Helleborus Thibetanus Research Articles

Non-deep simple epicotyl morphophysiological dormancy in seeds of Helleborus thibetanus (Ranunculaceae)

Helleborus thibetanus is a perennial herbaceous plant with high potential for medicinal and landscaping purposes. However, the dormancy characteristics of seeds and the conditions that help this species break dormancy have not been investigated. This study investigated embryo growth and the emergence of radicles and epicotyls in seeds over a range of GA3 pretreatment and temperature sequences. Upon reaching maturity, the seeds had underdeveloped embryos that developed fully at 15 °C warm stratification within 6 weeks. Immediately after embryo growth, radicles emerged during warm stratification. However, the epicotyls failed to emerge soon after the radicle emergence. Epicotyl emergence was observed in over > 13 % of seeds with an emerged radicle following 4 weeks of cold stratification at 4 °C. The effects of GA3 on radicle and epicotyl emergence were used to determine the specific types of seed dormancy. GA3 pretreatment partially substituted 15 °C warm stratification to promote radicle emergence and effectively served as a substitute for the 4 °C cold stratification to break the epicotyl dormancy. The overall temperature and GA3 requirements for breaking dormancy and seed germination indicate non-deep simple epicotyl morphophysiological dormancy in H. thibetanus.

Deep Simple Epicotyl Morphophysiological Dormancy in Seeds of Endemic Chinese Helleborus thibetanus

Helleborus thibetanus is an endemic species in China with important ornamental and medicinal value. However, the seeds have dormancy, and their germination percentage is low under natural conditions. This research was carried out to determine the seed germination requirements of H. thibetanus and to characterize the type of seed dormancy. The morphological post-ripening process of the seed embryo was studied according to the morphological anatomy, and the effects of temperature and gibberellic acid (GA3) on seed germination were investigated in H. thibetanus. The H. thibetanus seeds had a heart-shaped embryo at maturity. The embryo fully grew within the seed through warm stratification, and the embryo/seed ratio increased from 8.58% to 42.6%. The shortest time for a radicle to emerge (58.33 d) and the highest radicle emergence percentage (84.44%) were obtained at a temperature of 15 °C. The results showed that the H. thibetanus seeds had a morphophysiological dormancy. In addition, 300 mg/L GA3 treatments shortened the time of warm stratification and increased the radicle emergence percentage. Seeds with emerged radicles could not emerge epicotyl–plumule without cold stratification, which showed that the H. thibetanus seeds had epicotyl physiological dormancy. The length of the roots, cold stratification time, and GA3 markedly affected the release of the epicotyl physiological dormancy in H. thibetanus. The seeds with 2.5 cm roots required the shortest time to break their dormancy (50 d), and the epicotyl–plumule emergence percentage was the highest. Additionally, GA3 treatment also shortened the incubation time in cold stratification (5 °C) and successfully broke the epicotyl physiological dormancy. Our study showed that H. thibetanus seeds exhibited deep simple epicotyl morphophysiological dormancy. Temperature, GA3, and duration of stratification played vital roles in the seed germination of H. thibetanus. This research will provide valuable data for seed germination and practical dormancy-breaking techniques and will promote the cultivation and conventional crossbreeding of H. thibetanus.

Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography.

In this study, a combination approach involving macroporous resin (MR) column chromatography and gradient countercurrent chromatography (CCC) was employed to enrich and purify bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch. Initially, a D101 MR-packed column chromatography was utilized for fractionation and enrichment of the bufadienolides, which were effectively eluted from the column using a 60% ethanol solution. CCC was subsequently introduced to separate the enriched product using the ethyl acetate/n-butanol/water (EBuWat, 4:1:5, v/v) and EBuWat (5:0:5, v/v) solvent systems in a gradient elution mode. As results, five bufadienolides, including 6.1mg of hellebrigenin-3-O-β-D-glucoside (1), 2.2mg of tigencaoside A (2), 8.3mg of deglucohellebrin (3), 3.5mg of 14 β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (4), and 3.0mg of 14β-hydroxy-3β-[(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (5), were effectively separated from 300mg of the enriched product. The respective high-performance liquid chromatography purities were as follows: 95.2%, 75.8%, 85.7%, 82.3%, and 92.8%. This study provides valuable insights for the efficient enrichment and separation of bufadienolides from Helleborus thibetanus Franch.

