjected to vacuum liquid chromatography in a sintered glass Buchner funnel, which
was connected to a Buchner flask. Silica gel (230–400 mesh) was used as an
adsorbent. The vacuum (100–200 mmHg) was applied to suck the eluate. The mobile
phase was selected based on the TLC profile of crude extract. The TLC method of
crude extract was optimized to have forskolin’s R
f
value around 0.30. For TLC,
aluminum-backed silica gel GF
254
was used as the stationary phase and anisaldehyde-
sulfuric acid reagent was used as the spray reagent. The mobile phase which gave the
forskolin’s R
f
value around 0.30 was a combination of toluene and ethyl acetate
(80:20 % v/v). Instead of toluene, n-hexane also can be used. Elution was carried out
first with 100 % v/v of low-polar solvent (toluene or n-hexane) to elute the impurities
out. Then, exhaustive elution was carried out with the selected mobile phase.
After monitoring by TLC, similar fractions were combined and the solvent
was evaporated. The residue obtained thus was brownish yellow in color.
This crude forskolin was repeatedly crystallized in a mixture of n-hexane and ethyl
acetate (75:25 % v/v) to obtain white or off-white crystals of pure forskolin
[Unpublished reports].
4 Isolation by Charcoal Column Chromatography
Activated charcoal, a nonpolar adsorbent, has been extensively used for the
separation and purification of organic compounds for its easy regeneration,
low cost, high adsorption capacity. It is mainly used in the fractionation of
monosaccharide-oligosaccharide mixtures and in the isolation of sugars and their
polar derivatives using aqueous solvents. The adsorbent surface consists mainly of
carbon, which enables adsorption to take place on the principle of dispersion
forces [21]. Adsorption on charcoal is largely governed by the molecular size of
the sample. Charcoal more strongly adsorbs higher molecular weight dissolved
organic molecules than lower molecular weight molecules and nonpolar molecules
than polar molecules [22]. Recently, several applications of charcoal column
chromatography to separate low-polar compounds with nonaqueous solvents have
been reported [17,23,24].
Charcoal column chromatography was used to purify forskolin [17]. Activated
charcoal used in this study was untreated, granular carbon prepared from
chemically activated wood with a particle size of less than 75 mm (80–90 %)
(100–400 mesh). One hundred grams of powdered root material was extracted
with toluene. The toluene extract was concentrated to 15 ml under reduced pressure
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3329
at 40 C. To this extract, 150 ml of n-hexane was added slowly under continuous
stirring to obtain crude forskolin (brown solid precipitate) in powder form (1.6 g).
This crude forskolin was placed on a column (7.5 cm 1.7 cm i.d.), packed with
activated charcoal (6 g), placed on a Buchner flask of suitable size connected to
a vacuum source (100–200 mmHg), eluted with methanol and acetonitrile:methanol
(50:50, v/v). Forskolin was detected by TLC as a major spot in the eluates obtained
with acetonitrile:methanol (50:50, v/v) and as a minor spot in the eluates obtained
with methanol. Hence, the yellowish brown colored eluates obtained with acetoni-
trile:methanol (50:50, v/v) were combined and concentrated under vacuum at 40 C
to obtain a residue (500 mg). This residue was stirred with n-hexane to remove
n-hexane soluble impurities, and n-hexane was removed by filtration. After drying
the residue, it was dissolved in 5 ml of chloroform. To this solution, 50 ml of
n-hexane was added with continuous stirring to remove most of the coloring matter
and crude forskolin was obtained as a pale yellow precipitate. This precipitate was
crystallized in ethyl acetate:light petroleum (b.p. 40–60 C) mixture (25:75, v/v) to
obtain the forskolin. This crystallization step was repeated once to get forskolin as
off-white color precipitate. This precipitate was again crystallized with diethyl
ether:n-hexane (1:15, v/v) to obtain pure forskolin (yield – 0.0971 % w/w with
respect to the weight of dried root powder; purity – 96.9 % w/w) [17].
