Root Cultures of Coleus forskohlii Briq.

Int J Pharm Sci Nanotech Vol 5; Issue 2 • July−September 2012 International Journal of Pharmaceutical Sciences and Nanotechnology

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Volume 5 • Issue 2• July – September 2012

Research Paper

MS ID: IJPSN-3-4-12-VEERESHAM

Strategies to Improve the Production of Forskolin from Hairy Root Cultures of Coleus forskohlii Briq. C.S. Reddy, Ch. Praveena and C. Veeresham* Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences, Kakatiya University, Warangal-506009, India Received March 4, 2012; accepted April 30, 2012 ABSTRACT The aim of this study was to elucidate the effect of elicitors and precursors on the production of forskolin from the hairy root cultures of Coleus forskohlii Briq. Hairy root cultures were established from leaf explants by infecting with Agrobacterium rhizogenes strain A4 on MS basal medium. Suspension cultures of hairy root cultures were initiated in MS medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L). We investigated the growth of biomass and forskolin production in suspension cultures of hairy roots. The production of forskolin was parallel to the growth of biomass. The maximum production of forskolin was observed after 5 weeks. With the objective to increase the yield of forskolin, abiotic elicitors such as salicylic acid (100 μM and

500 μM), copper sulphate (100 μM and 500 μM), methyl jasmonate (100 μM and 500 μM) and precursors such as αketoglutaric acid (0.2 mM and 1.0 mM), L-phenylalanine (0.2 mM and 1.0 mM) were added to hairy root cultures on different days of incubation period and evaluated their effects on production of forskolin. Elicitor, methyl jasmonate (500 μM) and the precursor, L-phenylalanine (1 mM) on day14 addition significantly enhanced the production of forskolin over the control hairy root cultures C. forskohlii. Given forskolin’s limited commercial supply, this study provides avenues for improving the production of forskolin in the hairy root culture of C. forskohlii.

KEYWORDS: C. forskohlii; elicitors; forskolin; hairy roots; precursors.

Introduction Coleus forskohlii (Willd.) Briq. [Synonym C. barbatus (Andr.) Benth.], family Lamiaceae (Labiatae) is an important ancient root drug in Indian Ayurvedic Medicine (Shah et al., 1996), containing an important secondary metabolite labdane diterpenoid forskolin which occurs exclusively in this plant (Ammon et al., 1982, Desouza et al., 1988, Shah et al., 1980). Forskolin has several biological and pharmacological activities that have been linked to its role as an activator of adenylate cyclase (Ammon et al., 1985). It is reported to be useful in conditions such as eczema, asthma, psoriasis, cardiovascular disorders and hypertension where a decreased intracellular cAMP level is believed to be a major factor in the development of the disease process (Rupp et al., 1985). Forskolin content of the roots obtained from natural habitats showed a range of variation from 0.04 to 0.44% dry cell weight (Vishwakarma et al., 1988). The total synthesis of forskolin reported involves 30 steps (Delpech et al., 1996). The sole source of forskolin is the roots of wild or cultivated C. forskohlii plants. Biotechnological approaches especially plant tissue culture are found to have potential as a supplement to traditional agriculture

in the industrial production of bioactive plant metabolites. Several valuable phytochemicals of pharmaceutical importance have been isolated from plant cell cultures and/or transformed cultures such as taxol, camptothecin, vinblastine and vincristine, podophyllotoxin, morphine and codeine, diosgenin, digoxin etc (Vanisree et al., 2004) and some of which are commercially produced. Hairy roots, the result of genetic transformation of Agrobacterium rhizogenes, have the attractive properties for secondary metabolites production. They often grow as fast as or faster than plant cell cultures (Charlwood et al., 1991; Flores et al., 1999) and do not require hormones in the medium. The advantage of hairy roots is that they often exhibit about the same or greater biosynthetic capacity for secondary metabolite production compared to their intact plants (Banerjee et al., 1998; Kittipongpatana et al., 1998) coupled with genetic stability (Flores et al., 1985; Flores et al., 1999). There are few reports on the in vitro production of forskolin suspension cultures, root cultures (Mersinger et al.,1988; Sen et al., 1992; Reddy et al., 2001) and transformed cultures (Krombholz et al., 1992; Zhouli et al., 1996; Mukherjee et al., 1996; Sasaki et al., 1998; Mukherjee et al., 2007) of C. forskohlii. Because of the

