A high-frequency micropropagation protocol for Anoectochilus roxburghii (Wall.) Lindl. was developed via protocorm regeneration, induction and proliferation of protocorm-like bodies (PLBs), shoot regeneration, in vitro rooting, and acclimatization. Silver nanoparticles (AgNPs) were used for surface disinfection of floral-buds to regenerate protocorm which was divided into node, intercalary meristem, and internode to induce PLBs under the influence of plant growth regulators-PGRs (auxin and cytokinin), and silver nanoparticles (AgNPs). In addition, the effect of organic supplements (coconut water, potato starch and peptone) on PLB proliferation and shoot regeneration, and the influence of AgNPs on in vitro rooting, acclimatization, and kinsenoside accumulation were also evaluated. After 30 days, flower-bud explants regenerated protocorms a rate of 83.00% and 3.45 protocorms/explant on Schenk & Hildebrandt (SH) medium supplemented with 1.0 mg/L 6-Benzylaminopurine (BAP) or Thidiazuron (TDZ) only after 60 days. PLBs were induced predominantly at the node-intercalary meristem junction from protocorms cultured on SH medium containing BAP, alpha-naphthaleneacetic acid (NAA), and 1.5 mg/L AgNPs. The highest PLB proliferation (50.33 PLBs/explant) and shoot regeneration (35 shoots/PLB cluster) were achieved on SH medium supplemented with 20% coconut water, 1.0 mg/L BAP, 0.5 mg/L NAA, and 1.5 mg/L AgNPs after 90 days. Plantlet growth (plantlet height, leaf number and width, root number and length, fresh/dry weight, and SPAD index-chlorophyll content) was maximal on SH medium with 2.0 mg/L AgNPs. Plantlets from media containing 2.0 g/L AgNPs were acclimatized in a peat moss: perlite (1:1) substrate and reached 85% survival after 12 months in the greenhouse. Kinsenoside contents increased with cultivation time (3, 6, 9, 12 months) peaking at 2.591% in the whole plant after 12 months. This study establishes an efficient micropropagation protocol for A. roxburghii via protocorm and PLB regeneration.

Đặt vấn đề: Sâm đất Côn Đảo từ lâu đã được người dân sống ở Côn Đảo dùng như một loại thảo dược để cải thiện, tăng cường sức khỏe. Tại Việt Nam, chưa có nhiều công trình nghiên cứu về cây Sâm đất Côn Đảo để làm rõ thành phần hóa học của loài này. Mục tiêu nghiên cứu: Nghiên cứu này được thực hiện với mục đích nhằm phân lập và xác định cấu trúc của các hợp chất trong cao DCM chiết từ rễ Sâm đất Côn Đảo, làm tiền đề cho các thử nghiệm sinh học cũng như tiêu chuẩn hóa dược liệu. Đối tượng và phương pháp nghiên cứu: Rễ Sâm đất Côn Đảo thu hái tại vườn Quốc gia Côn Đảo vào tháng 5/2018. Dược liệu được chiết với cồn 96%, lắc phân bố lỏng với các dung môi có độ phân cực tăng dần. Các kỹ thuật sắc ký cột được sử dụng cho mục đích phân tách và phân lập hợp chất tinh khiết. Các hợp chất phân lập được xác định cấu trúc bằng kỹ thuật phổ MS, NMR. Kết quả: Từ cao DCM đã phân lập và xác định cấu trúc 2 hợp chất là: Maslinic acid và Pouzolignan N. Kết luận: Các hợp chất Maslinic acid và Pouzolignan N đã được phân lập từ phân đoạn cao DCM của rễ Sâm đất Côn Đảo và đây cũng là lần đầu tiên các hợp chất này được tìm thấy ở trong loài Parietaria debilis G.Forst.