5 Isolation by Immunoaffinity Column Chromatography
Yanagihara et al. developed an immunoaffinity column chromatography method
to isolate forskolin from the roots of C. forskohlii, by using the anti-forskolin
monoclonal antibody [25]. Anti-forskolin monoclonal antibody was prepared
by injecting the forskolin-bovine serum albumin conjugate into BALB/c mice
followed by cell culture techniques. Purified IgG in phosphate buffered saline
(PBS) solution was added to the slurry of CNBr-activated Sepharose 4B in coupling
buffer (0.1 M NaHCO
3
containing 0.5 M NaCl), and used as the affinity gel. The
dried powder of tuberous root was extracted with diethyl ether. After evaporation of
solvent, the residue was dissolved in PBS containing 6 % methanol and subjected to
the immunoaffinity column separation. Elution was carried out with PBS containing
45 % methanol. The forskolin content was estimated by ELISA. The recovery of
forskolin was found to be 95.6 % w/w. However, forskolin purified in this way by
the immunoaffinity column was still contaminated by a small amount of 7-deacetyl
forskolin because this compound has cross-reactivity against anti-forskolin
monoclonal antibody. Therefore, the mixture was treated with pyridine and acetic
anhydride at 4 C for 2 h to give pure forskolin [25].
6 Isolation by Hydrotropic Extraction
Hydrotropes are highly water-soluble small molecular weight organic salts
(e.g., sodium salicylate and sodium cumene sulfonate). They possess ability to
3330 M. Saleem A.
dissolve other organic compounds in aqueous solutions in a concentration-dependent
manner. Hydrotropes exhibit their solubilization capacity for organic compounds
above a characteristic minimum hydrotrope concentration (MHC). Hydrotropic
solubilization technique is one of the methods used to enhance the aqueous solubility
of insoluble or slightly soluble bioactive compounds. Hydrotropes dissolve well
the organic compounds above their MHC in aqueous solution. Hence, if the
hydrotrope solution containing organic compound is diluted below its MHC, then
the solubility of that organic compound in aqueous solution decreases and the organic
compound may separate out [26–29]. Gaikar et al. employed this technique to isolate
piperine [30], boswellic acids [31], diosgenin [32], and andrographolide [33]. This
technique was successfully employed to isolate forskolin by Mishra et al. [34].
Pulverized roots of C. forskohlii were suspended in aqueous hydrotrope solution
of sodium cumene sulfonate and agitated vigorously. After the extraction, the
solution was subsequently filtered under vacuum. A clear brown color solution
was obtained as filtrate, while the insoluble sticky solid portion was collected as
residue. The filtrate was diluted with water to the respective minimum hydrotrope
concentration (MHC) of the hydrotrope. Solid brown color crystals of forskolin that
precipitated out from the hydrotrope solutions were isolated by centrifugation or
filtration. The purity of isolated forskolin was 85 % w/w with maximum extraction
of 70 % w/w [34].
7 Isolation by Microwave-Assisted Extraction
Microwave is used to irradiate the plant material during the extraction process.
Microwave irradiation is reported to cause rupture of cells, thereby facilitating
the action of solvents on the cells [35]. The usage of microwave irradiation was
employed for the isolation of curcumin from turmeric [36–39], piperine from Piper
nigrum [40], solanesol from tobacco leaves [41], and artemisin from Artemisia
annua [42].
Microwave irradiation was employed to extract forskolin from the roots of
C. forskohlii by Devendra et al. [35]. The raw material was soaked in water before
subjecting it to microwave irradiation and it was irradiated with microwave, after it
was evenly and thinly spread over a Petri dish, in a microwave oven. After the
irradiation, the material was extracted by suspending it in 150 ml of methanol
with vigorous agitation. The methanolic extract was concentrated by evaporating
methanol up to 80 % of its original volume. Crude forskolin was crystallized from
the concentrated solutions by simultaneous addition of petroleum ether and water
in a 1:1 volumetric ratio. Forskolin crystallized from the above process was a
free-flowing and brown colored powder with 30–35 % w/w purity. The crude
forskolin was dissolved in acetonitrile and further decolorized by adsorption on
an alumina column. When forskolin extract in acetonitrile was passed over an
alumina bed, all brown colored organic impurities of the crude extract were
strongly retained by the column. The light yellow eluate from the column contained
78 % (w/w) forskolin, 15 % (w/w) of 7-deacetylforskolin, and 6 % (w/w) of
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3331
1,9-dideoxyforskolin. No separation of forskolin and its analogues was, however,
achieved with the alumina column, but the product contained no other impurities.
