Antioxidant Effects and Phytochemical Properties of Seven Taiwanese Cirsium Species Extracts.

In the present investigation, we compared the radical-scavenging activities and phenolic contents of seven Taiwanese Cirsium species with a spectrophotometric method. We further analyzed their phytochemical profiles with high-performance liquid chromatography-photodiode array detection (HPLC-DAD). We found that the flower part of Cirsium japonicum var. australe (CJF) showed the best radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and the hypochlorite ion, for which the equivalents were 6.44 ± 0.17 mg catechin/g, 54.85 ± 0.66 mmol Trolox/g and 418.69 ± 10.52 mmol Trolox/g respectively. CJF also had the highest contents of total phenolics (5.23 ± 0.20 mg catechin/g) and phenylpropanoids (29.73 ± 0.72 mg verbascoside/g). According to the Pearson's correlation coefficient, there was a positive correlation between the total phenylpropanoid content and ABTS radical-scavenging activities (r = 0.979). The radical-scavenging activities of the phenylpropanoids are closely related to their reducing power (r = 0.986). HPLC chromatograms obtained in validated HPLC conditions confirm that they have different phytochemical profiles by which they can be distinguished. Only CJF contained silicristin (0.66 ± 0.03 mg/g) and silydianin (9.13 ± 0.30 mg/g). CJF contained the highest contents of apigenin (5.56 ± 0.09 mg/g) and diosmetin (2.82 ± 0.10 mg/g). Among the major constituents, silicristin had the best radical-scavenging activities against DPPH (71.68 ± 0.66 mg catechin/g) and ABTS (3.01 ± 0.01 mmol Trolox/g). However, diosmetin had the best reducing power and radical-scavenging activity against the hypochlorite anion (41.57 ± 1.14 mg mmol Trolox/g). Finally, we found that flavonolignans (especial silicristin and silydianin) and diosmetin acted synergistically in scavenging radicals.

LED Lights Affecting Morphogenesis and Isosteroidal Alkaloid Contents in Fritillaria cirrhosa D. Don-An Important Chinese Medicinal Herb.

Investigations were carried out to study the effects of light-emitting diode (LED) lights on growth and development of isosteroidal alkaloids in embryogenic calli of Fritillaria cirrhosa D. Don, an important traditional Chinese medicine herb. Calli were cultured in glass bottles, each containing 100 mL of Murashige and Skoog's basal medium supplemented with 2% sucrose and 0.4% gellan gum powder, a gelling agent. These bottles were incubated in a specially designed plant growth chamber equipped with eight different LED lights consisting of single or combinations of four different light spectra emitting blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm) light. After three months of incubation, morphological changes in embryogenic calli were recorded, and LC-MS/MS analysis of cultures was carried out for peimisine, sipeimine, peiminine, and peimine. The highest number of somatic embryos and the maximum fresh weight was recorded in calli incubated under red (9R), infrared (9IR), and a combination of red+blue+infrared (3R3B3IR), respectively, in decreasing order. The highest contents of peimisine, peiminine, and peimine were recorded under red (9R) and infrared (9IR) lights, respectively. Eight LED lights had significant effects on the morphogenesis of embryogenic calli of F. cirrhosa D. Don and contents of isosteroidal alkaloids.

The protective ability and cellular mechanism of Koelreuteria henryi Dummer flower extract against hydrogen peroxide-induced cellular oxidative damage

BACKGROUND: Koelreuteria henryi Dummer is an indigenous plant in Taiwan. The species has been used in traditional folk medicine for the promotion of liver functions and for treating malaria and urethritis. The present study investigated the antioxidant activity of the flower extract of Koelreuteria henryi Dummer. The extraction conditions were optimized by the contents of total phenolic acids and total flavonoids, and antioxidant activity assays. Moreover, an in vitro study for investigating antioxidant activity of K. henryi flower extract was demonstrated by hydrogen peroxide (H2O2)-induced apoptosis. RESULTS: K. henryi flower extracted for 150 min showed high contents of total phenolic acids and total flavonoids. In an in vitro model, L929 cells were pretreated with K. henryi flower extract, and then treated with H2O2 to induce oxidative damage. Results demonstrated that H2O2-induced apoptosis was inhibited by the treatment of 200 µg/ml K. henryi flower extract through the mitochondria-mediated pathway and mitogen-activated protein kinase (MAPK) pathway. The caspase 8/9 activity and expression of p-p38 and pERK were repressed by K. henryi flower extract. In addition, the prevention of H2O2-induced apoptosis by K. henryi flower extract activated the nuclear factor-erythroid 2-related factor (Nrf2) stress response pathway to transcript heme oxygenase 1 (HO-1). Also, K. henryi flower extract prevented H2O2-induced apoptosis through HO-1 production, as evident by the use of HO-1 inhibitor. CONCLUSIONS: The present study demonstrated that K. henryi flower extract could inhibit the H2O2-induced apoptosis in L929 cells through the activation of the Nrf2/HO-1 pathway.