ABBREVIATIONS: FW- Fresh weight; IBA-Indole butyric acid; MJ-Methyl jasmonate; SA- Salicylic acid.

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Reddy et al: Strategies to Improve the Production of Forskolin from Hairy Root Cultures of Coleus forskohlii Briq.

relatively modest content of forskolin in the plant which limited its development as drug, and reports of production of forskolin through different plant tissue culture techniques, inspired us to study the strategies for enhanced production of forskolin from hairy root culture. However, to date there are no reports on the effect of elicitors and precursors on the production of forskolin from hairy root cultures. In the present paper, we described the establishment of hairy roots from leaf explants with A4 strain and the effects of elicitors and precursors on the production of forskolin from these transformed cultures.

Materials and Methods Plant Material Young leaves of C. forskohlii were washed under running tap water followed by treatment with surfactant Tween-20 10% v/v for 2-3 minutes. The excess of surfactant was removed by washing thoroughly with running tap water and finally with double distilled water. Then the leaves were surface sterilized by immersion in 2% v/v sodium hypochlorite for 10 minutes with occasional shaking for every 2-3 minutes in a laminar air flow chamber and finally leaves were thoroughly rinsed with sterile double distilled water to remove excess sodium hypochlorite.

Bacterial Strains Agrobacterium rhizogenes strains 15834, k599, LBA9402 and A4 were procured from the Institute of Microbial Technology, Chandigarh. The former three bacterial cultures on LB medium (sodium chloride 10 g/L, yeast extract 5 g/L, typtone 10 g/L, agar 15 g/L, pH 7) and A4 on YMB medium (Dipotassiumhydrogenphosphate dihydrate 0.655 g/L, magnesium sulphate heptahydrate 0.2 g/L, sodium chloride 0.1 g/L, yeast extract 0.4 g/L, D-mannitol 10 g/L, agar 15 g/L, pH 7) were maintained and sub cultured at 3-week intervals. Prior to infection the bacterial strains were grown for 48 hours in the respective liquid media containing 10 μM acetosyringone (Flores et al., 1987) at 25oC on rotary shaker at 120 rpm in the dark.

Induction and Establishment of Hairy Root Cultures The surface sterilized leaves were infected by injecting suspension cultures of Agrobacterium rhizogenes strains 15834, k599, LBA9402 and A4 near the veins for 48 hours. Thereafter, explants were cocultivated on a hormone-free MS medium with 3% sucrose, solidified with 1.1% agar (pH 5.6-5.8). After 2 days of co-cultivation in the dark at 25 + 2oC with the individual bacterial strain, the leaf explants were transferred onto hormone free MS medium containing cefatoxime (500 mg/L) to eliminate free-living bacteria and then incubated at 25 + 2oC. Similar type of explants, pricked with a sterile needle devoid of bacterial

