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Near infrared (NIR) spectroscopy was employed to perform a quantitative analysis of gentiopicroside, the bioactive component of the medicinal plant Gentiana scabra Bunge. Modified partial least squares regression (MPLSR) and stepwise multiple linear regression (SMLR) calibration models were built using 94 plant tissue culture samples and 136 grown plant samples, respectively, over the full wavelength range (400–2498 nm) and the silicon charge-coupled-device (CCD) sensing band (400–1098 nm). For tissue culture, the smoothing, first-derivative MPLSR model can produce the best effect [calibration set ( R c ) = 0.868, standard error of calibration (SEC) = 0.606%, standard error of validation (SEV) = 0.862%] in the wavelength ranges of 900–1000, 1200–1300, and 1600–1700 nm. By contrast, for grown plant samples, the smoothing, second-derivative MPLSR model can produce the best effect ( R c = 0.944, SEC = 0.502%, SEV = 0.685%) in the wavelength ranges of 400–500, 1100–1200, 1600–1800, and 2200–2300 nm. With the silicon CCD sensing band, the smoothing, second-derivative, four-wavelength (670, 786, 474, and 826 nm) SMLR model showed best predictability ( R c = 0.860, SEC = 0.775%, SEV = 0.848%). This study successfully built spectral calibration models for determining gentiopicroside content at different growth stages of G. scabra Bunge. The specific wavelengths selected within the silicon CCD sensing band can be used in combination with multispectral imaging as a powerful tool for monitoring or inspecting the quality of G. scabra Bunge during cultivation.

In this study, optimization of smashing tissue extraction (STE), preliminary chemical characterization and antioxidant activity in vitro of crude polysaccharides (CPS) from Gentiana scabra bge (G. scabra) were investigated. The optimal extraction conditions were determined as follows: sample particle size of 80mesh, solid/liquid ratio of 1:34, extraction voltage of 157.09V and extraction time of 130.38s. Under these conditions, the extraction yield of CPS had reached 15.03±0.14% (n=3). Chemical composition analysis indicated CPS was mainly composed of mannose, rhamnose, galacturonic acid, glcose, galactose, arabinose and fucose in a molar ratio of 1.00:9.89:51.59:35.37:38.06:99.13:21.34, respectively. The average molecular weight of CPS was estimated to be 3.8×104Da. In addition, the potential antioxidant activity of CPS extracted by STE were demonstrated by DPPH radical scavenging assay, superoxide anion radical scavenging assay, hydroxyl radical scavenging assay and ferric reducing power assay. Overall, this study provided an effective extraction technique for G. scabra polysaccharides which would be explored as a promising natural antioxidant agent applied in functional foods or medicines.

Medicinal plants have played a significant role in maintaining human health and improving the quality of life for thousands of years. In the past few decades, there has been an exponential rise in demand for herbal medicines, which in turn has resulted in an overexploitation and dwindling supply of medicinal plants in the wild. Therefore, it is imperative to explore all possible modes of plant propagation and large-scale cultivation. Taiwan is the home of many highly valued medicinal herbs used in the traditional Chinese medicines. In our laboratories, for more than two decades, tissue culture techniques have been used successfully for the propagation of several medicinally important plant species. The present article reviews the studies carried out on in vitro propagation of selected medicinal plants in Taiwan (Glossogyne tenuifolia, Saussurea involucrata, Gentiana scabra, and Drynaria fortunei). The article also includes the studies on induction of somatic embryogenesis in medicinal herb Peucedanum japonicum and influence of different ventilation closures on hyperhydricity (vitrification) in cultures of Scrophularia yoshimurae, Bupleurum kaoi, Gentiana scabra, and Glossogyne tenuifolia.

Glucagon-like peptide-1 (GLP-1) which secreted by enteroendocrine L cell have been issued as a therapeutic agent for type II diabetes mellitus, because of its function to stimulate insulin secretion in pancreatic β-cells. It is widely studied that sweet, bitter, and umami taste stimuli induce the secretion of GLP-1 in enteroendocrine L cell via G protein coupled receptor (GPCR) signaling pathway. Gentiana scabra (GS) is one of the bitter tasting herbal medicines which have been used in traditional oriental medicine to treat the diabetes mellitus. This study shows the GLP-1 secretion of the enteroendocrine L cell stimulated by stimulation of GS extractions through GPCR pathway. To examine the molecular mode-of-action, GLP-1 ELISA and microarray was performed. This study provides the understanding the possibility of GS used as a therapeutic herbal medicine for the type II diabetes.