In vitro propagation of bulblets and LC-MS/MS analysis of isosteroidal alkaloids in tissue culture derived materials of Chinese medicinal herb Fritillaria cirrhosa D. Don.

BACKGROUND: Fritillaria cirrhosa, an important Chinese medicinal herb, is a Class-III protected and highly exploited species by pharmaceutical industry. Dwindling wild populations of species are unable to meet market demand. Therefore, this study was carried out to develop an in vitro propagation method for bulblet production. Also, the study aimed to carry out LC-MS/MS analysis of tissue culture-derived bulblets and callus for the presence of isosteroidal alkaloids (peimissine, verticine, and verticinone), and compare its quantities with commercially available crude drug samples. RESULTS: In vitro seed germination (91%) of F. cirrhosa was achieved on Murashige and Skoog's basal medium (MSBM) supplemented with 6-benzylaminopurine (1 mg L-1) and α-naphthalene-acetic-acid (0.4 mg L-1). On transfer of germinated seeds from Petri-dishes to glass bottles containing hormone-free MSBM, 37.5% of seedlings developed bulblets after 3 months of incubation. Regeneration and multiplication of bulblets were achieved by culture of transverse sections of bulblets on 1/2 X MSBM. By repeated subcultures at an interval of 2 months, 3072 bulblets weighing 1270 g could be produced at the end of 5th subculture. LC-MS/MS analysis showed a significant presence of peimissine in in vitro bulblets while callus incubated in the dark showed presence of peimissine and verticine. CONCLUSION: The study reports an efficient in vitro propagation method of bulblets production of F. cirrhosa and presence of some isosteroidal alkaloids in tissue culture-derived bulblets and callus. The study could be of immense help in production of F. cirrhosa bulblets and callus under laboratory conditions round the year. Also, these results can be used further to investigate production of isosteroidal alkaloids in bioreactors at commercial scale using liquid and cell suspension cultures. Thus, we not only can reduce our dependence on collections from natural habitats, but also can help in in situ conservation of this important species.

Sophora Tomentosa Extract Prevents MPTP-Induced Parkinsonism in C57BL/6 Mice Via the Inhibition of GSK-3ß Phosphorylation and Oxidative Stress.

Sophora species are used as dietary medicines in aging-associated symptoms. Sophora tomentosa L. (ST) is a native medicinal plant in Southeast Asia; however, there is no pharmacological literature about ST extract. The present study evaluates the antioxidant phytoconstituent contents and radical scavenging capacities of ST extract. The further investigation was to clarify the neuroprotective mechanism of ST extract against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism by assaying the activities of the dopaminergic system and antioxidant defenses, glycogen synthase kinase 3ß (GSK3-ß) phosphorylation, and α-synuclein levels in C57BL/6 mice. The results show that ST extract alleviated the motor deficits in MPTP-induced Parkinsonism with four behavioral tests, including a rearing locomotor, catalepsy test, balance beam walking test, and pole test. ST extract reversed the number of tyrosine hydroxylase (TH)-positive neurons in substantia nigra (SN) that had decreased by MPTP. ST extract also restored the decreased levels of dopamine and the expression of tyrosine hydroxylase (TH) in the striatum. Furthermore, ST extract restored the levels of glutathione (GSH) and the activities of antioxidant enzymes, and decreased the elevated levels of malondialdehyde (MDA) in mouse striatum. ST extract also decreased α-synuclein overexpression and GSK-3ß phosphorylation in mouse striatum. In vitro, ST extract exerted higher 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging capacities through its higher phenolic contents, especially protocatechuic acid and epicatechin. These results suggest that ST extract has the potential to counteract MPTP-induced motor deficit. The neuroprotective mechanism of ST extract against MPTP-induced Parkinsonism might be related to decreasing GSK-3ß phosphorylation and restoring the activities of striatal antioxidant defenses to restore the nigrostriatal dopaminergic function and decrease α-synuclein accumulation.