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suspension, were cultured under similar conditions as controls. Induction of hairy roots was observed within 1-4 weeks in A4 strain infected leaf explants. The emerging hairy roots were subsequently transferred to MS solid media containing cefetoxime (500 mg/L) for three subcultures at an interval of 3 weeks, for further proliferation. Once established, the roots were placed in antibiotic free media. For initiation of suspension cultures of hairy roots, hairy roots were transferred to MS liquid medium supplemented with IBA 1.0 mg/L, casein hydrolysate 600 mg/L and without hormones. The cultures were kept on a rotary shaker at 120 rpm at 25°C in the dark. Maintenance of hairy root cultures Hairy roots were cultured initially on full, half and one-fourth strengths of MS liquid medium containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L) and sucrose (3% w/v) and incubated in a NBS shaker incubator at 25 + 2oC and 120 rpm to find out the optimal medium for the growth of hairy roots (Archana et al., 2001). Confirmation of transgenic nature of hairy roots A detection of opines by paper electrophoresis was done to confirm the transformation (Petit et al., 1983). Fresh hairy roots of 100 mg were macerated with 100 μl of 1% hydrochloric acid in 2 ml eppendorff’s tubes and incubated for 5 minutes at 100°C. The extracts were centrifuged at 10,000 rpm for 5 minutes. After centrifugation, 10 μl of supernatant was spotted on whatmann 3 mm paper, along with standard mannopine, then dried with hot air flow and subjected to electrophoresis (100 V/cm, 10-15 minutes). The buffers used were formic acid/acetic acid/water (30/60/910) (Petit et al., 1983). The paper was dried and stained with alkaline silver nitrate for visualization of opines (Trevelyan et al., 1950). Growth and production kinetics For growth kinetic studies 50-60 mg actively growing hairy roots, consisting of five root tips of 40-50 mm long from 7 day old culture, were transferred to 100 ml conical flasks containing 25 ml of ¼th MS basal liquid medium supplemented with IBA (1 mg/L), casein hydrolysate (600 mg/L), sucrose (30 g/L). The flasks were incubated on a NBS shaker at 25 + 2oC and 120 rpm. The samples of hairy root cultures were withdrawn at the interval of 7 days till 42 days of growth. The contents of the flask were harvested, blotted dry and weighed. The hairy roots were kept in deep freezer for 24 hours with three volumes of dichloromethane for extraction of forskolin. The extracts were allowed to stand at room temperature and the dichloromethane layer was separated and filtered. The same process was repeated thrice for the complete extraction of forskolin. The extracts were pooled together and evaporated to dryness. The residue was dissolved in 1 ml methanol (HPLC grade) and subjected to TLC and HPLC analysis (Inamdar et al., 1984).

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Int J Pharm Sci Nanotech

Analysis

A4

strain

on

MS

Vol 5; Issue 2 • July−September 2012

media.

The

other

strains

of

Agrobacterium rhizogenes 15834, k599, LBA9402 were

The HPLC analysis of extracts and standard was done according to the method of Sasaki et al., 1998. The HPLC system consisted of solvent delivery module Shimadzu LC-10AT model equipped with C-18 column (Tracer Analitica Nucleosil-100, 25 cm x 0.4 cm diameter, 5 μ) with a Shimadzu Photo Diode Array (SPD-M10AVP model) detector. The mobile phase consisted of acetonitrile-water (60:40), (pH 2.5) degassed using bath sonicator. The injection volume of the standard solution and each of extracts was 20 μl injected using a Hamilton syringe. The chromatography was performed isocratically at a flow rate of 1 ml/minute at room temperature and forskolin was detected at 218 nm (Sasaki et al., 1998). The compound was detected on the basis of retention time Rt and comparison of UV spectra with the authentic standards.

failed to induce hairy roots. This is in agreement with the earlier reports where bacterial strain specificity was found to play a determining role in establishing hairy roots (Byrne et al., 1987; Porter et al.1991; Zehra et al., 1999). Leaf explants treated with a sterile needle devoid of bacterial suspension induced neither roots nor callus. To study the effect of acetosyringone on transformation efficiency, bacterial culture media with and without acetosyringone was used. The frequency of hairy root induction was enhanced with the addition of acetosyringone 10 μM to bacterial culture. Acetosyringone is known to be the activator of the vir genes of the Ti plasmid that would aid the successful transfer of T-DNA (Stachel et al., 1985). After 2-4 weeks numerous hairy roots appeared at the inoculated site. The obtained roots were maintained in media containing antibiotic cefatoxime (500 mg/L). The elimination of bacterial strain was successful after three subcultures done at 3 week intervals. Then the roots were placed in antibiotic free media. The success in clearing the bacterial growth from the roots is achieved on solid media but the roots did not develop well on hormone-free media hence the roots were transferred to liquid media containing IBA (1.0 mg/L), casein hydrolysate (600 mg/L) for the roots to grow faster, profuse branching with lateral rooting (Krombholz et al., 1992). The hairy roots were white in colour and highly branched and the percentage of initiation was 90.