Flower senescence of the potted gentian ( Gentiana scabra ) ‘Shinbisei’ was investigated in relation to ethylene sensitivity and production. ‘Shinbisei’ flowers were used for all experiments except for those with inflorescences. Exposure to ethylene at 0.5 μL L −1 or higher concentrations for 24 h markedly accelerated flower senescence, indicating that G. scabra flowers are highly sensitive to ethylene. Treatment with 0.2 or 0.5 mM silver thiosulfate complex (STS) and 2 μL L −1 1-methylcyclopropene (1-MCP), ethylene action inhibitors, and 50 mM α-aminoisobutyric acid, an inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, did not delay flower senescence. However, treatment with 1 mM l -α-(2-aminoethoxyvinyl) glycine, an inhibitor of ACC synthase, slightly delayed flower senescence. Pollination significantly accelerated petal senescence of G. scabra flowers. Ethylene production of petals, gynoecium, and stamens in unpollinated flowers slightly increased during senescence. Pollination significantly increased ethylene production of petals, gynoecium and stamens 1 day after pollination. To clarify whether 1-MCP delays senescence of cut gentian inflorescences, cut G. scabra ‘Yuki-hotaru’, G. scabra × Gentiana triflora ‘Aoi-kaze’, and G. triflora ‘Koharu’ inflorescences with various stages of flowers, including buds with colored petals, were treated with 2 μL L −1 1-MCP for 24 h. 1-MCP treatment delayed flower wilting of cut inflorescences of ‘Aoi-kaze’ and ‘Yuki-hotaru’ more than that of ‘Koharu’, suggesting that there is species variation in the effect of 1-MCP in delaying flower senescence of cut gentian inflorescences.

Development of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) agents is pivotal to prevent the reemergence of the life-threatening disease, SARS. In this study, more than 200 extracts from Chinese medicinal herbs were evaluated for anti-SARS-CoV activities using a cell-based assay that measured SARS-CoV-induced cytopathogenic effect (CPE) in vitro on Vero E6 cells. Six herbal extracts, one each from Gentianae Radix (龍膽 lóng dǎn; the dried rhizome of Gentiana scabra), Dioscoreae Rhizoma (山藥 shān yào; the tuber of Dioscorea batatas), Cassiae Semen (決明子 jué míng zǐ; the dried seed of Cassia tora) and Loranthi Ramus (桑寄生 sāng jì shēng; the dried stem, with leaf of Taxillus chinensis) (designated as GSH, DBM, CTH and TCH, respectively), and two from Rhizoma Cibotii (狗脊 gǒu jǐ; the dried rhizome of Cibotium barometz) (designated as CBE and CBM), were found to be potent inhibitors of SARS-CoV at concentrations between 25 and 200 μg/ml. The concentrations of the six extracts needed to inhibit 50% of Vero E6 cell proliferation (CC50) and 50% of viral replication (EC50) were determined. The resulting selective index values (SI = CC50/EC50) of the most effective extracts CBE, GSH, DBM, CTH and TCH were > 59.4, > 57.5, > 62.1, > 59.4, and > 92.9, respectively. Among these extracts, CBM and DBM also showed significant inhibition of SARS-CoV 3CL protease activity with IC50 values of 39 μg/ml and 44 μg/ml, respectively. Our findings suggest that these six herbal extracts may have potential as candidates for future development of anti-SARS therapeutics. Abbreviations SARS, severe acute respiratory syndrome CoV, coronavirus CPE, cytopathogenic effect TCM, traditional Chinese medicine

In traditional Chinese medicine, the rhizomes of Gentiana scabra are used to treat hepatitis, stomatitis, and inflammatory diseases. This study aimed to examine the protective effects of G. scabra aqueous extract (GS) and polyphenols in liver of CCl4-intoxicated mice. Antioxidant activities of GS were also evaluated. Results showed that GS exhibited anti-lipid peroxidation, 2,2-diphenyl-1-picrylhydrazyl, and superoxide radical scavenging activities with IC50 values of 45.84, 183.38, and 56.25 μ/mL, respectively. High-performance liquid chromatography analysis revealed that the major polyphenolic constituents of GS were kaempferol, ellagic acid, and quercetin. Daily oral administration of 500 mg/kg GS (GS500) and 1000 mg/kg GS (GS1000) significantly prevented the elevation of glutamic pyruvic transaminase and thiobarbituric acid reactive substances levels, while enhancing the levels of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and serum total antioxidant activity in mice with hepatoxicity. At 1000 mg/kg, GS was as effective as 100 mg/kg silymarin in reducing oxidative stress and preventing liver injury. Histopathological studies further confirmed the hepatoprotective activity of GS. Taken together, these results show that the antioxidant activities and polyphenolic compounds (kaempferol, ellagic acid, and quercetin) of GS may have contributed to its hepatoprotective activity in CCl4-intoxicated mice, and its mechanism of action could be mediated through the reduction of oxidative stress in liver tissue.

When the influence of host species, inoculum density, temperature, leaf wetness duration, and leaf position on the incidence of gentian brown leaf spot caused by Mycochaetophora gentianae, was examined, the fungus severely infected all seven Gentiana triflora cultivars, but failed to infect two cultivars of G. scabra and an interspecific hybrid cultivar. Inoculum density correlated closely with disease incidence, and a minimum of 102 conidia/mL was enough to cause infection. In an analysis of variance, temperature and leaf wetness duration had a significant effect upon disease incidence, which increased with higher temperature (15–25°C) and longer duration of leaf wetness (36–72 h). No disease developed at temperatures lower than 10°C or when leaf wetness lasted 50% of conidia germinated at temperatures >15°C after 24-h leaf wetness. More appressoria formed at higher temperatures (15–25°C) with extended duration of leaf wetness (24–72 h). At 48-h leaf wetness, appressorium formation was 0, 8, 26, and 73% at 10, 15, 20, and 25°C, respectively. These results suggest that temperature and leaf wetness duration were important factors for infection of gentian leaves.