Chemical constituents of Helleborus thibetanus

The chemical constituents of Helleborus thibetanus were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative RP-HPLC, and the structures of all compounds were identified by modern spectrographic technology(MS, NMR). The MTT method was used to measure the cytotoxicity of compounds 1-8. Twelve compounds were isolated from the roots and rhizomes of H. thibetanus and were identified as(25R)-22β,25-expoxy-26-[(O-β-D-glucopyranosyl)oxy]-1β,3β-dihydroxyfurosta-5-en(1), β-sitosterol myristate(2), β-sitosterol lactate(3), β-sitosterol 3-O-β-D-glucopyrannoside(4), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(5), 1,3,5-trimethoxybenzene(6), 7,8-dimethylbenzo pteridine-2,4(1H,3H)-dione(7), 1H-indole-3-carboxylic acid(8), p-hydroxy cinnamic acid(9), lauric acid(10), n-butyl α-L-arabinofuranoside(11) and methyl-α-D-fructofuranoside(12), respectively. Among them, compound 1 is a new compound and named thibetanoside L; compounds 2, 5-8, 11 are first isolated from the family Ranunculaceae; compound 12 is isolated from the genus Helleborus for the first time. The results of MTT assay showed that the IC_(50) values of compounds 1-8 against HepG2 and HCT116 cells were greater than 100 μmol·L~(-1).

Exploring the Catalytic Flexibility and Reversibility of Plant Glycosyltransferase HtUGT72AS1 for Glycodiversification of Phenolic Compounds.

Plant bioactive metabolites such as flavonoids are usually present in glycosylated forms by the attachment of various sugar groups. In this study, a catalytically flexible and reversible glycosyltransferase (HtUGT72AS1) was cloned and characterized from Helleborus thibetanus. HtUGT72AS1 could directly accept six sugar donors (UDP-glucose/-arabinose/-galactose/-xylose/-N-acetylglucosamine/-rhamnose) to catalyze the 3-OH glycosylation of flavonols. It also catalyzed the 4' and 7-OH glycosylation of other types of flavonoids, which lacked the 3-OH group. Additionally, the HtUGT72AS1-catalyzed reaction was highly reversible when using 2-chloro-4-nitrophenyl glycosides as substrates, which could be used for one-pot or coupled production of bioactive glycosides. It is the first reported UGT for the synthesis of arabinosides and galactosides using a transglycosylation platform. Based on structural modeling and mutagenetic analysis, the mutation of Tyr377 to Ara enhanced the catalytic efficiency of HtUGT72AS1 toward UDP-N-acetylglucosamine, and the V146S mutant gained an improvement in the regioselectivity toward 7-OH of flavonoids.

Paleo Distribution and Habitat Risks under Climate Change of Helleborus thibetanus

As an endemic species and the only Helleborus species in China, Helleborus thibetanus is highly valued in medicinal and ornamental applications, and basic research is needed for its further resource conservation and utilization. Considering the interesting disjunct distribution of the genus Helleborus, we focus on the distribution pattern of H. thibetanus in this research. Based on species distribution models using three different algorithms (MaxEnt, RF, and FDA), we constructed a robust ensemble model and predicted potential distributions under different scenarios: current situation, paleo periods since the Last Glacial Maximum, and simulations of climate change in the 2070s. The habitat suitability of H. thibetanus across geography and scenarios was further analyzed by calculating regional areas and centroids. The results showed that H. thibetanus is currently distributed in southern Shaanxi and northern Sichuan, while central and southern Sichuan used to be suitable 14 thousand years ago but gradually became unsuitable, which may reflect the population decrease in Sichuan and the population expansion in Shaanxi over the last 14 thousand years. Our results showed that current populations are under limited extinction pressure in the soft climate change scenario (ssp126), but most populations in Shaanxi are under extinction pressure in the hardy situation scenario (ssp585). Fortunately, northern Sichuan is predicted to be relatively stable under climate change (both ssp126 and ssp585), and regions in western Sichuan and eastern Qinghai are predicted to become newly suitable for H. thibetanus. These findings should be helpful for the further conservation and utilization of H. thibetanus and also help us understand the history of the conjunct distribution pattern of the Helleborus genus.

