De Novo Regeneration of Scrophularia yoshimurae YAMAZAKI (Scrophulariaceae) and Quantitative Analysis of Harpagoside, an Iridoid Glucoside, Formed in Aerial and Underground Parts of In Vitro Propagated and Wild Plants by HPLC
Abhay P. SAGARE, Chao-Lin KUO, Fu-Shin CHUEH, Hsin-Sheng TSAY
Biological and Pharmaceutical Bulletin
The family Scrophulariaceae comprises of some important medicinal plants such as Bacopa monnieri, 1) Digitalis spp., 2) Rehmannia glutinosa 3) and Scrophularia spp. Species of the genus Scrophularia have been used as crude drug in traditional medicine.     Scrophularia ningpoensis HEMSLEY, commonly known as "Xuanshen" in Chinese, is an important medicinal plant. The roots of S. ningpoensis have been used in the traditional Chinese medicine in the treatment of inflammation,
... , tonsillitis, abscesses of carbuncles 8) and constipation. 9) It can lower blood pressure 4,10,11) and blood sugar levels and also has antibacterial and antioxidant effects. 10,11) Small doses of S. ningpoensis are cardiotonic 8) and it is commonly used in combination with other herbs as nutrient and health strengthening agent. 10) The root contains iridoid glycosides, which are active principles, and small amounts of essential oils, alkaloids, flavanoids, and p-methoxycinnamic acid. 10) Scrophularia yoshimurae YAMAZAKI, a herbaceous perennial plant 40-60 cm tall, is indigenous to Taiwan. S. yoshimurae is used as "Xuanshen", a substitute for S. ningpoensis, in traditional Chinese medicine in Taiwan. 12) Populations of S. yoshimurae are distributed in the central mountain range of Taiwan and they are adapted to a narrow set of environmental conditions, found only at middle elevations (altitude about 1000-1300 m). 13) It is very difficult to locate plants of S. yoshimurae in wild. In Taiwan, the processed roots of Scrophularia ningpoensis are imported from mainland China and used as crude drug as S. yoshimurae is not cultivated on commercial scale and the roots collected from plants growing naturally in the mountains of Taiwan are insufficient to meet the local demand. Development of a rapid in vitro propagation system for S. yoshimurae would help in commercial cultivation and conserving the germplasm of this medicinally important species. A protocol for de novo regeneration and rapid micropropagation of Scrophularia yoshimurae (Scrophulariaceae) has been developed. Multiple shoot development was achieved by culturing the shoot-tip, leaf-base, stemnode and stem-internode explants on Murashige and Skoog (MS) medium supplemented with 4.44 m mM N 6 -benzyladenine (BA) and 1.07 m mM a a-naphthaleneacetic acid (NAA). Stem-node and shoot-tip explants showed the highest response (100%) followed by stem-internode (74.4%) and leaf-base (7.7%) explants. The shoots were multiplied by subculturing on the same medium used for shoot induction. Shoots were rooted on growth regulator-free MS basal medium and the plantlets were transplanted to soil and acclimatized in the growth chamber. The content of harpagoside, a quantitatively predominant iridoid glycoside, in different plant material was determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of harpagoside in the aerial and underground parts of S. yoshimurae was significantly higher than the marketed crude drug (underground parts of Scrophularia ningpoensis).