Abstract Panax vietnamensis (Vietnamese ginseng, Ngoc Linh ginseng) is an endemic Panax species of Vietnam. From the methanol extract of the leaves of Panax vietnamensis, five compounds (1–5) were isolated, including one new sesquiterpene lactone such as panaxolide (1) and four known compounds. The structures of the compounds (1-5) were elucidated by spectral techniques such as 1 D NMR (1H NMR, 13C NMR), 2 D NMR (COSY, HSQC, HMBC, NOESY) and mass spectrometry. The absolute configuration of 1 was determined based on the Cotton effects in the CD spectrum. All of the five compounds were screened for their in vitro growth inhibitory activities against cancerous cells (HepG2) and normal cells (fibroblast) using the SRB assay. Panaxolide (1) showed the highest potential for the growth inhibition of cancerous cells HepG2 with the IC50 values of 63.8 μM. Graphical Abstract

A new benzofuran derivative, nervione (1), was isolated from Nervilia concolor (Blume) Schltr. (Orchidaceae). Eight previously reported compounds were also isolated: 5,7-dimethoxyflavone (2), 3,5,7-trimethoxyflavone (3), 7-methoxyflavone (4), 3,7-dimethoxy-5-hydroxyflavone (5), tetramethylscutellarein (4',5,6,7-tetramethoxyflavone) (6), 5,7-dimethoxy-4'-hydroxyflavone (7), rhamnetin (8), and 5,7-dihydroxy-3',4'-dimethoxyflavone (9). The structures were elucidated by 1D, 2D NMR, and HRESIMS spectroscopy in addition to the literature. The relative configuration of 1 was defined using DP4+ probability while its absolute configuration was defined by comparison of the ECD spectrum of 1 with those of previously reported compounds. All isolated compounds were evaluated for alpha-glucosidase inhibition, revealing weak or no activity.

Huperzine A, isolated from Huperzia serrata, is a potent, specific, and reversible inhibitor of acetylcholinesterase with high efficiency and low toxicity. To evaluate the presence of huperzine A in Vietnamese H serrata, a reliable capillary zone electrophoresis method was developed. The analytical conditions were established using 80 mM ammonium acetate buffer, pH 6.0, hydrodynamic injection at 50 mbar for 5 s, applied voltage of 20 kV, temperature at 25 °C, uncoated fused-silica capillary, 56 cm (50 cm effective length) × 70 µm inner diameter, and ultraviolet detection at 310 nm. The recovery rates ranged from 98.05% to 100.64%, with a relative standard deviation <2%. Good linear regression was observed in the concentration range of 1 to 500 µg/mL, with a correlation coefficient of 0.9994. The limit of detection and limit of quantification were 0.33 and 1.0 µg/mL, respectively. These results demonstrate that this method is simple, selective, and suitable for performing quality control for huperzine A derived from Vietnamese H serrata.

Bioactive-guided phytochemical investigation of Euphorbia antiquorum L. growing in Vietnam led to the isolation of five ent-atisanes, one seco-ent-atisane, and one lathyrane (ingol-type). The structures were elucidated as ent-1α,3α,16β,17-tetrahydroxyatisane (1), ethyl ent-3,4-seco-4,16β,17-trihydroxyatisane-3-carboxylate (2), ent-atisane-3-oxo-16β,17-acetonide (3), ent-3α-acetoxy-16β,17-dihydroxyatisane (4), ent-16β,17-dihydroxyatisane-3-one (5), calliterpenone (6), and ingol 12-acetate (7). Their chemical structures were unambiguously determined by analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high resolution mass spectrometry, as well as by comparison with literature data. Among them, 1 is a new compound while 2 is an ethylated artifact of ent-3,4-seco-4,16β,17-trihydroxyatisane-3-carboxylic acid, a new compound. Isolates were evaluated for alpha-glucosidase inhibition. Compound 3 showed the most significant inhibitory activity against alpha-glucosidase with an IC50 value of 69.62 µM. Further study on mechanism underlying yeast alpha-glucosidase inhibition indicated that 3 could retard the enzyme function by noncompetitive.