Two New Spirostanol Glycosides from the Roots and Rhizomes of Helleborus thibetanus Franch.

Two New Spirostanol Glycosides from the Roots and Rhizomes of Helleborus thibetanus Franch.

Steroidal components from the roots and rhizomes of Helleborus thibetanus

Three new steroidal components hellebogenins A and B (1 – 2) and thibetanoside I (3) together with four know ones ( 4 – 7 ) were isolated from the roots and rhizomes of Helleborus thibetanus . Their structures were elucidated by chemical methods and spectroscopic analysis. Among them, compound 1 possessed three double bonds at the positions of 1(2), 5(6) and 25(27), while compound 2 had an α , β unsaturated ketone carbonyl on the A ring. In addition, compounds 1 and 2 showed cytotoxic activities against HCT116 cells with IC 50 values of 42.0 ± 1.3 μM and 67.3 ± 1.5 μM, respectively. • Three new steroidal components ( 1 3 ) were isolated from Helleborus thibetanus . • Compound 1 possessed a rare type of 1(2), 5(6) and 25(27) double bonds. • Compound 2 had an α , β unsaturated ketone carbonyl on the A ring. • Compounds 1 and 2 showed cytotoxicities against HCT116 cancer cell lines.

The co-occurrence of bufadienolides and podophyllotoxins from Helleborus thibetanus

Phytochemical investigation of Helleborus thibetanus led to the isolation of two new bufadienolides with a trans A/B ring fusion mode (1–2) and two known ones (3–4), three podophyllotoxins (5–7), two flavones (8 and 9), one phytoecdysteroid (10), one spirostane (11) and one pseudo-spirostane (12). The structures of the new compounds were identified using extensive spectroscopic analyses and the known compounds were identified by comparing the spectral data with those previously reported. Isolation of bufadienolides with a trans A/B ring fusion mode could be chemotaxonomic marker for the differentiation of H. thibetanus from the toad venom and other plants of this genus, on the other hand, co-occurrence of podophyllotoxin from H. thibetanus supported the inclusion of Berberidaceae and Ranunculaceae in the same monophyletic order Ranunculales based on the outgroup comparison and parsimony analysis.

Cardiac Glycoside Compound Isolated from Helleborus thibetanus Franch Displays Potent Toxicity against HeLa Cervical Carcinoma Cells through ROS-Independent Autophagy.

The current study aimed to examine the anticancer activity of HTF-1, a cardiac glycoside (CG) isolated from Helleborus thibetanus Franch, using a cell-based model and to discover the underlying mechanisms with specific focus on autophagy. We found that HTF-1 was able to potently decrease the viability of several cancer cell lines especially for HeLa cervical carcinoma cells. It was discovered that HTF-1 dose dependently induced overproduction of ROS in HeLa cells, and the cell viability can be rescued when adding ROS scavenger N-acetyl-l-cysteine (NAC). More, we found that HTF-1 induced ROS-independent autophagy in concentration- and time-dependent manners in HeLa cells. This can be collectively verified by LC3-II and p62 abundance and also eGFP-LC3 puncta assay, bafilomycin clamp experiment, and acidotropic dye fluorescent labeling experiment. Additionally, TEM examination showed more autophagic vacuoles for HTF-1-treated HeLa cells. In HeLa cells, pretreatment with wortmannin (an inhibitor of the initial stages of autophagy to block autophagosome formation, thus, it should weaken the autophagy induction effect of HTF-1) decreased the autophagic flux and partially antagonized cell death induced by HTF-1, indicating that autophagy induced by HTF-1 played a cancer-suppressing role. Furthermore, coadministration of BAF (as a distal inhibitor of autophagy) with HTF-1 demonstrated a synergistic anticancer effect against HeLa cells. We believe that our work will enrich the understanding of CGs and especially anticarcinoma activity, also, pave the way for natural-product-based anticancer drug development.