This fraction was further purified to isolate forskolin by using a chloromethylated
polystyrene polymer loaded with diethanolamine [35].
8 Isolation by Using Selective Adsorbent Designed
by Molecular Simulation
Devendra et al. reported the usage of selective adsorbent designed by the molecular
simulation technique to isolate forskolin and its analogues based on the interaction
between the adsorbent and the target molecules [35].
Diethanolamine was loaded on chloromethylated polystyrene matrix by
chemical synthesis. Diethanolamine was selected as a ligand for interacting
with the target molecules based on the hypothesis that the hydroxyl groups of
diethanolamine would form hydrogen bonding with the hydroxyl groups of
forskolin and its analogues in a differential manner because of relative spatial
positions of different hydroxyl groups on the structures of interacting molecules.
This hypothesis was tested in silico by using a computer modeling software,
Material Studio 3.2 (MS, Acclerys, USA). Each solute and two units of
the amine-loaded polystyrene were allowed to interact in different solvent environ-
ments. In the case of interaction studies in the solvated state in acetonitrile, none of
the functional groups of forskolin showed any interaction with the amine-loaded
polymer, whereas in the case of 7-deacetylforskolin, all the four hydroxyl
groups interact with the hydroxyl groups of the amine-loaded polymer. In the
case of 1,9-dideoxyforskolin, the acetyl group at seventh position interacts with
the hydroxyl groups of the amine-loaded polymer. These theoretical calculations
indicated the possibility of separating forskolin from its analogues by selective
adsorption of the latter on a diethanolamine-loaded polystyrene matrix from
acetonitrile solutions.
Devendra et al. performed the experimental work to confirm the predictions of
in silico molecular simulation. The methanolic extract of the root was concen-
tratedbyevaporatingmethanolupto80%of its original volume. Crude forskolin
was crystallized from the concentrated solutions by simultaneous addition of petro-
leum ether and water in a 1:1 volumetric ratio. Forskolin crystallized from the above
process was a free-flowing and brown colored powder with 30–35 % w/w
purity. The crude forskolin was dissolved in acetonitrile and further decolorized
by adsorption on an alumina column. The eluate obtained after decolorization of
the crude forskolin extract through an alumina column mainly contained forskolin
and its analogues and, therefore, was used as a feed solution for adsorption on the
amine-loaded polymer, loaded in a column to investigate their adsorption behavior.
The uptake by the polymer from the acetonitrile solution was found to be
maximum for 1,9-dideoxyforskolin (91 %), intermediate for 7-deacetylforskolin
(37 %), and minimum for forskolin (12.2 %) until five bed volumes. These differen-
tial uptakes validated the hypothesis from molecular simulation that analogues shall
3332 M. Saleem A.
be adsorbed more preferentially than forskolin on the polymer. Almost 90 % w/w
of forskolin was recovered from this technique and the purity of forskolin
was 94 % w/w.
In another study, Devendra et al. [43] used different selective polymers designed
by molecular simulation to have interaction with forskolin to isolate forskolin.
N-propinoyl aspartic acid (NPAA), phenyl glycine-o-carboxylic acid (PGOCA),
and phenyl glycine-p-sulfonic acid (PGPSA) were used as the ligands for selective
adsorption of forskolin. Extraction and decolorization processes of crude extract
were carried out as per the previous report [35]. The ligands were synthesized and
loaded on chloromethylated polystyrene matrix cross-linked with divinylbenzene in
dimethyl sulfoxide (DMSO) solutions. The ligand-loaded polymer was packed in a
glass column. The decolorized extract solution was pumped through the packed
adsorption bed by a peristaltic pump. Samples were collected at regular time intervals
at the exit of the column. The PGPSA-loaded polymer gave almost 98 % w/w pure
forskolin during desorption, with the yield of 16 % w/w. About 95 % w/w pure
forskolin was achieved with 46.54 % w/w recovery with PGOCA-loaded polymer.
