Development of Coleus forskohlii as a medicinal crop

Virbala Shah and B.S. Kalakoti
Research Centre, Hoechst Marion Roussel Limited
Mulund, Bombay 400 080, India

Abstract

Coleus forskohlii belongs to the family Labiatae (Lamiaceae) and grows wild in arid and semi-arid regions of India. In a targeted plant screening programme at Hoescht AG the species was discovered to have pharmacological activities of lowering blood pressure and producing positive inotropic activity. The pharmacological activities were attributed to forskolin, a labdane diterpene, located in root tubers. Forecasts of the requirements of forskolin for drug development indicated the need for a sustained supply of root material in quantities that would threaten the survival of the species in nature. Concern for species conservation and a sustained supply of the root material led to consideration of developing C. forskohlii as a medicinal crop. The focus of the development studies was the increased yield of root tubers and forskolin. The development research entailed studies on natural species populations, evaluation of intraspecific variation, identification of suitable agroclimatic regions, standardization of growth conditions, and genetic improvement of elite genotypes. As a consequence of this development, C. forskohlii is now being cultivated as a source of forskolin.

Introduction

Development of a new medicinal plant in the pharmaceutical industries generally is taken up subsequent to the drug development. Our strategy at Hoescht AG in the case of C. forskohlii, differed from this standard strategy in that the crop development and the drug development activities were concurrent. Although the development activities were spread over 13 years, the species was commercialized in 1986, subsequent to identification of the superior strain and suitable agroclimatic zones.

· discovery of C. forskohlii as a potential drug (1974-1981)

· natural habitats and intraspecific variation (1982-1985)

· collection of germplasm and its evaluation (1983-1985)

· identification of suitable agroclimatic regions (1985-1986)

· optimization of growth conditions (1985-1987)

· genetic improvement (1989-1994)

Discovery of C. forskohlii as a potential drug

Coleus forskohlii Briq. (synonyms, C. barbatus Benth., Plectranthus forskohlii Willd., P. barbatus Andr. and P. comosus Willemse) belonging to the family Labiatae was collected in 1973 from Dehra Dun in North India for targeted pharmacological screening. The collection rationale was its phylogenetic relationship to a medicinal herb, C. amboinicus Lour. Methanol extract of root tubers exhibited blood pressure lowering and positive inotropic activities in animal models (de Souza 1977). Bioactivity-guided purification of the active extract provided an active labdane diterpene forskolin (Bhat et al. 1977). Subsequent research revealed forskolin to have many more pharmacological activities, namely, antiglaucoma, antiplatelet aggregation, antiinflammatory, antithrombotic (Rupp et al. 1985) and a biochemical activity of adenylate cyclase stimulant increasing intracellular cyclic AMP (Metzger & Lindner 1981,Seamon et al. 1981). As a consequence, demand increased for forskolin as a bioactive molecule for drug development and as a research tool for the study of the cyclic AMP dependent physiological phenomena. The increased demand warranted synthesis of the forskolin molecule or a sustained supply of the plant source. Since total synthesis of forskolin is not a practical route for meeting the demands of the compound, the latter option was considered. Concern for the sustained supply of the root material and conservation of the natural C. forskohlii led us to develop the species with increased root production and forskolin content.