Ngoc Linh ginseng or Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) of the Araliaceae family, a endemic plant in Ngoc Linh mountain, is the 20th type of ginseng found in the world (1985). The content of saponin of Ngoc Linh ginseng is much higher than that of other ginsengs in the world such as Panax ginseng (Korean ginseng), anax quinquefolium(American ginseng) and anax notoginseng (Chinese ginseng). Until now, from roots, rhizomes and leaves of Ngoc Linh ginseng, there are 63 saponin which were isolated and determined structures. In addition, polyacetylenes, polyphenols, fatty alcohols and amino acids were also reported. Some of them showed diverse biological activities. In this research, from Ngoc Linh ginseng leaves, six compounds were isolated and structural elucidated by 1D and 2D NMR, MS spectroscopic methods and comparison of the spectral data with those in literature. They were one triterpene, squalene (1), one quinone terpenoid, a-tocopherolquinone (2), two fatty alcohols, tetradecanol (3), docosanol (4) from the -hexane extract and a steroid glycoside, daucosterol (5) and one flavonoid, kaempferol (6) from the methanol extract. All six compounds were reported for the first time from Ngoc Linh ginseng leaves.

From the leaves ofMarkhamia stipulatavar.canaenseV.S. Dang, one new phytosphingolipid, named markhasphingolipid A (6) together with five known compounds, 4',7-O-dimethylapigenin (1), narigenin (2), tectoquinone (3), mollic acid (4), 1-hexadecanoyl-sn-glycerol(5)were classified by various chromatographic methods. Their structures were designated by IR, UV, HR-ESI-MS, HR-ESI-MS/MS and NMR experiments. All compounds were recognized for the first time from this species. The cytotoxicity of alln-hexane fractions and isolated compounds (5&6)against three human cancer cell lines (HeLa, HepG2, and MCF-7) were evaluated by SRB assay. Alln-hexane fractions expressed cytotoxic effect on three tested cancer cell lines (at the concentration of 100 mu g/mL, percent of cytotoxicity ranged from 55.81% to 95.83%) as well as compound5(IC(50)ranged from 48.51 to 63.30 mu M) whereas fraction H.I and compound6did not show activity.

Four flavonols, including rhamnazin (1), nervilifordizin A, B (2, 3) and nervilifordin B (4) were isolated from the whole plant of Nervilia aragoana. All the compounds (1-4) were found for the first time from this species. Their structures were elucidated by 1D, 2D-NMR and compared with published data.

Introduction: Panax vietnamensis Ha et Grushv. (Ngoc Linh ginseng) – a new species recently discovered in Vietnam – has received much interest due to its rich content of saponins, including those unknown. This study assessed the effects of the Ngoc Linh ginseng extract fractions on proliferation and differentiation of cultured mouse neural stem cells. Methods: Whole brains were harvested from E13.5-14 Swiss mouse fetuses. Isolated cells were floating seeded to form spheroid bodies. Neurospheres were treated with one in fractions of ethanol 200-500 mg/mL, or nbutanol 200 mg/mL, or aqueous 200-500 mg/mL for 5 days. Neural stem cells could persistently generate secondary spheres. Neurospheres strongly expressed nestin, CD24 and deriving cells could differentiate into the GFAP-positive astrocyte-like cells. Results: Ginseng fractions significantly promoted neurosphere growth rate. Particularly, 200 mg/mL ginseng ethanol fraction significantly increased the neurosphere size (28.00 +/- 3.00%, p<0.0001) not showing degeneration to the 5th day. However, n-butanol and aqueous fraction could not sustain the sphere structure. Ginseng ethanol fraction also elevated in the G2/M proportion (28.73+/-0.45%, p<0.0001), up-regulated proliferation mRNA ki67 (4.605+/-6.48 fold-change, p<0.05), cycA1 (12.61+/-4.65 fold-change, p<0.0001), cycD1 (22.47+/-8.18 fold-change, p<0.0001), cycC (9.53+/-2.63 fold-change, p<0.0001) compared with those of the n-butanol or aqueous fraction-treated neurospheres. Shorten G0/G1 phase (47.08 +/-0.16, p<0.0001), up-regulation of sox2 (71.25+/-27.24 fold-change, p<0.0001) mRNA levels indicated self-renewal effect of the ginseng ethanol fraction; however, those of n-butanol and aqueous fraction-treated neurospheres suggested an inhibiting effect on the cell proliferation. Conclusion: Panax vietnamensis extract fractions had a positive effect on the proliferation of cultured neural stem cells. The ethanol fraction at 200 mg/mL could significantly promote the growth rate while still sustained the integrity of treated spheres.