9 Extraction by Three-Phase Partitioning
Three-phase partitioning (TPP) is a bioseparation technique useful for the fraction-
ation and concentration of proteins from plant materials and microorganisms. It
involves the partitioning of hydrophilic constituents, proteins, and hydrophobic
constituents in three phases comprising of water, ammonium sulfate, and organic
solvent [44,45]. TPP was utilized for extraction of forskolin from C. forskohlii
roots. Aqueous slurry was prepared by mixing 5 g of C. forskohlii root powder in
50-ml distilled water by mild stirring with a magnetic stirrer. Calculated quantities
of ammonium sulfate was added to the aqueous slurry prepared, followed by
addition of known amount of t-butanol. The extraction was carried out for 1 h by
gentle stirring with magnetic stirrer. The mixture was allowed to stand for 1 h for
the formation of three phases. The three phases so formed were separated by
centrifugation. The upper organic layer was collected and the solvent (t-butanol)
was evaporated to dryness. Forskolin content was estimated in the extract thus
obtained by HPLC. A maximum of 30.83 % recovery of forskolin was obtained
under the optimized conditions. Ultrasonication and enzyme pretreatment with
commercial enzyme preparations of Stargen
®
002 (contains Aspergillus kawachi
alpha-amylase expressed in Trichoderma reesei and a glucoamylase from T. reesei
that work synergistically to hydrolyze granular starch substrate to glucose) and
Accellerase
®
1500 (mixture of cellulase and glucosidase that works synergistically
to hydrolyze cellulosic substrate to glucose) followed by TPP gave 79.95 % and
83.85 % recovery (when compared to conventional soxhlet extraction which is
taken as 100 %), when used individually within 4 h [46]. This study only described
extraction procedure for the extraction of crude extract which contains forskolin
and not the isolation of pure forskolin from C. forskohlii roots [46]. The summary of
all isolation procedures is given in Table 109.1.
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3333
10 TLC Analysis of Forskolin
Generally, 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 [47]. 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.
A validated TLC method was reported by Ahmad et al. [48]. This method was
developed on TLC aluminum plates precoated with silica gel 60F
254
using
solvent system benzene:methanol (9:1, v/v), which gave compact spot of
forskolin (R
f
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. This method was applied
for determination of forskolin in C. forskohlii root and in capsule dosage
forms,whichshowed0.18%and0.57%w/wofforskolin[48]. In a stability
test, forskolin was subjected to acid and alkali hydrolysis, oxidation,
photodegradation, and heat degradation. It was observed that the drug was
susceptible to acid, base hydrolysis, oxidation, photo-oxidation, and heat
degradation. This TLC method effectively resolved the forskolin from compo-
nents of C. forskohlii root, from excipients of capsule as well as the degradation
products of forskolin [48].
Table 109.1 Overview of isolation of forskolin by several methods
Isolation method
Principle of
separation/isolation
Yield from plant
roots Purity Reference
Column
chromatography
Adsorption 0.103 % w/w [7]b [7–9,18,19]
Vacuum liquid
chromatography
Adsorption a b [Unpublished
reports]
Charcoal column
chromatography
Adsorption 0.0971 % w/w 96.9 % w/w [17]
Immunoaffinity column
chromatography
Antigen-antibody
interaction
ab[25]
Hydrotropic extraction Solubility a 85 % w/w [34]
Microwave-assisted
extraction
Solubility/partition/
adsorption
ab[35]
Selective adsorbent
designed by molecular
simulation
Adsorption a 94 % w/w
[35], 98 %
w/w [43]
[35,43]
