Industrial production forskolin by plant tissue culture

1. Pharmacognosy
Rajasekhar Reddy Alavala
M. Pharm., (PhD)
Assistant Professor,
K L COLLEGE OF PHARMACY, Vaddeswaram, Guntur, A.P-522502
Industrial production of Forskolin

2. ◦ Industrial production, estimation and utilization of the following
phytoconstituents:
Forskolin Sennoside
Artemisinin Diosgenin
Digoxin Atropine
Podophyllotoxin Caffeine
Taxol Vincristine and Vinblastine

3. Need of the hour
◦ Plants are capable of synthesizing
a variety of low-molecular-weight
organic compounds, called
secondary metabolites, usually
with unique and complex
structures.
◦ Plant secondary metabolites are
of tremendous importance, both
for the plant itself (for plant–
environment interactions) and to
humans, for their biological
activities that can have
therapeutic value.

4. ◦ Compared to the main molecules found in the plants,
these secondary metabolites were soon defined by
their low abundance, often less than 1% of the total
carbon, or storage, usually occurring in dedicated
cells or organs.
◦ India’s booming export trade in medicinal plants has
risen almost three-fold during the last decade.
◦ This boom in local use and export trade is depleting
many species from the wild, bringing some to the
edge of extinction.
◦ The major problem in conventional procurement of
MAPs is that only a few are cultivated; over 95% of
the medicinal plants used in India are collected from
the wild.

5. Need of the hour
◦ Some species are becoming difficult to obtain
in sufficient amounts to meet increasing
demands.
◦ Destruction of natural habitats and technical
difficulties in cultivation are also driving the
drastic reductions in plant availability.
◦ For the production of desirable medicinal
compounds from plants, biotechnological
approaches, specifically plant tissue culture,
has the potential to supplement traditional
agriculture in the industrial production of
bioactive plant metabolites.

6. Need of the hour
◦ Large-scale plant tissue culture is found to be an attractive
alternative approach to traditional methods of plantation,
as it offers a controlled supply of biochemicals
independent of plant availability.
◦ In vitro propagation and complete plant regeneration can
be a viable method of ex situ conservation of these
important species.
◦ Biotechnological production of important
phytopharmaceuticals (secondary metabolites) can
indirectly help in the conservation of important plants, by
reducing the demand for raw materials from the wild.

7. Forskolin
◦ Forskolin, an important secondary metabolite in Coleus forskohlii
(Lamiaceae), has several biological and pharmacological activities.
◦ Central Drug Research Institute (CDRI), Lucknow, India, in 1974,
revealed the presence of a hypotensive and spasmolytic component of C.
forskohlii that was named coleonol.
◦ Further investigation (Saksena et al., 1985) determined the exact chemical
structure of this labdane diterpene and its name was changed to forskolin
(7b-acetoxy- 8,13-epoxy-la,6b,9a-trihydroxy-labd-14-en-11-one)
◦ It shows positive inotropic, positive chronotropic and hypotensive
activity, inhibits thrombocyte aggregation, and decreases intraocular
pressure.

8. Forskolin
◦ Micropropagation and in vitro
culture for production of forskolin
◦ Forskolin synthesis in transformed
cultures:
gall calluses,
cell suspension cultures,
rhizogenic calluses, and
rooty and shooty teratomas
◦ Transformed cell suspension
culture
◦ Transformed hairy root cultures
◦ Molecular cloning

9. Transformed hairy root cultures
Establishment of C. forskohlii hairy root culture
◦ The shoots (2 cm in length) were surface-sterilized with 75% ethanol for 30 s and NaClO2 (available chlorine
concentration of 2%) for 10 min.
◦ Shoot buds (ca. 1 cm) excised from the sterilized shoots were incubated on hormone-free 1/2 Murashige and Skoog
(1962) (MS) solid medium containing 3% sucrose, and cultured at 25°C in 16 h light/day.
◦ The axenic plantlets of C. forskohlii were used as explants for the induction of hairy roots. Agrobacterium rhizogenes
strain MAFF 03-01724 subcultured on YEB liquid medium, was inoculated by needle on a newly cut surface of the
plantlets.
◦ After 2–3 weeks, several hairy roots appeared at the inoculated sites.
◦ Tips of the hairy roots were cut off and cultured on woody plant (WP) solid medium containing 3% sucrose and 0.5 g/l
Claforan for the elimination of bacteria.

10. Transformed hairy root cultures
Establishment of C. forskohlii hairy root culture
◦ The axenic hairy roots thus obtained were transferred to hormone-free WP solid medium containing 3% sucrose, in the
dark at 25°C. Insertion of T-DNA into the plant genome was determined by the detection of opine (mikimopine).
HPLC analysis of forskolin
◦ Fifty milligrams of lyophilized and powdered hairy roots was extracted with 5 ml CH2Cl2 for 30 min at 40°C. The
extracts were reduced to dryness in vacuo, and dissolved with 1 ml methanol, then filtered through a 0.45-µm
membrane filter.
◦ The filtrate was subjected to HPLC using TSKgel ODS-120A (4.6 mm internal diameter ×25 cm; Tosoh, Tokyo, Japan)
at 40°C and eluted with 50% acetonitrile at a flow rate of 1.0 ml/min. The eluate was monitored at 218 nm with a UV
detector (UV-8000; Tosoh).

11. Major salts (macronutrients )/ 1L
Ammonium nitrate (NH4NO3) 1,650 mg/l
Calcium chloride (CaCl2 · 2H2O) 440 mg/l
Magnesium sulphate (MgSO4 · 7H2O) 370 mg/l
Monopotassium phosphate (KH2PO4) 170 mg/l
Potassium nitrate (KNO3) 1,900 mg/l.
Minor salts (micronutrients)/ 1L
Boric acid (H3BO3) 6. 54 mg/l
Cobalt chloride (CoCl2 · 6H2O) 0.025 mg/l
Ferrous sulphate (FeSO4 · 7H2O) 27.8 mg/l
Manganese(II) sulphate (MnSO4 · 4H2O) 22.3 mg/ll
Potassium iodide (KI) 0.83 mg/l
Sodium molybdate (Na2MoO4 · 2H2O) 0.25 mg/l
Zinc sulphate (ZnSO4·7H2O) 8.6 mg/l
Ethylenediaminetetraacetic acid ferric sodium (NaFe-EDTA)
constituting 5 ml/l of a stock solution containing 5.57 g
FeSO4.7H2O and 7.45 g Na2-EDTA per litre of water.
Copper sulphate (CuSO4 · 5H2O) 0.025 mg/l
Vitamins and organics/ 1L
Myo-Inositol 100 mg/l Nicotinic Acid 0.5 mg/l
Pyridoxine · HCl 0.5 mg/l
Thiamine · HCl 0.1 mg/l Glycine 2 mg/l
Lactalbumin Hydrolysate (Edamin) (optional) 1 g/l
Indole Acetic Acid 1-30 mg/l Kinetin 0.04-10 mg/l
Murashige and Skoog (1962) (MS) solid medium: