High Performance Thin Layer Chromatography was a sensitive and accurate method for detection and estimation of forskolin in roots and by tissue culture of Coleus forskohlii. Maximum absorption was observed at 315 nm using fluorodensitometric analyis. Forskolin production was observed in callus cultures from leaf, stem and root origin as well as roots of in vitro grown plants.
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PAP, S MALATHY, J S

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Journal of Spices and Aromatic Crops 8 (2) : 153-157 (1999) Monitoring of forskolin production from roots and callus by HPTLC in Coleus forskohlii Briq. S MALATHY & J S PAP Food and Fermentation Technology Division Department of Chemical Technology University of Mumbai Matunga, Mumbai - 400 019, India. Abstract High Performance Thin Layer Chromatography was a sensitive and accurate method for detection and estimation of forskolin in roots and by tissue culture of Coleus forskohlii. Maximum absorption was observed at 315 nm using fluorodensitometric analyis. Forskolin production was observed in callus cultures from leaf, stem and root origin as well as roots of in vitro grown plants. Key words : Coleus forskohlii, forskolin, tissue culture. Abbreviations BA : Benzyl adenine 2,4-D : 2,4-Dichlorophenoxyacetic acid HPTLC: High Performance Thin Layer Chromatography IAA : Indole-3-acetic acid IBA : Indole-3-butyric acid Kn : Kinetin MS : Murashige and Skoog's medium NAA : a-N aphthaleneacetic acid TLC : Thin Layer Chromatography Introduction Coleus forskohlii Briq. (Labiateae), an aromatic herb found in many parts of India, is the only known source of forskolin (Shah et al. 1980) which is used in the treatment of glaucoma, congestive cardiomyopathy and asthma. The plant is generally propagated by 1 Corresponding author planting terminal stem cuttings from rapidly growing plants during Novem-ber-December. Protocols for rapid micropropagation ofthe plant are avail-able. A comprehens~ve review on the in vitro culture of the plant has been published (Petersen 1994). Production of forskolin has been reported in 154 suspension cultures by Mersinger et al. (1988) who recorded an yield of 0.031% and later by Sen & Sharma (1991) with 0.009% yield. Forskolin production in root callus cultures giving an yield of 0.075% has been reported by Tripathi et al. (1995). Forskolin was detected in Agrobacterium mediated tumorous cal-lus (0.002%), and in rhizogenic (0.011%) and root cultures (0.014%) (Mukherjee et al. 1996). While TLC has been reported to be useful for semiquantitative estimation (Inamdar et al. 1984), HPLC and GC have been used for accurate quantification of forskolin (Petersen 1994). In a recent report, forskolin has been estimated by dual wave length transmode Shimadzu CS-1910' TLC scanner at 212 nm with hexane-ethyl acetate (78:32) on silica ge160F254 plates (Tripathi et al. 1995). HPTLC offers a sensitive, accurate and reliable method for routine detection and quantification of compounds in a mixture such as in plant material. This paper reports the quantification of forskolin in roots as well as callus of C. forskohlii by fluores-cence mode using HPTLC. Materials and methods Chemicals and reagents Authentic sample of forskolin was ob-tained from Hoechst-Marion Roussel (India). Chromatographic grade rea-gents were obtained from Sisco Re-search Labs, Mumbai and precoated silica gel plates 60F254 from EMerck (India) Ltd. Instrumentation Standard forskolin and other samples were spotted on precoated silica gel plates as narrow bands of 4 mm width at a con~tant 'rate of 8 sec/~I uSIng a Camag Linomat IV model applicator under a nitrogen atmosphere. A mixture Malathy' & Pdi of benzene and ethyl acetate (85:15) was used as the mobile phase. The length of the chromatogram was 90 mm and 15 min were required for each run. The plates were sprayed with anisaldehyde, sulphuric acid reagent (0.5 illl anisaldehyde + 1 ml H2S04 + 50. ml acetic acid) and heated at 110°C for 5 min. Orange fluorescence was observed at 366 nm which was optimally detected and quantified at 315 nm using the Camag TLC scanner with CATS 3.17 software for quantificatiol1 of the sep,,-rated compounds on the chromatogram. Callus initiation from the plant Terminal stem pieces about '10. cm in length and young roots were excised from an actively growing 1 year old plant. The stem and. root pieces were washed in tap water and surface steri-lized by immersing in 98% ethanol for 30 sec. After rinsing with sterile dis-tilled water, the explants were treated with 0.1% HgC12 ·Jor4 min, washed thrice with sterile distilled water and dried on sterile filter paper. The nodal segments, leaves and root pieces w~re cut into 1 cm segm~l1t~ and placed on MS medium supplemellted with auxins NAA, 2,4-D, IBA, IAA and cytokinins BA and Kn. The cultures were grown at . 