Natural habitats and intraspecific variation

C. forskohlii grows wild on sun-exposed arid and semi-arid hill slopes of the Himalayas from Simla eastward to Sikkim and Bhutan, Deccan Plateau, Eastern Ghats, Eastern Plateau and rainshadow regions of the Western Ghats in India (fig. 1). Latitudinal and altitudinal range for the occurrence of the species is between 8^o and 31^o N and 600-800 m respectively. The species was studied for its ecological preferences in its native habitats throughout its distribution range excluding Eastern Plateau, Sikkim and Bhutan. Before the botanical studies were undertaken, the species was studied in the regional floras and herbarium specimens were examined in seven zonal herbaria of the botanical survey of India at Dehra Dun (Himalayan flora), Allahabad (Central India flora), Shillong (northeastern India flora), Jodhpur (Rajasthan flora), Pune (western India flora), Coimbatore (southern India flora) and Port Blair (Andaman and Nicobar group of islands flora), as well as at the Forest Research Institute, Dehra Dun and the Blatter Herbarium in Bombay. Eleven representative ecogeographic areas were selected for habitat and population studies (fig. 1); between 1982 and 1985, 27 botanical trips were made for the purpose. Coleus-growing areas in the Himalayas in Uttar Pradesh were visited every month from April to December, and the other areas were visited at least twice during the blooming period. The following is the summary of the observations made on different populations and habitats of C. forskohlii (Shah 1989).

· C. forskohlii is a subtropical and warm temperate species naturally growing at 600-1800 m elevation

· The species grows on sun-exposed hill slopes and plateaus in arid and semi-arid climatic zones

· The species inhabits loamy or sandy-loam soil with 6.4 to 7.9 pH

· The species is herbaceous with annual stems and perennial rootstock

· Populations from different ecogeographic areas vary greatly in their morphology

· Growth habit is strikingly variable being erect, procumbent or decumbent (fig. 2)

· Shoot height varies from 15.0 to 120 cm

· Lamina length varies from 1.5 to 15.5 cm² (fig. 3)

· Inflorescence length ranges from 3 to 40 cm

· Root morphology in different populations is fascinatingly diverse, being tuberous, semi-tuberous or fibrous (fig. 2)

· Fresh root yield in different populations ranges from 1 to 500 g per plant

· Forskolin content in roots varied from 0.07 to 0.58% of dry matter within and between the populations

Collection of germplasm and its evaluation

Initially 30 inter- and intra-populational ecotypes, including 2 cultivars (11 from the Himalayas, 18 from peninsular India and 1 from Sri Lanka) were collected for evaluation of root yields and forskolin content. Clones of these diverse ecotypes were grown vegetatively under uniform cultural conditions and at a single site on our factory campus in Bombay. This transplanting experiment proved the ecotypes to be genetic in nature. Based on growth habit, root and shoot morphlogy, root production, forskolin content and geographic location, the number of genotypes was reduced to 12-a manageable number for evaluation. Original locations of the 12 genotypes are shown in figure 1. Evaluation studies performed in Bombay identified a non-flowering cultivar to be superior in both parameters-forskolin content (about 0.4%) and root tuber yield (300-500 g plant^-1, and rarely 2000 g plant^-1). The other two varieties that were superior for one of the two parameters were a naturally growing variety, superior for forskolin content (about 0.4%) but with poor root tuber production, 50-200 g plant^-1, and another cultivar with poor forskolin content of about 0.1% or less but root tuber production as good as that of the non-flowering cultivar. These three cultivars were selected for further evaluation.

Figure 1. Distribution of Coleus forskohlii and research sites on the Indian subcontinent.

Figure 2. Diverse growth forms of Celus forskohlii.

Figure 3. Leaf variation in Celus forskohlii.

Identification of suitable agroclimatic regions

Suitable agroclimatic regions for commercialization of C. forskohlii were identified by the pilot cultivation at the sites listed in table 1, having different agroclimates. The investigation included the superior non-flowering cultivar variety and one or both of the two the varieties characterized above.

Table 1. Cultivation sites in India for Coleus forskohlii

The three cultivars under investigation at the above sites (table 1) reacted differently to the range of agroclimates. Forskolin content improved by 80-100% under the agroclimatic conditions of Bangalore and Salem, demonstrating these areas suitable for commercialization of C. forskohlii.

Optimization of growth conditions

A project with the Olericulture Department of the Tamil Nadu Agricultural University at Coimbatore was sponsored to determine optimal growth conditions: spacing, fertilizer (NPK) needs, planting time and harvest period. Cultural practices adopted for the experiment were as those followed in Gujarat, where the species is cultivated as a minor crop (site 12). Root tubers are consumed as a pickle in Gujarat and certain parts of Karnataka (site 11). The summary of the various trials is as follows.

