Peucedanum japonicum Thunb, an important medicinal herb is reported to possess pharmacological properties such as anti-obesity, anti-oxidant, anti-inflammatory, anti-bacterial, anti-diabetic and anti-platelet aggregation. The present study aimed to develop an in vitro plant regeneration system of P. japonicum via somatic embryogenesis and to analyse chlorogenic acid and rutin contents in a few commercially available plant products of P. japonicum in Japan and Taiwan markets, and tissue culture plants derived from somatic embryos. Induction of somatic embryogenesis could be achieved when root derived calli after three subcultures were transferred from Murashige Skoog’s salts and vitamins (MS basal) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1–5 mg/L) to a medium with abscisic acid (ABA) (0.5–4 mg/L), or exposed to eight different light spectra provided by light-emitting diode (LED) sources. Concentrations of ABA and LED light spectra had an influence on number of somatic embryos induced and proliferation of callus. Development of secondary somatic embryos and conversion of embryos to plantlets was achieved on a medium with ABA, or their exposure to red or blue lights in a special incubation chamber. Four months old tissue culture plants derived from somatic embryos showed significantly higher levels of chlorogenic acid (10.5 mg/g dw) compared to commercial product sold in Japanese market (0.55 mg/g dw). However, rutin was absent in tissue culture plants in contrast to commercial sample (0.33 mg/g dw). In this report, we describe in vitro plant regeneration system in P. japonicum via somatic embryogenesis and production of chlorogenic acid in tissue culture plants. The present study has application in further tissue culture propagation of elite plant material with high chlorogenic acid content, and identification of high yielding plants with the LC–MS method.

Wogonin (5,7-dihydroxy-8-methoxyflavone) is a flavone constituent of Scutellaria baicalensis with various beneficial biological activities and it has been shown to have tumor therapeutic potential in vitro and in vivo. The purpose of this study was to investigate the effects of wogonin in a human osteosarcoma cell line (U-2 OS). Results showed that a dose- and time-dependent reduction occurred in cell viability after exposure to wogonin in U-2 OS cells. Increasing the levels of reactive oxygen species (ROS) and Ca(2+) but decreasing the levels of mitochondrial membrane potential (Delta psi m) were examined in wogonin-treated U-2 OS cells. Flow cytometric assay indicated that wogonin induced sub-G1 phase (apoptosis) and increased caspase-3 activity in examined cells. Wogonin-induced apoptosis in U-2 OS cells was also confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining. Also, results from Western blotting indicated that wogonin increased the levels of Bad, Bax, cytochrome c, cleaved caspase-9, cleaved caspase-3, A IF, Endo G, Fas/CD95, caspase-8, GADD153, GRP78, ATF-6 alpha, calpain I, calpain 2 and caspase-4 then leading to cell apoptosis. In conclusion, wogonin induced ROS production and intracellular Ca(2+), and altered the levels of anti- (Bcl-2) and pro- (Bad and Bax) apoptotic proteins. Wogonin-induced apoptosis in U-2 OS cells was through the activation of caspase-3. In conclusion, these are the first findings to show wogonin-induced cytotoxic effects through induction of apoptotic cell death and ER stress in U-2 OS cells. The potent in vitro antitumor activities suggest that wogonin could be developed for the treatment of human osteosarcoma in the future

Background: Saussurea involucrata (Kar. et Kir.) commonly known as ‘snow lotus’ or ‘Xue Lian’ is an important plant in the traditional Chinese system of medicine. The plant contains flavonoids such as syringin and rutin. These compounds have been reported to be anti-rheumatic, anti-inflammatory and dilate blood vessels, lower blood pressure, prevent cardiovascular diseases, enhance immunity, and act as anti-aging, anti-cancer, and anti-fatigue agents. The species has become endangered due to the excessive collection of S. involucrata plants in the wild, slower plant growth and ecological destruction of natural habitats. There is a severe shortage of plant material, while the market demand is ever increasing. Hence, it is very important to apply tissue culture technique for plant propagation and production of the bioactive compounds of this species. Results: Multiple shoot induction and proliferation in shoot base explants derived from in vitro raised seedlings of S. involucrata was achieved on 3/4 strength of Murashige and Skoog’s (MS) basal medium (MSBM) supplemented with 1.0 mg/L⁻¹ BA and 1.5 mg/L⁻¹ NAA. Rooting was induced in 100 % shoots cultured on 1/2X MSBM supplemented with 1.0 mg/L⁻¹ IBA for one week and then transfer to auxin free medium. The plantlets could be acclimatized successfully by sachet technique and established in the greenhouse. Maximum callus induction and proliferation in leaf segments was achieved on 1/2X MSBM supplemented with 0.5 mg/L⁻¹ BA, 0.5 mg/L⁻¹ NAA, 0.4 % gelrite and on incubation at 20 °C. Container closures had an influence on the quality and quantity of callus and production of the active compounds. The HPLC analysis showed much higher syringin content in in vitro shoots and callus as compared to commercially available market crude drug. Conclusion: The present study describes an in vitro culture protocol of Saussurea involucrata. The bioactive compounds, syringin and rutin could be produced through tissue culture technique without sacrificing the endangered Saussurea involucrata plants in the wild.

In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC₅₀ value of 100 μM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (ΔΨ_m) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.