Addition of Precursors and Elicitors to Hairy Root Cultures

Confirmation of Transgenic Nature of Hairy Roots

Hairy root cultures were cultured in a 50 ml flask containing 15 ml of ¼th MS medium supplemented with IBA (1 mg/L), sucrose (3% w/v) and casein hydrolysate (600 mg/L) and these cultures were incubated at 25 + 2oC and 120 rpm for one week. One week old inoculums (~50 mg) were transferred to fresh media of same composition and used for precursor and elicitor studies. Precursors such as α-ketoglutaric acid (0.2 mM and 1 mM) and Lphenylalanine (0.2 mM and 1.0 mM); elicitors such as salicylic acid (100 μM and 500 μM), copper sulphate (100 μM and 500 μM) and (100 μM and 500 μM) were added to each flask of hairy root cultures separately on day 14 or 28. For each concentration three culture flasks were used while running the suitable control. The flasks were incubated in a refrigerated shaker incubator at 25 ± 2°C and 120 rpm. The hairy root cultures of C. forskohlii, after 35 days of incubation with different concentrations of precursors and control cultures were extracted and analyzed.

Transgenic nature of hairy roots was confirmed by opine assay using non-transformed roots as control. Mannopine detected in transformed roots while it was absent in non-transformed roots indicating the transformation of hairy roots from leaves of C. forskohlii.

Thin Layer Chromatography (TLC) The concentrated extracts of hairy roots were applied on a precoated silica gel G plate (Merck) along with standard forskolin. The chromatogram was run using solvent system toluene-ethylacetate (85:15). After developing the chromatogram, the plates were air dried and the spots were detected by spraying anisaldehydesulphuric acid reagent, followed by heating at 140oC for 3 to 4 minutes.

High-Performance Liquid Chromatography (HPLC)

Results and Discussion Induction of Hairy Root Cultures The hairy root cultures were successfully induced from leaf explants within 1 week after inoculation with

Maintenance of Hairy Root Cultures The hairy roots were transferred into different strengths (full, half, one-fourth) of MS liquid medium with IBA (1.0 mg/L), casein hydrolysate (600 mg/L) and incubated on a NBS shaker at 120 rpm at 25 ± 2°C and sub-cultured at two week interval onto to the fresh medium. The growth of hairy roots was similar on full, half and one-fourth MS mediums. Thus, on economical grounds, one-fourth strength MS medium was used for the further experiments.

Growth and Production Kinetics Rapid growth started after 2 weeks continued until fifth week as shown in Figure 1. Maximum growth index (6.16 + 0.70) was observed after 5 weeks. The production of forskolin was parallel with the growth indicating that forskolin is a growth-associated product, accumulated to maximum levels during the exponential growth phase in parallel with the biomass. This is in agreement with


earlier reports (Payne et al., 1991, Bhandra et al., 1997; Toivonen et al., 1990). The maximum production of forskolin (1.449 ± 0.023 mg/gm FW) from hairy roots was observed after 5 weeks. However, the growth of hairy root cultures was sustained at the same rate after 5 weeks but the forskolin content was declined at 6th week when compared with 5th week. So, thirty-five days of incubation is ideal for the production experiments.

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Effect of Precursors and Elicitors Precursors such as L-phenylalanine, ∝-ketoglutaric acid and elicitors such as SA, Copper sulphate and MJ were added to hairy root cultures to study their effect on the production of forskolin. The results pertaining to effects of precursors and elicitors are depicted in Figures 2 and 3.