Steroidal constituents from Helleborus thibetanus and their cytotoxicities.

Thibetanosides E-H (1-4), four new steroidal constituents including three rare sulfonates (2-4), were isolated from the roots and rhizomes of Helleborus thibetanus, together with nine known steroidal compounds (5-13). Their structures were elucidated by detailed spectroscopic analysis, including 1D and 2D NMR techniques and chemical evidence. In this study, compounds 2-13 were evaluated for their cytotoxic activities against HCT116, A549 and HepG2 tumor cell lines in vitro. Among them, compound 8 (thibetanoside C) showed cytotoxicities against A549 cells(IC50 39.6 ± 1.9 μmol·L-1) and HepG2 cells(IC50 41.5 ± 1.1 μmol·L-1), respectively. Compound 9 (23S, 24S)-24-[(O-β-D-fucopyranosyl)oxy]-3β, 23-dihydroxy-spirosta-5, 25(27)-diene-1β-ylO-(4-O-acetyl- α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside) showed cytotoxicity against HCT116 cells(IC50 33.6 ± 2.1 μmol·L-1).

Effect of neighboring seeds associated with habitats on the seed removal of two coexisting myrmecochorous plants in central China

Although neighboring plants can influence animal-seed dispersal interactions, little is known about the effect of neighboring seeds and the influence of habitat on seed dispersal by ants. Here we investigated the influence of neighboring seeds on seed removal in two coexisting myrmecochorous species (Epimedium pubescens and Helleborus thibetanus) from temperate deciduous forests of Qinling Mountains, central China, by examining (1) the potential role of ants and rodents and (2) whether the neighboring seed effect differed between forest edge and interior. We found that, presence of the higher-ESMR (elaiosome: seed mass ratio) E. pubescens did not significantly affect seed removal of the lower-ESMR H. thibetanus. By contrast, the presence of H. thibetanus decreased the seed removal rates of E. pubescens, with only a significant effect in rodent exclusion (ant alone) rather than in both ant exclusion (rodent alone) and full access (rodent+ant). Moreover, we found that those effects were not significantly different between the forest edge and the interior, which may be attributed to a similar pattern of overall seed-dispersing ant abundance in the two habitats. This suggested that neighboring seeds could influence seed removal of the focal plant depending on the absence of rodents; when rodents were present, the interaction of rodents and ants rendered no influence of neighboring seeds on seed removal. Our results show that the neighborhood effect was regulated by both dispersers and predators, and this effect was not context-dependent at a small spatial scale. This study highlights the importance to understand the effect of shared seed-removing animals and habitat context to assess the neighboring seed effect on plant-animal interactions.

Three new spirostanol glycosides from Helleborus thibetanus

An ongoing chemical investigation on n-BuOH extract of roots and rhizomes of Helleborus thibetanus afforded three new spirostanol glycosides (1–3). Their structures were elucidated by extensive analysis of 1 D, 2 D NMR spectra, together with IR and MS methods and acid hydrolysis. This is the first report of the isolation of spirostanol glycoside with xylose at C-24 of the aglycone in Helleborus.

New cytotoxic bufadienolides from the roots and rhizomes of Helleborus thibetanus Franch

Three new bufadienolides 14β, 16β-dihydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (1), 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→4)-(β-D-glucopyranosyl)oxy]-5α-bufa-20, 22-dienolide (2) and hellebrigenin-3-O-β-D-glucosyl-(1→4)-β-D-glucoside (3), together with eight known bufadienolides (4–11) were isolated from the roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by extensive spectroscopic methods and acid hydrolysis. Compounds 1–7 were evaluated for their cytotoxic activity against HCT116, A549 and HepG2 tumor cell lines. Compound 1 exhibited moderate cytotoxicity against HepG2 cells with IC50 value of 15.1 ± 1.72 μM. Compounds 5 and 6 exhibited moderate cytotoxicity against HCT116 cells with IC50 values of 15.12 ± 0.58 μM and 13.17 ± 2.34 μM, respectively.