a – yield not stated in the literature; b – purity not stated in the literature
3334 M. Saleem A.
Another TLC method was reported by our group, which was mainly used as
a qualitative identification method [7,17]. TLC was performed with precoated
plates of silica gel 60F
254
(Merck, Darmstadt, Germany) in toluene:ethyl acetate
(80:20, v/v) as mobile phase. Anisaldehyde-sulfuric acid was used as the spraying
reagent (1 ml concentrated H
2
SO
4
was added to 0.5-ml anisaldehyde in 50-ml
acetic acid). After developing the plate, the plate was dried and heated to
110–120 C for 5–10 min in a hot air oven with careful and periodical monitoring
to avoid excessive heating and subsequent charring of the plate. After the plates
were taken out from the hot air oven, the plates were allowed to cool to room
temperature and scanned immediately in a digital photoscanner and the scanned
image was stored in a computer. Furthermore, the plate was preserved by wrapping
with cellophane tape. It was observed that if the plates were left exposed to air, the
spots disappeared after few days. R
f
value of 0.27 was obtained for forskolin as
a single, compact dark violet/purple spot (Fig. 109.3). Similar TLC method was
reported by Selima et al. [49] but with a different R
f
value of forskolin (0.48).
There is a monograph available in Indian Pharmacopoeia Commission (IPC)
website (http://www.ipc.gov.in) for the characterization of Coleus dry extract [50].
A TLC method is described in that monograph. The procedure consisted of coating
the plate with silica gel GF
254
and developing in a mobile phase consisting of
a mixture of 40 volumes of ethyl acetate and 60 volumes of hexane. Test solution
was prepared by dissolving about 500 mg of the extract under examination
with 10-ml methanol and filtered. Reference solution was prepared by making
a 0.1 % w/v solution of forskolin RS in methanol; 10 ml of each solution was
applied to the plate as bands of 10 mm by 2 mm. The mobile phase was allowed to
rise up to 8 cm. The plate was air dried and sprayed with anisaldehyde-sulfuric acid
reagent solution. The plate was heated at 110 C for 10 min and examined under
365 nm and under day light. The chromatogram obtained with the test solution
showed a band corresponding to the band obtained by using reference solution,
indicating the presence of forskolin.
Fig. 109.3 Thin layer chromatogram of pure forskolin
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3335
11 HPTLC Analysis of Forskolin
HPTLC analysis of crude extracts for the quantitation of marker compounds has
gained wider acceptance by the industrial scientists in the recent days for the
relative ease of the technique and its capability to analyze multiple samples at the
same time in a single plate [51]. An automated multiple development HPTLC
method for the separation of several forskolin derivatives was developed and
validated. The HPTLC development used a 25-step gradient with a polarity range
of methylene chloride–methanol to hexane and detection by chlorosulfonic acid
reagent [52].
Pushpa et al. described a HPTLC method in which benzene 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 R
f
value of 0.45 [53].
Vijay et al. described a HPTLC method for estimation of forskolin in ophthalmic
preparations. A stock solution of forskolin (marker compound) equivalent to
1mgml
1
was prepared and different quantities of this solution, namely, 1, 2, 3,
4, 5 ml, were spotted onto precoated silica gel GF plates (10 10 cm size) using
automatic sample syringe (Linomat IV Application mode) of the CAMAG-HPTLC
equipment in order to develop calibration between 1 and 4 mg drug concentrations.
The plate was placed in the twin-trough development chamber, which was
presaturated with the mobile phase consisting either of a mixture of toluene:ethyl
acetate (7:3) or chloroform:methanol (8:2) separately. The plate was developed
for about 20 min and dried with a current of hot air. The plate was then
scanned at 292 nm in the densitometer, and the area under the curve (AUC),
of each concentration, was determined. Calibration curve was developed by
plotting a graph between the concentration and the area under the curve [54].
Several other HPTLC methods were also reported in the literature [55–58].
An overview of HPTLC parameters for the analysis of forskolin is given
in Table 109.2.
12 HPLC Analysis of Forskolin
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 [47].
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
temperature was held constant at 30 C, and the retention time of forskolin was
approximately 6.8 min. The samples were extracted with acetonitrile by sonication.