Fig. 1. Growth index and production kinetics in hairy root cultures of

C.forskohlii.

Fig. 2. Effect of precursors on the production of forskolin in hairy root cultures of C. forskohlii.

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Fig. 3. Effect of elicitors on the production of forskolin from hairy root cultures of

C.forskohlii.

The addition of L-phenylalanine 1 mM on day 14 to the hairy root cultures significantly improved the production of forskolin (3.054 ± 0.099 mg/gm FW), with a ~2 fold increase over control hairy root cultures (1.4401 ± 0.023 mg/gm FW). The addition of 0.2 mM L-phenylalanine on day 14 showed less effect compared to 1 mM Lphenylalanine. The effect of L-phenylalanine 0.2 mM and 1 mM on day 14 or 28 was significant on the production of forskolin, compared to control hairy root cultures. Forskolin is biosynthesized through acetate-mevalonate pathway. The overall improvement of forskolin may be due to the incorporation of L-phenylalanine as a precursor in the biosynthetic pathway or reducing the competition of L-phenylalanine to be used for primary and secondary metabolism. The enhanced production of secondary metabolite by the supplementation of phenylalanine was observed with tissue cultures of Datura stramonium (Ballica et al., 1993), Capsicum frutescens (Lindsey, 1986), Cephaelis ipecacuanha (Veeresham et al., 1994), Taxus cuspidata (Fett-Neto et al., 1994a,b) Taxus wallachiana (Veeresham et al., 2003), Taxus chinensis (Srinivasan et al., 1996), Nothapodytes foetida (Sundarvelan et al., 2002) and Azadirachta indica (Balaji et al., 2003). The addition of α-ketoglutaric acid 1 mM on day 14 to the hairy root cultures significantly improved the production of forskolin (2.226 ± 0.037 mg/gm FW) over the control hairy root cultures (1.440 ± 0.023 mg/gm FW). The addition of 0.2 mM on day 14 showed less effect compared to the 1 mM of α-ketoglutaric acid. There was ~1.5 fold increase in forskolin production by supplementation of α-ketoglutaric acid 1 mM on day 14 over the control hairy root cultures. However, there is no significant effect of α-ketoglutaric acid 0.2 mM on day 14 or 28 on the production of forskolin, compared to the control hairy root cultures. The improvement in yield of forskolin over the control cultures might be due to involvement of α-ketoglutaric acid in the biosynthetic pathway of forskolin.

The addition of SA 500 μM on day 14 and 28 to the hairy root cultures improved the forskolin production by two fold (2.556 ± 0.097 mg/gm FW), (1.947 ± 0.154 mg/gm FW) respectively over control hairy root cultures (1.440 ± 0.023 mg/gm FW). However, SA 100 μM additions on day 14 (1.599 ± 0.027 mg/gm FW) and day 28 (1.726 ± 0.072 mg/gm FW) does not have any significant effect on the production of forskolin from hairy root cultures of C. forskohlii. The effect of SA on forskolin production was showed in dose response manner. The overall improvement of forskolin in hairy root cultures of C. forskohlii may be due to inhibition of catalyse activity leading to increased levels of hydrogen peroxide, that may stimulate the enzymes involved in the biosynthesis of forskolin. These present investigations are in agreement with earlier reports of Sandra et al., 2000 that SA increased significantly (2-12 fold) the release of alkaloids scopolamine and hyoscyamine in hairy root cultures of Brugmansia candida. Addition of copper sulphate 500 μM on day 14 to the hairy root cultures of C. forskohlii significantly enhanced the production of forskolin (2.787 ± 0.105 mg/gm FW) over control hairy root cultures (1.440 ± 0.024 mg/gm FW). Copper sulphate 500μM on day-28 also enhanced the production of forskolin (2.331 ± 0.112 mg/gm FW) over control hairy root cultures (1.440 ± 0.024 mg/gm FW). However, no significant effect was observed with the addition of copper sulphate 100 μM either on day 14 or 28. The overall improvement of forskolin may be due to the cellular damage caused by copper ions especially to membranes, could release endogeneous elicitors that increase the production of secondary metabolites as a stress response to the damage (Parry et al., 1994). Similar kinds of reports were found with Atropa belladonna (Lee et al., 1998), Hyoscyamaus albus (Masanori et al., 1998). Addition of MJ 500 μM on day 14 to the hairy root cultures of C. forskohlii improved the forskolin production by 2.7 fold over control hairy root cultures.