25°C and 65% RH with a 12 h photo period. Extraction of forskoliri from samples Samples of roots (tissue cultured roots and roots from market) and callus (1 month old)· were dried at 50°C and extracted with benzene in a soxhlet apparatus for 8. h. The solvent was evaporated and the residue was dis-soh'ed in i ml of benzene. A fixed amount {5 mD was then spotted. on the precoated TLC Plates using theCam'lg Linomat applicator .. Production of forskolin Standard curve of forskolin in benezene A stock solution of forskolin with a concentration of 1 mg/ml was prepared in benzene and further diluted with the solvent to obtain a final concentration of 100 ng/m!. Appropriate quantities of this solution were applied to obtain spots in the range of 50-500 ng. The standard curve was plotted with.concen-tration against area. Results and discussion Optimization of chromatogram A mixture of benzene and ethyl acetate (85:15) was used as the mobile phase and optimum resolution was obtained at Rf 0.33 + 0.01. The chromatogram was scanned at different wave lengths from 300-400 nm for ",max. It was observed that though at 365 nm and 315 nm, the peak areas were similar for a particular concentration of forskolin, at 315 nm other peak areas were minimum and therefore forskolin was the predomi-nant compound that was estimated. Along with forskolin, at Rf= 0.7 another similar spot was observed with an orange floresecence. This could be 1,9-dideoxforskolin (DDF), which was ob-served in all samples. No other spot gave orange fluorescence and therefore was not detected at 315 nm. Production of DDF in quantities 1.6 to 2.5 times less than forskolin was earlier reported in suspension cultures (Mersinger et al. 1988). A linear regression was obtained for standard forskolin with regression coefficient = 0.9929 and the equation y = 7126 x. The lowest concentration of forskolin that was accurately detected was 25 ng. Organogenesis in callus cultures Organogenesis was observed from cal-lus of leaf nodal segments and roots 155 though callus of stem origin exhibited a greater potential for organogenesis (Table 1). In the medium where regen-eration was observed, the callus was green with dark green spots of growth which later gave rise to shoots and roots. Cline (1996) observed that in Coleus spp. exogenous auxin did not repress lateral bud outgrowth as com-pared to many other genus where the Thimann - Skoog experiment was con-firmed. Auxin treatments were carried out on 10 different species of plants including Coleus. Exogenous auxin did repress lateral bud outgrowth in· most species but not in Coleus and Arabidopsis spp. In our experiments shoot induction was observed from callus cultures with a combination of IAA and IBA without the presence of cytokinins. Forskolin production in roots and callus cultures Forskolin content of stem callus was higher than leaf callus. Callus from roots were slow growing as can be seen from the low dry weight. It has been reported that forskolin production could only be observed after induction of differentiation into roots (Mersinger et al. 1988). Differentiation capacity of stem callus into shoots and roots was observed to be greater as compared to leaf callus (Table 1). Mter growing in the soil for 1 year, the roots of tissue cultured plants produced 0.02% forskolin. In comparison, 0.01% forskolin was detected in roots of 1 month old in vitro grown plants (Sen & Sharma 1991). Variation in forskolin content has been reported from 0.01 - 0.4% in roots, 0.1% being most common in roots grown at higher altitudes (Shah 1996). The higher forskolin content of market roots which are generally cultivated in hill slopes could be due to this difference 156 Table 1. Organogenesis in callus