Mycochaetophora gentianae, the causal agent of brown leaf spot on gentian (Gentiana scabra), is characterized by its hyaline besom-like sporophore, although its conidiogenesis and phylogenetic position have so far remained unknown. We isolated the causal fungus from a new host, G. triflora, in Iwate, Japan. Both the G. triflora isolate and the ex-type M. gentianae isolate produced symptoms on G. triflora but not on G. scabra. Microscopic observations of the diseased leaves indicated that conidiogenesis was blastic from short conidiophores, and schizolytic secession of conidia left unthickened and inconspicuous conidial scars on the conidiogenous cells. Conidia were catenate, in branched acropetalous chains; secondary conidia were blastically produced from the first or second cell at the base of primary conidium. The G. triflora isolate was identified as M. gentianae because of its identity to the ex-type in characteristics of culture, pathogenicity, and conidia. Phylogenetic analyses using three ribosomal DNA (rDNA) sequences combined [small subunit (SSU) + large subunit (LSU) + 5.8S rDNA] indicated that both isolates clustered with Rhexocercosporidium carotae, and the cluster was placed within Helotiales–Rhytismatales. Additional analyses using internal transcribed spacers including 5.8S rDNA sequences revealed that both isolates were monophyletic and that they were closely related to three helotialean Pseudocercosporella-like hyphomycetous genera: Helgardia, Rhexocercosporidium and Rhynchosporium.

リンドウの褐斑病は1984年に岡山県で発生し，葉に多数の病斑を形成し，激しい場合には全体を褐変枯死させ，その被害の激しさから問題となっている．病原菌は病葉内部から病斑上に立ち上がった菌糸より毛鞠状ないし箒状の胞子塊を形成し，その所属は未決定であった．分生子懸濁液の噴霧接種により無傷でも潜伏期間が短く，発病後の進展も速やかであることが再確認された．病原菌の形態および分生子塊形成過程は，1931年に原・小川によって設立されたケヤキ円星病菌Mycochaetophora japonicaに酷似したが，分生子の大きさ，隔壁の数などが異なり，ケヤキには病原性を示さなかったことから，同属の新種としてM. gentianae Tak. Kobay., Kasuyama et Nasuと命名記載した．

High‐speed countercurrent chromatography (HSCCC) was applied to the separation and purification of 8‐hydroxy‐10‐hydrosweroside, swertiamarin, and trifloroside from the Chinese medicinal herb Gentiana scabra Bunge. Fifty milligrams of crude extracts were separated using n‐hexane‐1‐butanol‐methanol‐0.4% acetic acid in water (1.4:8:3:15.5, v/v) as the two‐phase solvent system yielding 8 mg of 8‐hydroxy‐10‐hydrosweroside, 18 mg of swertiamarin, 11 mg of trifloroside, and 4 mg of an unknown compound with the purity of 96.7, 98.4, 97.1, and 96.2%, respectively, in a one step separation. The chemical structures of these components were identified by ESI‐MS.

The heterodimer formation between B-class MADS-box proteins of GsAP3a and GsPI2 proteins plays a core role for petal formation in Japanese gentian plants. We previously isolated six B-class MADS-box genes (GsAP3a, GsAP3b, GsTM6, GsPI1, GsPI2, and GsPI3) from Japanese gentian (Gentiana scabra). To study the roles of these MADS-box genes in determining floral organ identities, we investigated protein-protein interactions among them and produced transgenic Arabidopsis and gentian plants overexpressing GsPI2 alone or in combination with GsAP3a or GsTM6. Yeast two-hybrid and bimolecular fluorescence complementation analyses revealed that among the GsPI proteins, GsPI2 interacted with both GsAP3a and GsTM6, and that these heterodimers were localized to the nuclei. The heterologous expression of GsPI2 partially converted sepals into petaloid organs in transgenic Arabidopsis, and this petaloid conversion phenomenon was accelerated by combined expression with GsAP3a but not with GsTM6. In contrast, there were no differences in morphology between vector-control plants and transgenic Arabidopsis plants expressing GsAP3a or GsTM6 alone. Transgenic gentian ectopically expressing GsPI2 produced an elongated tubular structure that consisted of an elongated petaloid organ in the first whorl and stunted inner floral organs. These results imply that the heterodimer formation between GsPI2 and GsAP3a plays a core role in determining petal and stamen identities in Japanese gentian, but other B-function genes might be important for the complete development of petal organs.