Aqueous Extract of Davallia mariesii Attenuates 6-Hydroxydopamine-Induced Oxidative Damage and Apoptosis in B35 Cells Through Inhibition of Caspase Cascade and Activation of PI3K/AKT/GSK-3ß Pathway.

The medicinal ferns of Polydiaceae and Davalliaceae species are called "Gusuibu" by Chinese physicians and used as antiaging dietary medicines. Our previous report revealed that Drynaria fortunei (Polydiaceae) protected against 6-hydroxydopamine (6-OHDA)-induced oxidative damage via the PI3K/AKT pathway in B35 neuroblastoma cells. The present study compares the antioxidant phytoconstituent contents and radical scavenging capacities of five Davalliaceae species. The further aim was to clarify the protective mechanism of Davallia mariesii (DM) against 6-OHDA-induced oxidative damage and apoptosis in B35 cells. The results show that Araiostegia perdurans (AP) and DM extracts have better radical scavenging capacities against 1,1-diphenyl-2-picryhydrazyl (DPPH) and reactive oxygen species (ROS) than other Davalliaceae species. However, only DM extract inhibited 6-OHDA autoxidation under cell-free systems and increased cell viability, compared to B35 cells solely exposed to 6-OHDA. DM extract decreased apoptosis and restored mitochondrial expression in 6-OHDA-treated B35 cells. Additional data indicated that DM extract decreased intracellular ROS and nitric oxide levels generated by 6-OHDA exposure. DM extract also restored glutathione (GSH) levels and the activities of glutathione peroxidase and reductase, and then decreased the elevated malondialdehyde (MDA) levels. Finally, DM extract regulated the protein expression of the caspase cascade and PI3K/AKT/GSK-3ß pathways. These results suggest that the protective mechanism of DM extract against 6-OHDA-induced oxidative damage and apoptosis might be related to its radical scavenging capacity, maintaining the mitochondrial function to inhibit the Bcl-2/caspase cascade pathway and activating intracellular antioxidant defenses (GSH recycling, HO-1 and NQO-1) by modulating the activation of the PI3K/AKT/GSK-3ß pathway.

Comparison of the Hepatoprotective Effects of Four Endemic Cirsium Species Extracts from Taiwan on CCl4-Induced Acute Liver Damage in C57BL/6 Mice.

Species of Cirsium (Asteraceae family) have been used in folk hepatoprotective medicine in Taiwan. We collected four Cirsium species—including the aerial part of Cirsium arisanense (CAH), the aerial part of Cirsium kawakamii (CKH), the flower part of Cirsium japonicum DC. var. australe (CJF), and Cirsii Herba (CH)—and then made extractions from them with 70% methanol. We compared the antioxidant contents and activities of these four Cirsium species extracts by a spectrophotometric method and high-performance liquid chromatography⁻photodiode array detector (HPLC-DAD). We further evaluated the hepatoprotective effects of these extracts on CCl4-induced acute liver damage in C57BL/6 mice. The present study found CAH possesses the highest antioxidant activity among the four Cirsium species, and these antioxidant activities are closely related to phenylpropanoid glycoside (PPG) contents. The extracts decreased serum ALT and AST levels elevated by injection with 0.2% CCl4. However, only CJF and CH decreased hepatic necrosis. Silibinin decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and hepatic necrosis caused by CCl4. CJF and CH restored the activities of hepatic antioxidant enzymes and decreased hepatic malondialdehyde (MDA) levels. CJF further restored the expression of hepatic antioxidant enzymes including Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), and glutathione S-transferase (GST) proteins. HPLC chromatogram indicated that CKH, CJF, and CH contained silibinin diastereomers (α and β). Only CJF contained diosmetin. Hence, the hepatoprotective mechanism of CJF against CCl4-induced acute liver damage might be involved in restoring the activities and protein expression of the hepatic antioxidant defense system and inhibiting hepatic inflammation, and these hepatoprotective effects are related to the contents of silibinin diastereomers and diosmetin.