· Experiments tested three levels of spacing (20, 40, and 60 cm) between the plants and 60 cm between the ridges. The dry root tuber yield after 5 months of growth was 3.2 kg, 2.9 kg and 2.6 kg per unit area respectively; the yield being proportional to the number of plants per unit area. No significant influence of planting density was observed on forskolin or root yields.

· Thirty combinations of NPK were tried. A combination of 40 kg of nitrogen, 60 kg of phophorus and 50 kg of potash was found to be optimal for maximum dry tuber yield. Enrichment of forskolin was not observed with this combination.

· Under Coimbatore conditions, June-July were found to be optimal months for planting the cuttings of C. forskohlii in terms of maximum dry tuber yield per unit area. The optimal period for harvesting tubers was 5.5 months later. December-April were found unsuitable for planting C. forskohlii. Leaving the crop unharvested beyond 5.5 months led to reduction in tuber yield.

Genetic improvement

Studies on genetic improvement (hybridization and polyploidy studies) of C. forskohlii, supported by Hoechst, were conducted at the Medicinal Plant Division of Indian Institute of Horticultural Research at Bangalore, under the supervision of Dr. R. Krishnan. Altogether 34 varieties-11 autotetraploids of diploids, 11 intervarietal hybrids, 11 autotetraploids of the hybrids, and 1 grafted variety-were developed over a period of 5 years from 1989 to 1994. An autotetraploid of the superior variety registered an increase of 23.1% in forskolin content, 1.3% over its diploid progenitor in pot studies. This tetraploid, using diploid progenitor as the check variety, was evaluated in a multilocational trial at four sites-three in Tamil Nadu and one in Karnataka for forskolin accumulation and root tuber yield. Percentage increases of forskolin in the tetraploid varied from site to site, being 2.3. 15, 17.7 and 51%. The root tuber yield, however, was decreased by 47.8, 46.1, 14 and 36.7% at the respective sites, indicating unstable growth characteristic of the autotetraploid and thereby making the autotetraploid unsuitable for commercialization. The diploid cultivar thus continues to be the variety of choice by the farmers.

Major accomplishments

· Through selection of a high forskolin-yielding variety and the determination of conditions for its optimum cultivation, forskolin yield has been increased eightfold.

· Natural resources of C. forskohlii were conserved through the development of a strategy to secure the plant source concurrently with the drug development, instead of the usual practice post-drug development.

Acknowledgments

I wish to thank Dr. W. Badziong, our former research director, and Dr. P. Hammann, the present director, for permission to present this paper; Dr. Bansi Lal, head of the Chemistry Department, for the encouragement; and my coworkers Mr. S.N. Godbole, Mrs. S. Mulye, Mr. R. Pereira and Mrs. Indira Sai for their assistance in preparation of the presentation.

References

Bhat S.V., Bajwa B.S., Dornauer H., Souza N.J. de & Fehlhaber H.W. 1977. Structure and stereochemistry of a new labdane diterpenoid from Coleus forskohlii Briq. Tetrahedron Letters 19:1669-1672.

de Souza N.J. 1977. p 83-92. In: Proceedings of the Third Asian Symposium on Medicinal Plants and Spices, Colombo, Sri Lanka. UNESCO.

Metzger H. & Lindner E. 1981. IRCS Medical Science 9:99.

Rupp R.H., de Souza N.J. & Dohadwalla A.N., eds. 1985. Proceedings of the International Symposium on Forskolin: its chemical, biological and medical potential. Hoechst India Limited, Bombay 400 080. p 19-30.

Seamon K.B., Padgett W. & Daly J.W. 1981. Proceedings of the National Academy of Sciences. U.S.A. 78:3363.

Shah V.C. 1989. Biosystematic studies on Coleus barbatus (Andr.) Benth. Ph.D. thesis. University of Bombay, Bombay, India.