Three new (1-3) and 17 known (4-20) iridoid and secoiridoid glycosides were isolated from a methanol extract of the rhizomes and roots of Gentiana scabra. Their chemical structures were elucidated from 1D and 2D NMR, IR absorption, and HR-ESI-MS spectra, as well as comparisons of these data with reported values. The effects of the isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells were investigated. Compounds 6, 10 and 20 exhibited significant inhibitory effects on LPS-induced IL-12 p40 and IL-6 production with IC50 values of 1.62-14.29 μM. Compound 10 also showed a strong inhibitory effect on the LPS-stimulated production of TNF-α with an IC50 value of 10.45 μM.

Cucumber mosaic virus(CMV) and Broad bean wilt virus 2(BBWV2) were isolated from Gentiana scabra plants showing typical mosaic and yellowing symptoms. When the inoculum of mosaic symptom propagated in Nicotiana benthamiana was inoculated to primary leaves of Vigna sinensis, the local lesions of different types was developed. Type one produced a small necrotic spot(SNS) of pinpoint type, while the other one showed a large necrotic spot(LNS) of halo type. LNS on primary leaves of V. sinensis was also induced by inoculum from yellowing symptom on G scabra. Single lesion from SNS induced a typical mosaic symptom on N. Benthamiana. On the other hand, LNS produced a chlorotic ring symptom on inoculated leaves and mosaic plus necrotic ringspot on upper leaves of N. benthamiana. An isolate of CMV from SNS and BBWV2 from LNS were detected by using dsRNA analysis, RT-PCR and agar gel double-diffusion test. Thus, our results should provide a tool of a simple method for discrimination from mixed infected plants by CMV and BBWV2.

Alginate beads containing axillary buds of in vitro-grown gentian (Gentiana scabra Bunge var. buergeri Maxim.), were successfully cryopreserved following 2 step-preculture with sucrose and desiccation. The optimal preculture conditions were as follows: axillary buds were excised from in vitro-grown gentian plants and precultured on semi-solid Murashige and Skoog (MS) medium containing 0.1 M sucrose for 10 days (25 °C, 16-h photoperiod) (first step). This was followed by incubation on semi-solid MS media containing 0.4 M (1 day) and then 0.7 M sucrose (1 day) (second step). After preculture, the buds were encapsulated in alginate beads and desiccated aseptically on silica gel for 9 h to a water content of 10% (fresh weight basis), followed by immersion in liquid nitrogen (LN). With this protocol, 87% of the gentian buds survived exposure to LN and showed normal development of shoots and roots in vitro and in vivo. Depletion of NH4NO3 in the regeneration medium did not improve survival following desiccation and exposure to LN. The results show that 2 step-preculture with sucrose is effectively applicable in encapsulation–desiccation based cryopreservation of gentian axillary buds. This preculture can replace the conventionally used lengthy cold-hardening treatment and is useful for routine cryopreservation of gentian germplasm.

One-step reverse transcription-polymerase chain reaction (one-step RT-PCR) was successfully adapted to detect Bean yellow mosaic virus in the crude sap of infected dwarf gentian plants (Gentiana scabra). The virus was detected directly from crude sap at dilutions up to 106-fold (W/V), which is 100 times more sensitive than indirect enzyme-linked immunosorbent assay or bioassay after mechanical inoculation, and equally as sensitive as immunocapture RT-PCR. This one-step RT-PCR method using crude sap was also useful for detecting 16 virus species from ten genera with isometric, filamentous, and rod-shaped particles from 32 plant species in 15 families.

A viral isolate, designated N-1 and obtained from a gentian (Gentiana scabra) plant that exhibited mosaic symptoms, was transmitted mechanically to nine plant species in six families. These plants are known as hosts of fabaviruses. The N-1 isolate was composed of isometric particles 30 nm in diameter and included two RNA molecules of approximately 6.0 and 3.6 kb in length, as estimated by agarose gel electrophoresis. The RNAs were encapsidated separately in two of the three types of particle. Each particle contained two distinct proteins with Mr values of 39.3 × 103 and 26.6 × 103, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of complete nucleotide sequences of the RNAs suggested that each encoded a single large polyprotein, in which putative functional proteins were arranged in a manner similar to those in Broad bean wilt virus 1 (BBWV-1) and Broad bean wilt virus 2 (BBWV-2), which are members of the genus Fabavirus (family Comoviridae). Analysis of the deduced amino acid sequences of the proteins indicated that those of isolate N-1 shared 38 to 66% identity with those of BBWV-1 and BBWV-2 but only 16 to 42% identity with those of a comovirus, Cowpea mosaic virus. Phylogenetic analysis, based on the amino acid sequences of RNA polymerase, placed isolate N-1 in a separate lineage from BBWV-1 and BBWV-2. In indirect-enzyme-linked immunosorbent assay, isolate N-1 exhibited distant serological relationship to BBWV-1, BBWV-2, and Lamium mild mosaic virus, another fabavirus. Our results suggest that N-1 represents a new species of Fabavirus. We propose the name Gentian mosaic virus for this new species.