cultures of Coleus forskohlii Explant Leaf callus Leaf callus Root callus Stem callus Stem callus Stem callus MS basal media + Hormone (mgl-') NAA (1.0) + BA (0.5) 1AA (1.0) + IBA (1.0) NAA (1.0) + BA (0.5) NAA (1.0) + BA (0.5) NAA (2.0) + BA (0.5) 1AA (1.0) + IBA (1.0) No. of days 20 20 30 20 25 25 No. of shoots 5 o 4 2 5 3 Malathy & Pai No. of roots 2 7 o o o 12 in habitat (Table 2). The forskolin content of callus from stem and leaf origin increased steadily with time as the dry weight of the callus also in-found to be accumulated (Abraham et al. 1988). Since callus cultures are less complex than differentiated tissues, the formation of vesicles and subsequent Table 2. Forskolin content in roots and callus of Coleus forskohlii Sample MS basal media + Dry weight Forskolin Hormone (mgJ-') (g) content (%) Leaf callus NAA (1.0) + BA (0.5) 0.267 0.010 Leaf callus 1AA (1.0) + 1BA (1.0) 0.100 0.007 Stem callus NAA (1.0) + BA (0.5) 0.060 0.009 Stem callus 1AA (1.0) + IBA (1.0) 0.133 0.010 Root callus NAA (1.0) + BA (0.5) 0.069 0.002 Tissue cultured roots 1.793 0.020 Market roots 2.000 0.040 '" 0.' o.~ __ dry wt g(laal) 0.' __ drywt g(stern) 0.06 -+-IOI$kolin %Qeaf) __ Iorskolln %(slem) ""' a; 0.15 0.00 i 0.05 " • 0.' 0'.04 0'.03 0,05 0.0'2 0.01 10 15 ro e w ~ ~ Days creased (Fig. 1). Production offorskolin was detected only after 21 and 18 days of growth in leaf and .stem callus, respectively. Differentiation of callus into shoots or roots was visible after 20 days of growth. Thus induction of organogenesis was necessary for the initiation of forskolin production. Pro-duction of forskolin was maximum after 40 days of growth in concentrations similar to market roots. Mature roots of C. forskohlii showed presence of cyto-plasmic vesicles in which terpenoids and other secondary metabolites were Fig. 1. Production of forskolin in callus cultures of Coleus forskohlii Production of forskolin translocation and segregation of the secondary metabolite as seen in the roots are not essential. Thus callus cultures could be a continuous and alternative source of the compound. HPTLC is a simple, sensitive, accurate and quick method for estimation of forskolin from roots and tissue culture of C. forskohlii and can be used as a routine method of analysis. References Abraham Z, Srivastava A K & Bagchi G D 1998 Cytoplasmic vesicles con-taining secondary metabolites in the root of Coleus forskohii (Willd.) Briq. Curro Sci. 57 : 1337-1339. Cline M G 1996 Exogenous auxin effects on lateral bud outgrowth in de-capitated shoots. Ann. Bot. (Lon-don) 78 : 255-266. Inamdar P K, Kanitakar P V, Reden J & De Souza N J 1984 Quantita-tive estimation of forskolin by TLC and HPLC. Planta Med. 50 : 30-34. Mersinger R, Dornauer H & Reinhard E 1988 Formation of forskolin by suspension cultures of Coleus forskohlii. Planta Med. 54 : 200-204. Mukherjee S, Ghosh B & Jba S 1996 Forskolin synthesis in vitro cul-tures of C. forskohlii Briq. trans-formed with Agrobacterium 157 tumefaciens. Plant Cell Rep. 15 : 691-694. . Murashige T & Skoog F 1962 A revised medium for rapid growth and bioassays with tobacco tissue cul-tures. Physiol. Plant. 15 : 473-497. Petersen M 1994 Coleus spp.: In vitro culture and the production of forskolin and rosmarinic acid. In: Bajaj Y P S (Ed.) Biotechnology in Agriculture and Forestry. Medici-nal and Aromatic Plants (pp. 69-92). Springer-Verlag, Berlin Heidelberg. Sen J & Sharma A K 1991 In vitro propagation of Coleus forskohlii Briq. for forskolin synthesis. Plant Cell Rep. 9 : 696-698. Shah V 1996 Coleus forskohlii-an over-view. In: Handa S S & Kaul M K (Eds.) Supplement to Cultivation and Utilization of Medicinal of Plants (pp. 385-412).' Regional Research Laboratory, Jammu. Shah V, Bhat S V, Bajwa B S, Dornauer H & De Souza N J 1980 The occurrence of forskolin in the Labiatae. Planta Medica 39 : 183-185. Tripathi C K M, Basu S K, Jain S & Tandon J S 1995 Production of coleonol (forskolin) by root callus cells of plant Coleus forskohlii. Biotech. Lett. 17 : 423-426. 

Monitoring of forskolin production from roots and callus by HPTLC in Coleus forskohlii Briq.

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Monitoring of forskolin production from roots and callus by HPTLC in Coleus forskohlii Briq.

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