3336 M. Saleem A.
The method was found to be precise and accurate. The response was linear through
zero from 6.3 to 630 mgml
1
with a correlation coefficient (R
2
) of 0.9998. Identity of
the marker compound was confirmed by a LC-MS experiment [61].
Another rapid reverse-phase HPLC method with evaporative light scattering
detection (ELSD) was reported for the determination of forskolin in weight-loss
multi-herbals products. The analysis was performed by water-acetonitrile gradient
elution at a flow rate of 1.0 ml min
1
. The evaporator tube temperature of ELSD
was set at 35 C, and with the nebulizing gas flow rate (pressure) of 3.0 bar. Good
linear relationships were obtained with correlation coefficients exceeding 0.9995.
The average recovery of forskolin ranged from 99.4 % to 100.4 % with RSDs
below 3 %. The percent relative standard deviations (% RSD) of intra- and inter-day
precision varied by less than 2.1 %. LOD and LOQ were 0.95 and 3.21 mgml
1
,
respectively. The method was found to be accurate, precise, and repeatable [62].
Several other similar HPLC methods were also reported in literature especially for
the analysis of forskolin in tissue culture studies and in formulations [63–72].
A HPLC method was used by our group to quantify the purity of isolated
forskolin. Actually, this method was adapted from a previously reported method
[61]. In this method, C18 (250 4 mm i.d., 5 m) column was used with a mobile
phase consisting of water (A):acetonitrile (B) (50:50, v/v) at a flow rate of
1.0 ml min
1
. The gradient elution was 50A/50B to 43A/57B in 10 min. The UV
detection was carried out at 210 nm. The retention time of forskolin was found to be
10.3 min [17]. An overview of various HPLC methods is given in Table 109.3.
13 GC Analysis of Forskolin
Usually gas chromatographic (GC) analysis requires an analyte to be volatile.
Forskolin is not naturally volatile. Hence, it needs to be converted into its volatile
Table 109.2 Overview of HPTLC conditions for the analysis of forskolin
Mobile phase
Stationary
phase
Detection/scanning
wavelength
R
f
value
of forskolin Reference
Benzene: Ethyl acetate (80:20 v/v) HPTLC
plate
anisaldehyde-
sulfuric acid/
560 nm
0.45 [53]
Toluene:ethyl acetate (7:3) or
chloroform: methanol (8:2)
Precoated
silica gel GF
plates
292 nm a [54]
Benzene:ethyl Acetate (85:15) Silica gel
60F
254
Vanillin-sulfuric
acid/550 nm
a[59]
Toluene:methanol (18:1.5, v/v) Silica gel
60F
254
Anisaldehyde
sulfuric acid/
545 nm
0.27 0.02 [60]
Benzene:ethyl acetate (75:25, v/v) Precoated
plates
200 nm 0.49 0.01 [58]
a–R
f
value not stated in the literature
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3337
derivatives. Since, this is a limiting factor especially for the analysis of crude
extracts, only one gas chromatographic method was reported in literature. A GLC
method was reported by Inamdar et al. for the quantitative estimation of forskolin in
crude extracts and in pharmaceutical formulations [74].
14 Analysis of Forskolin by ELISA
Immunoassay systems using monoclonal antibody (MAb) against natural products
have become an important tool for studies on receptor binding analysis, enzyme
assay, and quantitative and/or qualitative analytical techniques in animals or
plants [75]. Immunogenic forskolin-bovine serum albumin (BSA) conjugate was
synthesized via 7-deacetyl-7-hemisuccinyl forskolin. BALB/c male mice were
injected intraperitoneally with forskolin-BSA conjugate emulsified with Freund’s
complete adjuvant. Splenocytes were isolated from the hyperimmunized BALB/c
mice and fused with the P3-X63-Ag8-U1 myeloma cells. Six hybridomas
producing monoclonal antibodies (MAbs) reactive to forskolin were obtained.
Table 109.3 HPLC conditions for the analysis of forskolin
Stationary phase Mobile Phase Flow rate Detection Reference
C18 water-acetonitrile
gradient elution
1.0 ml min
1
ELSD evaporator
tube temperature at
35 C, nebulizing gas
flow rate (pressure) of
3.0 bar
[62]
C18 (150 4.6 mm
i.d., 5 m)
Water (A): Acetonitrile
(B) (50:50, v/v); The
gradient elution was
50A/50B to 43A/57B in
10 min.