An attenuated isolate of Bean yellow mosaic virus (BYMV), designated B-33, that caused barely any mottling on the leaves of gentian plants (Gentiana scabra) was isolated from dwarf gentian plants. Infection with this isolate did not cause severe symptoms in several leguminous plants or in prairie gentian (Eustoma grandiflorum) plants. Dwarf gentian plants that had been infected with an inoculum of partially purified B-33 were propagated vegetatively from cuttings without difficulty. The B-33 isolate could be distinguished from other, more virulent, isolates by comparing symptoms on prairie gentian plants and by examining restriction fragment length polymorphism after reverse transcription and amplification by the polymerase chain reaction. In cross-protection tests, dwarf gentian plants that had been infected with B-33 were protected from infection with the virulent BYMV isolate 35-1. Potted plants that had been inoculated with B-33 grew larger and produced more flowers than unprotected plants. Thus, B-33 is an effective agent for controling mosaic and atrophic diseases caused by BYMV in clonal dwarf gentian plants.

開花時期の異なるエゾ系リンドウ3系統とササ系リンドウ1系統を供試し，展葉期と休眠期の茎頂培養個体の花らい形成について検討した．次いで，節培養に及ぼす花らいの有無，節位，あるいはNAAとホルクロルフェニュロン添加の影響を調査し，花らい形成個体の増殖の可能性を検討した． 展葉期および休眠期由来の培養個体において，培養期間が105日間では花らい形成率は低く，特に越冬芽を用いると全系統で6％以下となった．培養期間が165日間に延びると，展葉期由来の個体の花芽形成率はいずれの系統でも50％以上となったが，休眠期由来の個体では，エゾ系リンドウでは全く花らいがみられなかった．節培養において植物成長調整剤を与えない場合，花らい有個体上位節での草丈と発根数は，花らい無個体に比べて大きく劣った．しかし，下位節由来の個体の成長は，花らい有個体由来の方がむしろ優れていたので，花らいを形成しても，下位節を用いることで増殖は可能であると考えられた．NAAとホルクロルフェニュロンの添加によって，置床したすべての節でカルスが形成され，そこから多数のシュートが発生した．発生シュート数は花らい無個体由来で多かったが，花らい有個体由来からのシュート数も，頂部節で8.6本に達した．草丈も花らいの有無や節位による有意差は無く，得られたシュートは十分に増殖個体として利用し得ることが示された．

Five triterpenoids, (20S)-dammara-13(17),24-dien-3-one, (20R)-dammara-13(17),24-dien-3-one, chirat-16-en-3-one, chirat-17(22)-en-3-one and 17beta,21beta-epoxyhopan-3-one, were isolated from the rhizomes and roots of Gentiana scabra together with five known ones, chiratenol, hop-17(21)-en-3-one, hop-17(21)-en-3beta-ol, lupeol and alpha-amyrin. The structures of new compounds were elucidated on the basis of spectroscopic studies.

Background Gentiana scabra Bunge commonly known as ‘Long dan cao’ in China has been used in traditional Chinese medicines for more than 2000 years. Dry roots and rhizome of the herb have been used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are the main bioactive compounds which attribute to the pharmacological properties of this plant. The species is difficult to mass propagate by seed due to the low percentage of germination and limited dormancy period. Wild populations in some locations are considered to be in the endangered category due to over exploitation. Results In the present study, we report an efficient micropropagation system. Shoot apices of six weeks old in vitro grown G. scabra plants were used as explants for the in vitro propagation. Induction of multiple shoots (9.1/explant) was achieved on the culture of shoot apices on half strength Murashige and Skoog’s basal medium (MSBM) containing 2.0 mg/L−1 6-benzylaminopurine (BA), 3% sucrose and 0.9% Difco agar. In vitro shoots induced profuse rooting on half strength of MSBM supplemented with 0.1 mg/L−1 1-naphthaleneacetic acid (NAA), 3% sucrose and 0.3% gelrite. A two-stage ventilation closure procedure during the in vitro culture, and transparent sachet technique enhanced the survival rate of G. scabra plantlets to 96% in the greenhouse. Tissue culture plants flowered after 5 months of transfer to pots. Conclusions A simple and an efficient in vitro propagation protocol of Gentiana scabra Bunge by optimizing the medium composition and ventilation closure treatments has been developed. The protocol can be very useful in germplasm conservation and commercial cultivation of G. scabra plants.

Two polysaccharide fractions, GSP I-a and GSP II-b, were isolated from Gentiana scabra Bunge roots. Both GSP I-a and GSP II-b comprised seven monosaccharides: fructose, mannose, rhamnose, galacturonic acid, glucose, galactose, and fucose. Ultraviolet and infrared analyses show that GSP I-a and GSP II-b are proteoglycans. In vitro evaluation of the antioxidant activity suggests that GSP I-a and GSP II-b scavenge 1,1-diphenyl-2-picrylhydrazyl radicals. However, the scavenging activity of the latter is stronger than that of the former. GSP I-a and GSP II-b have relatively low reducing powers and scavenging activities toward superoxide anions and hydroxyls. GSP I-a and GSP II-b significantly increase lymphocyte proliferation when lipopolysaccharide is used as a mitogen for lymphocytes, but only GSP I-a can significantly increase lymphocyte proliferation within the test-dosage range when concanavalin A is used as a mitogen.

A simple protocol for in vitro mass propagation of Gentiana davidii var. formosana (Gentianaceae) has been developed. Multiple shoot development was achieved by culturing the stem node explants on Murashige and Skoog (MS) medium supplemented with 4.44 microM N6-benzyladenine (BA). The shoots were multiplied by subculturing on MS medium supplemented with 1.07-10.74 microM alpha-naphthaleneacetic acid (NAA) and 8.88 microM BA. Shoots were rooted on MS basal medium supplemented with various auxins. Shoots rooted on growth regulator-free medium were transferred to peat moss:vermiculite mixture and acclimatized in the growth chamber. The contents of gentiopicroside and swertiamarin, the two important secoiridoid glucosides, in different plant material were determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of gentiopicroside and swertiamarin in the aerial and underground parts of G. davidii var. formosana was higher than the marketed crude drug (underground parts of G. scabra) and varied with the age of the plant.

The flavonoid 3′,5′-hydroxylase (F3′,5′H) gene is in the NADPH-Cytochrome P450 reductase family. A partial F3′,5′H cDNA was cloned by RT-PCR from corollas ofGentiana scabra. Activity of F3′,5′H was observed during flower development, and activity was highest at the stage prior to flower opening. The enzyme activity was increased with the addition of SO μM gibberellic acid (GA3) immediately prior to flower opening. Western blot analysis showed that purified F3′,5′H protein fromG. scabra was cross-reacted with rabbit anti-Gentiana triflora NADPH-Cyt P450 reductase antibodies. Only one 80 to 82 kDa protein was detected by western blot analysis of fresh microsomal extracts. The enzyme activity was not distinctly changed by adding 50 μM GA3 to corollas. Therefore, we conclude that GA3 controls the expression of the F3′,5′H gene at the transcriptional level, not at the translational level.

In order to find out anti-platelet activating factor (PAF) from natural resources, Korean medicinal plants used for the treatments of peripheral circulation disorders were tested for their possible protective effects on PAF-induced anaphylactic shock. From the above screening, the methanol extract of Gentiana scabra showed a potent antagonistic activity against PAF. Water suspension of the extract was partitioned with CH2Cl2 and EtOAc, successively. The EtOAc fraction which showed the highest activity was chromatographed on silica gel to yield 6 fractions. From the fraction which showed higher PAF-antagonistic activity than the other fractions, compound 1 was isolated by recrystallization. On the basis of spectral data, compound 1 was identified as 2-hydroxy-3-methoxybenzoic acid glucose ester. The compound prevented the mice from the PAF-induced death at a dose of 300 micrograms/mouse.

Axillary buds of in vitro-grown gentian ( Gentiana scabra Bunge var. buergeri Maxim.) were successfully desiccated and cryopreserved using a novel two-step preculture method: preculturing axillary buds excised from in vitro-grown gentian plants on Murashige and Skoog (MS) semi-solid medium containing 0.1 M sucrose for 8–13 days at 25°C under a 16 h-photoperiod (first step) and the subsequent short period incubations on 0.4 M sucrose (MS agar medium) for 1 day followed by 0.7 M sucrose (MS agar medium) for 1 day (second step). After preculturing, the buds were air-dried down to about 10% water content (fresh weight basis) without encapsulation, and then immersed in liquid nitrogen (LN). With this cryogenic protocol, we could obtain 78–90% of survival of axillary buds following desiccation and LN exposure. Most of the buds that survived showed normal shoot and root development. The most important part of this protocol was the preculture which circumvents the conventionally used lengthy cold-hardening treatment. The results show that induction of desiccation tolerance by preculturing was specific to a few species of sugars used in the medium and not merely caused by osmotic effects. The efficient desiccation–cryopreservation protocol shown here using the new preculture method appears promising for routinely cryopreserving gentian germplasm.

Background Gentiana scabra is commonly known as ‘Longdan’ is an important herb in traditional Chinese medicines, commonly used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are main bioactive compounds which attributed to the pharmacological properties of this plant. The use of hairy root cultures as an excellent alternative for the production of pharmaceutically important metabolites in less time period with ensured quality of raw materials. Results An efficient hairy root culture system of Gentiana scabra and influence of different plant growth regulators (PGRs) on the production of gentiopicroside, swertiamarin and loganic acid constituents were described. Leaf explants were infected with Agrobacterium rhizogenes, which induced hairy roots up to 21%. The transformed hairy root lines were confirmed by PCR using rolB and rolC gene-specific primers. Among various solid and liquid media, B5 liquid medium resulted maximum root biomass (36- fold higher) in 4-weeks. Quantitative analysis showed loganic acid was 6.6- fold higher in the presence of zeatin (1 mg/l) and gentiopicroside accumulation was 1.8- fold higher in the presence of naphthaleneacetic acid (NAA, 1 mg/l), as compared to the roots of plants grown in greenhouse. On the other hand, 1.4- and 2.5- fold higher gentiopicroside and swertiamarin were observed in the presence of 1.0 mg/l NAA as compared to commercial Gentiana herb No. 2. The result also showed iridoid and secoiridoid contents affected greatly by age, physiology and growing environment of the plant. Conclusions The use of hairy root cultures is an excellent alternative to harvesting natural or in vitro grown plants to produce pharmaceutically important metabolites in less time with ensured quality.

Cultivated gentian (Gentiana scabra var.buergeri) has been seriously damaged by pink root rot since the 1970s. Diseased plants finally collapse after wilting and stunting. More than 40 fungal genera were found to be associated with roots of mature and immature diseased plants or seeds. Among these fungi,Pyrenochaeta gentianicola sp. nov. andP. terrestris were almost always associated with the diseased plants. Their morphologies and temperature responses were compared, and their pathogenicity was also demonstrated by artificial inoculation tests.

There has been a renewed interest in the use of herbal medicines throughout the world due to toxicities and health hazards associated with synthetic drugs and antibiotics. The World Health Organization estimates that up to 80% of people still rely mainly on traditional remedies such as herbs for their medicine, resulting in the increasing demand for medicinal plants. About 85% of traditional medicines involve the use of plant extracts. However, a large number of medicinal plants are needed to be investigated for their possible pharmaceutical value. Many pharmaceutical industries harness wildly growing plant populations for the supply of raw materials for extraction of medicinally important compounds. Many of the medicinal plants are severely threatened owing to illicit and indiscriminate collection and destruction of natural habitat. Advanced biotechnological methods of culturing plant cells and tissue provide an alternative means for rapid propagation and conservation of rare and endangered and/or commercially important medicinal plants. This chapter reviews the work carried out by our group in Taiwan on in vitro propagation of Pinellia ternata , Momordica charantia , Gentiana scabra var Bunge and Taraxacum formosanum and production of secondary metabolites from the callus of Stephania tetrandra , hairy root of Salvia miltiorrhiza and cell suspension culture of Gentiana davidii and Dioscorea doryophora .

Five triterpenoids, (20S)-dammara-13(17),24-dien-3-one, (20R)-dammara-13(17),24-dien-3-one, chirat-16-en-3-one, chirat-17(22)-en-3-one and 17β,21β-epoxyhopan-3-one, were isolated from the rhizomes and roots of Gentiana scabra together with five known ones, chiratenol, hop-17(21)-en-3-one, hop-17(21)-en-3β-ol, lupeol and α-amyrin. The structures of new compounds were elucidated on the basis of spectroscopic studies.

Two new secoiridoid glycosides, 4'''-O-beta-D-glucopyranosyltrifloroside 1 and 4'''-O-beta-D-glucopyranosylscabraside 2, along with three known secoiridoids were isolated from the rhizomes of Gentiana scabra (Gentianaceae) in our recent phytochemical study. Their chemical structures were determined by spectroscopic data including 1D and 2D NMR spectra. The chemotaxonomic significance of the secoiridoid glycosides is briefly discussed.

In order to quality control for medicinal plants such as Aconitum charmicaelii, Rhemannia glutinosa, Atractyrodes lancea, Pinellia ternata, Panax species and Gentiana scabra were clonally micropropagated by tip tissue culture and embryogenetic techniques. Monoclonal antibodies against the bioactive compounds contained in the crude drugs were prepared and set up the ELISA as a high sensitive and quick determination method. A newly developed eastern blotting methodology can stain typically the only antigen molecule and the related compounds having a same aglycone. Knockout extract can be prepared by using an immunoaffinity column conjugated with monoclonal antibody, and its importance has been discussed. Single chain fragment-variable gene against solamargine was cloned and transformed to a host plant, Solanum chasianum resulting in the increase of antigen molecule as 2.5 to 3 times. Three biosynthetic enzymes regarding marihuana compounds, THCA-, CBDA-, CBCA-synthases were isolated. THCAsynthase was cloned, over expressed and confirmed its characteristics including FAD combining enzyme, and finally determined the structure by X-ray analysis. The distribution of THCAsynthase was also investigated using its and GFP hybrid gene. We found new functions for saffron like improving learning and memory and LTP for blocking by ethanol. A folk medicine in Taiwan, Anoectochilus formosanus was propagated in vitro and investigated opening new pharmacological activities in lipid metabolism.

