Tissue Culture Literation
| Plant latin name | Uncaria rhynchophylla (Miq.) Miq. |
| Literature code | Uncaria_rhynchophylla-Ref-1 |
| Reference | Kohda H. et al., Chemical and Pharmaceutical Bulletin 44: 352-357 (1996) |
| Summary | The effects of growth hormones and nutrients on growth and alkaloid production in callus cultures of Uncaria rhynchophylla (MIQ) MIQUEL (Rubiaceae) were investigated. Gamborg B5 (B5) medium supplemented with indoleacetic acid (10-4 M) and 6-benzylaminopurine (3×10-5 M) was optimized both for the growth (1.8g fresh weight/5 week) and alkaloid production (1.73 mg/g dry weight/5 weeks). The influence of sucrose concentration was examined, and 2% sucrose was found to be the most appropriate for the growth and alkaloid production. The effect of the composition of nitrogen sources was examined. Maximal growth and alkaloid production were obtained when 6 mM ammonium chloride and 25 mM potassium nitrate were added to the B5 medium. Hirsuteine, hirsutine, 3α-dihydrocadambine and ursolic acid were isolated from the callus. The 3α-dihydrocadambine concentration of callus was ca. 50-fold higher than that of the hooks and stem of U. rhynchophylla. |
| Objectives | Producing effective alkaloids from Uncaria rhynchophylla. |
| Materials | U. rhynchophylla plants cultivated at the Medicinal Plant Garden of Hiroshima University. |
| Explant | The leaves were cut into 5 mm squares. |
| Initial culture | (Callus induction) The leaves were surface-sterilized with 70% ethanol for 10s followed by treatment with 8% chlorinated lime and were then rinsed twice with sterilized water. They were cut into 5 mm squares and then placed on MS agar-gelled medium supplemented with 10-4 M NAA and 10-4 M -10-6 M Kin in the dark. |
| Shoot multiplication | (Callus growth and alkaloid production) The callus growth and alkaloid production were maximum when callus was cultured in B5 agar-solid medium supplemented with 10-4 M IAA and 3×10-5 M BAP, 2% sucrose and 6mM ammonium chloride and 25mM potassium nitrate. |
| Rooting | |
| Acclimation | |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
| Ingredients analyzed | Hirsuteine, hirsutine, 3α-dihydrocadambine and ursolic acid |
| Extraction | 50mg of lyophilized callus were extracted with 2ml of CH3CN-H2O-AcOH (50:100:1) overnight. |
| Analitical methods | After filtration, the alkaloid extract was analyzed by TLC and HPLC. TLC was carried out on silica gel plates using acetone-MeOH (20:1) and AcOEt-n-hexane (1:1) as solvents. The detection reagent used was a Dragen-droff reagent. HPLC was carried out TSKgel ODS-120T (250 mm×4.6 mm i.d., 5 μm) at a flow rate of 0.75 ml/min at 40℃. The mobile phase was CH3CN-H2O-AcOH (50:100:1). The effluent was detected at 254nm. |
| Notes |