For the first time, the cytotoxic and phytochemical investigation of the leaves of Stereospermum binhchauensis V.S. Dang, a new species discovered in Viet Nam were finalized and led to purify nine compounds, including one furancoumarin (1), one chromone (3), two triterpenoids (2, 4), two flavonoids (5, 8), two flavanoids (6, 7) and one iridoid (9) using various chromatography methods. Their structures were verified by HR-ESI-MS, NMR experiments and compared with previous literatures. For the first time, compounds (5–8) were realized from the genus Stereospermum, while compounds (1, 2, 3, 4 & 9) were designated from the species S. binhchauensis. Furthermore, the furancoumarin, chromone and flavanoid classes were notified for the first time from the genus Stereospermum.

For the first time, the phytochemical constituents of the leaves of Polyscias fruticosa (L.) Harms. cultivating in An Giang Province, Viet Nam were investigated and led to purify two new oleanane-type triterpenoid saponins, named polyscioside J (1) and polyscioside K (2) together with two known saponins, ladyginoside A (3) and chikusetsusaponin IVa (4) using variously chromatographic methods. Saponin (4) was reported for the first time from this species. Their structures were verified by IR, UV, HR-ESI-MS, NMR 1D and 2D experiments and compared with previous literatures.

Anoectochilus setaceus is a medicinal plant used as a traditional medicine as well as nutritious products for health care in China and many Asian countries. In conventional propagation, the the vessel type, material and volume lead to differences in the moisture content between the internal and external conditions which result in great impact on the quality of in vitro plantlets as well as subsequent growth at nursery. In this study, several factors affecting the multiplication and regeneration of A. setaceus cultured in vitro were investigated. The highest percentage of shoot regeneration (52.88%) from ex vitro stem nodes was obtained when explants were cultured on SH medium supplemented with 1 mg/l BA, 30 g/l sucrose with cotton wool plug as the substrate and pH of the medium was adjusted to 5.8 prior to autoclaving. In the present study, culture vessel volume and ventilation culture conditions indicated to have effect on shoot quality. Results showed that 3-liter Erlenmeyer flasks in combination with ventilation caps resulted in better shoot growth and development (shoot height: 10.75 cm; 9.45 leaves per shoot; fresh- and dry- weight of 2.45 and 0.219 g per shoot, respectively; leaf diameter: 2.85 cm and SPAD: 39.99). Results of chromatography (TLC) analysis indicated that explants gave the same Rf value as that of authentic Adenosine. Qualifying Adenosine using TLC showed that extracts of shoots cultured on solid and liquid medium (ventilation plastic bag), and that of shoots cultured in 3-liter vessel with liquid medium (with and without ventilation) gave the same pattern in the respects of color and Rf value.

In the present study, Hibiscus sagittifolius Kurz. seeds were used as the plant materials for studying on the effects of simulated microgravity (on a 2D clinostat) on seed germination, shoot multiplication, growth, development and secondary metabolite accumulation. After surface sterilization, seeds were cultured on MS medium supplemeted with 30 g/l sucrose and 9 g/l agar in Petri dishes (9 seeds per dish, the seed to seed distance of 1.5 cm and kept in the same direction), and maintained in a Clinostat (2 rpm). The results showed that simulated microgravity inhibited the growth and development of Hibiscus sagittifolius roots with root length of 11.83 cm, fresh and dry weight of 58.28 and 5.23 mg, respectively but it made an increase in germination rate (87%) and accumulation of secondary metabolites (the total saponins content of 53.00 mg/g and the total coumarin content of 25.67 mg/g) after 3 weeks of culture. In addition, the simulated microgravity also resulted in positive shoot multiplication (shoot height of 3.07 cm, 6.33 nodes per shoot, 3.33 shoots per explant, and the fresh and dry weight of 401.33 and 37.00 mg, respectively), and growth and development of Hibiscus sagittifolius shoots (plant height of 12.17 cm with 5.67 leaves per shoot together with the average root length of 1.77 cm, and the fresh and dry weight of 419.00 and 36.00 mg) after 4 weeks of culture. The results from this study could be attributed to future perspectives in research on plant breeding and accumulation of secondary metabolites in medicinal plants.