In order to quality control for medicinal plants such as Aconitum charmicaelii, Rhemannia glutinosa, Atractyrodes lancea, Pinellia ternata, Panax species and Gentiana scabra were clonally micropropagated by tip tissue culture and embryogenetic techniques. Monoclonal antibodies against the bioactive compounds contained in the crude drugs were prepared and set up the ELISA as a high sensitive and quick determination method. A newly developed eastern blotting methodology can stain typically the only antigen molecule and the related compounds having a same aglycone. Knockout extract can be prepared by using an immunoaffinity column conjugated with monoclonal antibody, and its importance has been discussed. Single chain fragment-variable gene against solamargine was cloned and transformed to a host plant, Solanum chasianum resulting in the increase of antigen molecule as 2.5 to 3 times. Three biosynthetic enzymes regarding marihuana compounds, THCA-, CBDA-, CBCA-synthases were isolated. THCAsynthase was cloned, over expressed and confirmed its characteristics including FAD combining enzyme, and finally determined the structure by X-ray analysis. The distribution of THCAsynthase was also investigated using its and GFP hybrid gene. We found new functions for saffron like improving learning and memory and LTP for blocking by ethanol. A folk medicine in Taiwan, Anoectochilus formosanus was propagated in vitro and investigated opening new pharmacological activities in lipid metabolism.

Pink-flowered gentian plants (Gentiana scabra) have been bred from spontaneous mutations of blue-flowered gentian plants, but the formation mechanism(s) is unknown so far. To investigate the process, two independent pink-flowered gentian plant lines were analyzed by a molecular biological approach. HPLC analysis showed that petals of the blue-flowered cultivar contained a small amount of cyanidin derivatives and major delphinidin derivatives, whereas pink petals had only a small amount of cyanidin derivatives. To find the causal factor(s) of this change, we focused on flavonoid 3',5'-hydroxylase (F3',5'H), which is a key enzyme for delphinidin biosynthesis in the flavonoid biosynthetic pathway. Molecular analyses confirmed that the loss of delphinidin synthesis could be attributed to the insertions of different transposable elements in the F3',5'H gene in each independent pink-flowered gentian plant. Sequence analysis showed that these transposable elements were classified into an hAT superfamily and terminal-repeat retrotransposon in miniature (TRIM), by which normal F3',5'H transcripts were interrupted. Southern blot analysis indicated that they belong to high copy number elements and are also found in a related gentian species (G. triflora). These results suggest that the transposable elements inserted in F3',5'H are the source of the mutations and may also play a substantial role in the genomic evolution of the genus Gentiana.

HPLC profile guided study led to the isolation of an acylated secoiridoid glycoside, named gentiotrifloroside (1), together with six known compounds, i.e., loganic acid (2), 6-O-beta-d-glucopyranosylgentiopicroside (3), swertiamarin (4), gentiopicroside (5), sweroside (6) and 2 -(o,m-dihydroxybenzyl)-sweroside (7) from Gentiana triflora and Gentiana rigescens. The structure of 1 was deduced from one- and two-dimensional NMR spectroscopic experiments. Compounds 1-7 were used successfully as chemical markers for the comparison of the four species of Gentiana used as Radix gentianae. Additionally, differentiation of Gentiana species mentioned and those used as adulterants was evaluated. The close similarity of chemical composition among the four genuine Gentiana species explain their popular usage as R. gentianae in Chinese medicine. We have also shown that the variation of chemical composition in R. gentianae and related adulterants agree well with their botanical phylogeny.

Micellar electrokinetic electrophoresis was employed to determine two active components, gentiopicroside (GE) and swertiamarin (SW) in one Tibetan preparation medicine named shiweilongdankeli, two Tibetan herbal medicines named Gentiana rhodantha and Gentiana kitag and three other Chinese Gentiana medicines named Gentiana scabra, Gentiana rigescens and Gentiana macrophylla. The dissociation constants of gentiopicroside and swertiamarin determined by MEKC were 7.71 and 6.25. The optimum buffer system was 70 mm borate-10 mm sodium dodecylsulfate (SDS) -6% (v/v) ispropanol (pH 9.0). The voltage was 15 kV and detection was at 254 run. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 3.86 mg L-1 for GE and 5.88 mg L-1 for SW. The relative standard deviation of the migration time and peak area of the GE and SW were 2.33, 2.47 and 1.27, 2.19%, respectively and the recoveries of the two compounds were 96-104% for GE and 92-102% for SW. Copyright (C) 2004 John Wiley Sons, Ltd.

Two new secoiridoid glycosides, 4'-O-beta-D-glucopyranosylgentiopicroside (1) and 6'-O-beta-D-glucopyranosylgentiopicroside (2), have been isolated from the rhizomes and roots of Gentiana scabra together with three known compounds, olivieroside, 1-O-beta-D-glucopyranosylamplexine, and benzyl alcohol O-alpha-L-arabinopyranosyl (1 --> 6)-beta-D-glucopyranoside. The structures of 1 and 2 were elucidated using chemical and physicochemical (MS and NMR) studies.

A new bitter acylsecoiridoid glucoside, rindoside, has been isolated along with a known iridoid glucoside, loganic acid, from rhizomes and roots of GENTIANA SCABRA var. BUERGERI. The structure of rindoside was established by chemical and spectral evidence.

A new strongly bitter acyl secoiridoid glucoside, scabraside, has been isolated along with two known secoiridoid glucosides, trifloroside and gentiopicroside (= gentiopicrin), from the rhizomes and roots of GENTIANA SCABRA var. BUERGERI. The structure of the scabraside was established by chemical and spectral evidence.