However, the addition of lower concentrations of elicitor 100 μM on day 14 (2.005 ± 0.011 mg/gm FW) or day 28 (1.951 ± 0.011 mg/gm FW) could not influence the production of forskolin in hairy root cultures of C. forskohlii. The overall improvement of forskolin may be due to de novo transcription and translation, ultimately leading to enhanced biosynthesis of secondary metabolites (Yukimune et al., 1996). Similar kinds of reports were reported with Hyoscyamus albus (Kuroyangi et al., 1998), Datura stramonium (Zabetakis et al., 1999), Catharanthus roseus (Sylvain et al., 2003) Narcissus confusus (Raul et al., 2004), Taxus baccata (Furmanowa et al., 1995) and Azadirachta indica (Balaji et al., 2003). Conclusions This is the first report on the effect of elicitors and precursors on the production of forskolin from hairy roots cultures C. forskohlii established with Agrobacterium rhizogenes A4 strian. The objective of attaining higher levels of forskolin from hairy root cultures of C. forskohlii has been achieved with abiotic elicitor MJ and precursor L-phenylalanine. However, these findings need to be scaled up for pilot plant production using bioreactors. Acknowledgements Mr. C. S. Reddy is thankful to UGC, New Delhi for the grant of SRF fellowship which enabled him to carry out this work. References Ammon HP and Kemper FH (1982). Ayurveda: 3000 years of traditional medicine. Med Welt. 32: 148-153. Ammon HP and Muller AB (1985). Forskolin: from an ayurvedic remedy to a modern agent. Planta Med, 46: 473-477. Archana G, Ravindra ST, Dhingra V, and Lakshmi Narasu M(2001). Influence of different strains of Agrobacterium rhizogenes on induction of hairy-roots and artemisinin production in Artemisia annua. Curr Sci 81: 378-382. Balaji K, Veeresham C, Srisilam K, and Kokate CK (2003). Azadirachtin, a novel biopesticide form cell cultures of Azadirachta indica. J. Plant Biotech. 5: 121-129. Ballica R, Ryu YDD, and Kado IC (1993). Tropane alkaloid production in Datura stramonium suspension cultures: Elicitor and precursor effects. Biotech. Bioengg. 41: 1075-1081. Banerjee S, Rahman L, Uniyal GC, and Ahuja PS (1998). Enhanced production of valepotriates by Agrobacterium rhizogenes induced hairy root cultures of Valeriana wallichii DC. Plant Sci. 131: 203–208. Bhandra R, and Shanks JV (1997). Transient studies of nutrient uptake, growth and indole alkaloid accumulation in heterotrophic cultures of hairy toot cultures of Catharanthus roseus. Biotechnol Bioeng. 55: 527-534. Byrne M, Mc Donnell R, Wright M, and Carnes M (1987). Strain and genotype specificity in Agrobacterium soyabean interaction. Plant Cell Tissue Organ Cult. 8: 3-5. Charlwood BV and Charlwood KA (1991). Terpenoid production in plant cell culture. In: Harborne JB and Tomas-Barberan FA (eds.), Ecological chemistry and biochemistry of plant terpenoids. Oxford: Clarendon Press, 95-132. Delpech B, Calvo D, and Lett R (1996). Total synthesis of forskolin. Tetrahedron Lett. 37: 1015-1018.

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