A cardiac glycoside HTF-1 isolated from Helleborus thibetanus Franch displays potent in vitro anti-cancer activity via caspase-9, MAPK and PI3K-Akt-mTOR pathways

Experiments have been undertaken and for the first time, we have identified that a new cardiac glycoside (CG) isolated from Helleborus thibetanus Franch. a plant endemic to China, bears potent anti-cancer activity. We have named it as HTF-1. By using in vitro cell models, we have found that HTF-1 induces apoptosis against several types of cancer cells in a concentration- and time-dependent manner. It is able to inhibit cancer cell in proliferation, migration and invasion. HTF-1 causes S cell cycle arrest. Further-on, we have identified that HTF-1 triggers caspase-9 dependent apoptosis pathway and double strand DNA breaks (DSBs). Additionally, HTF-1 activates JNK, but suppresses ERK and PI3K-Akt-mTOR pathways. Collectively, the above-mentioned mechanisms contribute to the anti-cancer activity of HTF-1. It is rare to discover novel anti-cancer CG during the past couple of decades. We believe that our work will enrich the understanding of CGs; also, pave the way for natural product-based anti-cancer drug development.

New minor spirostanol glycosides from Helleborus thibetanus

Phytochemical reinvestigation of the dried roots and rhizomes of Helleborus thibetanus afforded four new minor spirostanol glycosides (1–4) and four known spirostanol glycosides (5–8). Their structures were determined on the basis of spectroscopic analyses, including 1D and 2D NMR experiments, together with HR-ESI-MS and IR measurements and the results of acid hydrolysis.

Four new minor spirostanol glycosides from Helleborus thibetanus

Four new minor spirostanol glycosides (1–4) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were established by means of a combination of 1D and 2D NMR experiments, together with HRESIMS and IR measurements as well as the results of acid hydrolysis. The spirostanol glycosides with both a double bond at C-25 and glycosidation at 1-OH have seldom been reported.

Three new steroidal saponins from Helleborus thibetanus

Three new steroidal saponins including two spirostanol glycosides (1–2) and one furostanol glycoside 1-sulphate (3) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Structures of the compounds were determined on the basis of extensive use of 1-D and 2-D NMR experiments, together with HR–ESI–MS and IR measurements, as well as the results of acid hydrolysis. Compounds 1–2 represented steroidal saponins with an unusual substitution pattern, which possessed a double bond at C-25 and were glycosylated at 1-OH.

Six New Steroidal Saponins from Helleborus thibetanus

AbstractSix steroidal saponins, including five spirostanol glycosides, 1–5, and one furostanol glycoside 1‐sulfonate, 6, previously unknown in nature, together with three known compounds, 7–9, were isolated from dried roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by extensive 1D‐ and 2D‐NMR experiments, along with IR and HR‐ESI‐MS data, as well as the results of acid hydrolysis. Compounds 1–5 possessed a C(25)C(27) bond and were glycosylated at HOC(1), which was unusual in steroidal saponins.

Two new bufadienolides and one new pregnane from Helleborus thibetanus

Two new bufadienolides, 3β,14β,16β-trihydroxy-5α-bufa-20,22-dienolide (1) and 14β-hydroxy-3β-[β-D-glucopyranosyl-(1→6)-(β-D-glucopyranosyl)oxy]-5α-bufa-20,22-dienolide (2), one new pregnane, 3β-hydroxypregna-5,16-diene-20-one-1β-yl sulfate (3), along with one known pregnane (4) were isolated from the dried roots and rhizomes of Helleborus thibetanus. Their structures were elucidated by the extensive use of 1D and 2D NMR experiments, together with IR and HRESIMS spectra and the results of enzymatic hydrolysis.