Influence of LED light spectra on in vitro somatic embryogenesis and LC-MS analysis of chlorogenic acid and rutin in Peucedanum japonicum Thunb.: a medicinal herb.

BACKGROUND: 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. RESULTS: 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). CONCLUSION: 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.

In vitro culture and production of syringin and rutin in Saussurea involucrata (Kar. et Kir.) - an endangered medicinal plant.

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.

Protective effects of Drynaria fortunei against 6-hydroxydopamine-induced oxidative damage in B35 cells via the PI3K/AKT pathway.

In this study, we demonstrate the antioxidant and protective properties of the aqueous extract of two commercial Polydiaceae plants - Drynaria fortunei (DF) and Pseudodrynaria coronans (PC) against 6-hydroxydopamine (6-OHDA)-induced oxidative damage in B35 neuroblastoma cells. The contents of their phytochemical profiles were determined by spectrophotometric methods and high performance liquid chromatography using a photodiode array detector. DF extract showed better effects than PC extract in scavenging ROS and inhibiting 6-OHDA autoxidation. Following exposure to 6-OHDA, B35 cells showed a marked decrease in cell survival and the activation of intracellular antioxidant enzymes and the PI3K/AKT pathway, and then an increased level of lipid peroxidation. Pretreatment with DF extract blocked these 6-OHDA-induced cellular events. Naringin and epicatechin are major components of DF extract. These results show that DF extract exerts protective effects against 6-OHDA toxicity via radical scavenging activity and an increase in the activation of the PI3K/AKT pathway to elevate the levels of intracellular antioxidant enzymes including HO-1, NQO-1 and glutathione-related enzymes.

The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea.

Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.

In vitro propagation and analysis of secondary metabolites in Glossogyne tenuifolia (Hsiang-Ju) - a medicinal plant native to Taiwan.

BACKGROUND: Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Penghu Islands, Taiwan. The herb is a traditional anti-pyretic and hepatoprotective used in Chinese medicine. Several studies on G. tenuifolia have demonstrated its pharmacological values of antioxidation, anti-inflammation, immunomodulation, and cytotoxicity on several human cancer cell lines. Active compounds, oleanolic acid and luteolin in G. tenuifolia are affected by several factors, including climatic change, pathogens and agricultural practices. Plant population of G. tenuifolia has been severely affected and reduced considerably in natural habitat due to the use of herbicides by farmers. Also, collection of plant material from the natural habitat is restricted to a few months in a year. Therefore, the objective of the present study was to develop an efficient micropropagation protocol for G. tenuifolia. The study also aimed to investigate the influence of in vitro growth environment on the active compounds in in vitro shoots, tissue culture raised greenhouse plants; compare the values with wild plants and commercially available crude drug. RESULTS: Half-strength MS (Murashige and Skoog) basal medium supplemented with 0.1 mg/L 6-benzyladenine (BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA) induced the maximum average number of shoots (7.3) per shoot tip explant excised from in vitro grown seedlings. Induction of rooting in cent percent in vitro shoots with an average number of 6.6 roots/shoot was achieved on ½ strength MS medium supplemented with 3.0 mg/L indole-3-acetic acid (IAA). The rooted plantlets acclimatized successfully in the greenhouse with a 100% survival rate. HPLC analysis revealed that the quantity of oleanolic acid and luteolin in in vitro shoots, tissue culture plants in the greenhouse, wild type plants and commercial crude drug varied depending upon the source. The oleanolic acid and luteolin contents were found to be significantly higher (16.89 mg/g and 0.84 mg/g, respectively) in 3-month old tissue culture raised plants in greenhouse compared to commercially available crude drug (6.51 mg/g, 0.13 mg/g, respectively). CONCLUSIONS: We have successfully developed an in vitro propagation protocol for G. tenuifolia which can expedite its plant production throughout the year. The contents of oleanolic acid and luteolin in the tissue culture raised plants in the greenhouse were significantly higher than the marketed crude drug demonstrating the practical application of the tissue culture technology. These findings may be very useful in micropropagation, germplasm conservation and commercial cultivation of G. tenuifolia. So far, there is no published report on tissue culture propagation of this important medicinal plant species.