One new cycloartane triterpenoid, named markhacanasin C (1), together with three known triterpenoids, oleanolic acid (2), ursolic acid (3) and 6β,19α-dihydroxyursolic acid (4) were isolated by various chromatographic methods from the most cytotoxic fraction of the ethyl acetate extract of Markhamia stipulata var. canaense V.S. Dang leaves. Among them, 4 was reported for the first time from the genus Markhamia, while 2 and 3 were found for the first time from this species. Their structures were elucidated by IR, UV, HR-ESI-MS and NMR experiments. The cytotoxicity of isolated compounds (3 and 4) against three human cancer cell lines (HeLa, HepG2, and MCF-7) were evaluated. At the concentration of 100 μg/mL, 3 exhibited significant cytotoxic activity (86.36 ± 3.69%).

One new flavonol glycoside named visconoside C (1), together with seven known flavonol glycosides, quercetin 3-O-beta-D-glucopyranoside 7-O-alpha-L-rhamnopyranoside (2), quercetin 7-O-alpha-L-rhamnopyranoside (3), astragalin (4), kaempferol 3-O-(4-O-acetyl)-alpha-L-rhamnopyranoside (5), kaempferol 7-O-alpha-L-rhamnopyranoside (6), kaempferitrin (7) and kaempferol 3-O-beta-D-glucopyranoside 7-O-alpha-L-rhamnopyranoside (8) were isolated by various chromatography methods from the leaves of Cleome viscosa L. Their structures were elucidated by IR, UV, HR-ESI-MS and NMR (1D & 2D) experiments. The cytotoxicity and hepatoprotective activities using HepG2 human hepatoma cell line of 1 were measured by MTT assay. At the concentration of 25 mu M and 50 mu M, 1 showed cytotoxic activity against HepG2 cells (cell viability was decreased to 22.2 and 23.0%, respectively, compared with doxorubicin control), while at the concentration of 100 mu M, 1 showed hepatoprotective activity against CCl4-induced hepatotoxicity on HepG2 cells (34.3%, compared with quercetin control). [GRAPHICS] .

Background:Ginseng has been known since ancient time for its unique pharmaceutical effects on human health. Modern studies indicated that extracts of ginseng could improve a broad range of pathological conditions including neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. Panax vietnamensis, a new ginseng species recently discovered in Vietnam, has received much interest due to its high composition of both known and new saponins whose therapeutic effects have not yet fully evaluated. In this study, the proliferative and differentiative effects of different fractions of the extract of Panax vietnamensis on cultured neural stem cells would be assessed. Materials and methods: Fractions from the Panax vietnamensis were prepared following a successive extraction of the raw material using ethanol, n-butanol, ether ethylic, and water, respectively. Potential toxicity and proliferative effects of each fraction were assessed based on the increase in the sphere diameter in before further analyses. The percentage of cells in active phases, cell cycle- or differentiation-associated genes were analyzed using flow cytometry and real-time RT-PCR. Results:Our results revealed that supplementing media with 200 µg/ml of ethanol Panax vietnamensis fraction significantly increased diameters of neurospheres, also maintained the growth rate of the neurospheres compared with the control within the five-day period. Moreover, the results also demonstrated that the ginseng ethanol fraction and a known neurotrophic protein, NGF, affected the differentiation of the neural stem cells. Conclusion:200 µg/ml ethanol fraction from the extract of Panax vietnamensis most significantly induced the proliferation of cultured neural stem cells. However, the higher concentration of the fraction seemed not to maintain the growth of neurospheres. Regarding differentiation, the ethanol fraction of Vietnamese ginseng possibly shared similar mechanisms with the neurotrophic growth factor.

From the most cytotoxic fraction of the ethyl acetate extract of Markhamia stipulata var. canaense V.S. Dang leaves, two new cycloartane-type triterpenoids, named Markhacanasin A (1) and Markhacanasin B (2); were isolated by various chromatographic methods Their structures were elucidated by IR, UV, HR-ESI–MS, NMR (1D & 2D) experiments. The cytotoxicity of two new compounds against five human cancer cell lines (HeLa, HepG2, MCF-7, Jurkat and NCI-H460) were evaluated by SRB assay. As results, 1 exhibited significant cytotoxic activity against all cancer cell lines (IC50 ranged from 14.72 to 29.55μM) while 2 did not show activity.