1.0 ml min
1
210 nm [61]
C18 (250 4.6 mm
i.d., 5 m)
Acetonitrile:water
(50:50, v/v)
1.6 ml min
1
220 nm [59]
C18 (250 4.6 mm
i.d., 5 m)
Acetonitrile:methanol
(80:20, v/v)
1.0 ml min
1
254 nm [58]
C18 (250 4.6 mm
i.d., 5 m)
Acetonitrile:water
(45:55, v/v)
1.0 ml min
1
220 nm [67]
C18 (250 4mm
i.., 5 m)
Water (A): Acetonitrile
(B) (50:50, v/v); The
gradient elution was
50A/50B to 43A/57B in
10 min.
1.0 ml min
1
210 nm [17]
C18 (300 3.9 mm
i.d.)
Acetonitrile:water
(65:35, v/v) at pH 2.5
by adding
orthophosphoric acid
1.0 ml min
1
202 nm [73]
3338 M. Saleem A.
One of the original clones secreted anti-forskolin antibody which was purified by
protein A affinity column chromatography and its purity was confirmed by MALDI
mass spectrometry. The MAb was bound to polystyrene microtitration plates
precoated with a forskolin-human serum albumin (HAS). The full measuring
range of the assay extended from 6 ng to 200 ng ml
1
of forskolin [68,76]. The
ELISA system established in this study was more sensitive compared to TLC [47],
GLC [74] or HPLC [47,77].
15 Hyphenated Techniques
Hyphenated techniques like GC-MS and LC-MS techniques are mainly used for
identification of the compound and confirmation of its structure and stereochemis-
try. A LC-MS technique was employed for the identification of forskolin in crude
extracts and in marketed formulations. A C18 column (150 4.6 mm, 5 mm)
was used at ambient temperature. The mobile phase consisted of water (A) and
acetonitrile (B). At a flow rate of 0.5 ml min
1
, the gradient elution was
programmed to change from 50A/50B to 43A/57B in 10 min and to remain constant
at 43A/57B for next 10 min. The detection wavelength was 210 nm, and the
injection volume was 5 ml. Best results were obtained in positive electrospray
ionization (ESI) mode, with ionization voltage set to 25 V, source voltage to
3.0 kV, and probe temperature to 350 C[61].
16 Conclusion
Several diversified techniques are summarized for the isolation of forskolin from
the tuberous roots of C. forskohlii. However, the purity of the isolated forskolin
remains a matter of concern for the biologists, since forskolin with high purity is
required in biological tests. Higher the purity of a bioactive compound, lesser would
be the effect of the impurities present and the observed pharmacological effect can
be safely attributed to the major compound. Only few techniques like charcoal
column chromatography, selective adsorption using phenyl glycine-p-sulfonic
acid–loaded polymer, and selective adsorption using phenyl glycine-o-carboxylic
acid–loaded polymer could achieve purity greater than 95 % w/w. Even though
hydrotropic extraction process seems to be simple to perform, the purity of
forskolin was only 85 % w/w, thus disabling its potential application. Further
research is required to identify processes which will yield high purity forskolin in
less amount of time in an environment-friendly manner. One of those approaches
may focus on using molecular simulation technique to identify a suitable
hydrotrope or adsorbent for separating forskolin from its structurally similar
analogues.
Standardized crude extract of C. forskohlii and forskolin are marketed as
weight-loss products. Several analytical techniques were reported in the literature
for the quantification of forskolin from crude extracts as well as in formulations.
109 Methods of Isolation and Analysis of Forskolin from Coleus forskohlii 3339
Among them, many were HPLC methods. In addition, HPTLC technique was also
reported in many papers. These indicate the popularity of HPLC and HPTLC
techniques in the analysis of forskolin. The analysis of forskolin by ELISA
technique appears to be more sensitive than other methods and may be further
explored for regular utilization.
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