Yellow light promotes the growth and accumulation of bioactive flavonoids in Epimedium pseudowushanense.

The plant species of the genus Epimedium L. are well-known traditional Chinese medicinal herbs with special therapeutic effects on human beings and animals in invigorating sexuality and strengthening muscles and bones. In large-scale cultivating Epimedium that is a typical shade plant species, they are arbitrarily covered with black colored shade nets. However, their optimal growth conditions, especially light, are still less understood. During the investigation of different light qualities on the growth of Epimedium pseudowushanense, it was found that, all the values of plant growth characteristics (except shoot number) and photosynthetic characteristics were lower under red, yellow, or blue light treatment than under white light treatment. However, yellow light treatment had beneficial effects on shoot number, dry biomass (per plant) as well as net photosynthesis rate (Pn) and maximal apparent quantum efficiency (AQY) in E. pseudowushanense when compared with red or blue light treatment. More importantly, we found that E. pseudowushanense accumulated higher levels of bioactive flavonoids under yellow light treatment than under white, red, or blue light treatment. Furthermore, both RNAseq and qPCR analyses revealed that yellow light could highly up-regulate the expression levels of flavonoid biosynthetic genes, in particular CHS1, F3H1, PT_5, and raGT_5 that possibly contributed to the enhanced accumulation of bioactive flavonoids in E. pseudowushanense. Taken together, our study revealed that yellow light is the optimal light for the growth of E. pseudowushanense. Our results provided key information on how to improve the cultivation condition and concurrently enhance the accumulation of bioactive flavonoids in E. pseudowushanense.

A Widely Metabolomic Analysis Revealed Metabolic Alterations of Epimedium Pubescens Leaves at Different Growth Stages.

Epimedium folium is the major medicinally-used organ of Epimedium species and its metabolic changes during the leaf growth have not been studied at the metabolomic level. E. pubescens is one of five recorded species in the Pharmacopoeia of the People's Republic of China and widely grows in China. A UPLC-ESI-MS/MS-based targeted metabolomic analysis was implemented to explore the metabolite composition in E. pubescens leaves under the cultivation condition and further to investigate their temporal variations among four representative growth stages. A total of 403 metabolites, including 32 hitherto known in Epimedium species, were identified in E. pubescens leaf, of which 302 metabolites showed the growth/development-dependent alterations. Flavonoid-type compounds were the major composition of the metabolites identified in this study. Most flavonoids, together with tannin-type and lignans and coumarin-type compounds, were up-regulated with E. pubescens leaf growth and maturation after the full flowering stage. Our results not only greatly enriched the existing Epimedium phytochemical composition database and also, for the first time, provided the metabolomics-wide information on metabolic changes during E. pubescens leaf growth and development, which would facilitate in the choice of an optimum harvest time to balance a higher biomass yield of Epimedium folium with its better medicinal quality.

[Effects of plant growth regulators and nitrogen on germination of Epimedium pseudowushanense after its stems and leaves reaped].

In order to explore appropriate measures to promote germination after the harvest of Epimedium pseudowushanense, 6-BA, urea, ammonium bicarbonate and GA3 were chosen to spray on the root and rhizomes, and then the biological indicators such as branches, leaf length, leaf width, plant height and so on, were measured in different periods, and the contents of epimedin A, epimedin B, epimedin C and icariin in the dry leaves were detected by HPLC. Results showed that 6-BA 90 mg·L⁻¹(B1), 6-BA 60 mg·L⁻¹(B2),6-BA 30 mg·L⁻¹+urea 300 mg·L⁻¹ (C1), 6-BA 60 mg·L⁻¹+urea 300 mg·L⁻¹(C2),6-BA 60 mg·L⁻¹+ ammonium bicarbonate 300 mg·L⁻¹(C4) significantly increased bud germination in the early period, and the plants quickly set up new system of photosynthesis, the branches in a month of which were higher than the control group respectively by 165.9%, 115.76%, 103.86%, 104.50%, 81.67%.However the branches developed the next year and the dry weight of leaves per plant in group B1 and B2 were much lower than that in control group. The groups that use 6-BA and nitrogen at the same time reaped a good yield of leaves even though the treatment had no significant influence on the branches developed the next year. The dry weight of leaves of C1, C2, C4 treatments were 36.80%, 32.84%, 45.97% more than the control group respectively. Therefore, C1, C2 and C4 treatments are the more appropriate to promote recovery after harvest. Furthermore, different groups, except 10 mg·L⁻¹ 6-BA treatment significantly reduced the content of epimedin C, other groups didn't have any significant effect on the contents of such flavonoids.

Effects of Light Intensity on the Growth, Photosynthetic Characteristics, and Flavonoid Content of Epimedium pseudowushanense B.L.Guo.

Epimedium pseudowushanense B.L.Guo is used in traditional medicine as an aphrodisiac and to strengthen muscles and bones. Several recent reports have shown that flavonoids from Epimedium also significantly affect the treatment of breast cancer, liver cancer, and leukemia. However, few studies have examined the medicinal-ingredient yield of Epimedium, a light-demanding shade herb, under different light intensities. To investigate the effects of light intensity on medicinal-ingredient yields, Epimedium was exposed to five levels of light intensity until harvest time. Leaf dry biomass under L4 was the highest among different light treatments. L4 was also associated with the highest net photosynthetic rate. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that L3 produced the highest amount of epimedin C, and that flavonoid content responded to light levels differently. Results indicated that L3 and L4 were the optimal light levels for medicinal-ingredient yield.

Exploring the best model for describing light-response curves in two Epimedium species.

Photosynthetic responses to light environment can be useful measurements to determine favorable habitat conditions for the conservation and cultivation of medicinal species. The nonrectangular hyperbola model, rectangular hyperbola model, modified rectangular hyperbola model, exponential model, modified exponential model are used to explore the best model for describing light-response curves in two Epimedium species. The results show that the light-response curves of E. wushanense and E. acuminatum were best described by the modified exponential model, the test MSE and MAE of the modified exponential model were smaller, and the LSP and Pmax calculated by the modified exponential model were more accurate than those calculated by other models in E. wushanense and E. acuminatum. This model may be widely applicable to light-response curves of other Epimedium species.

The growth and medicinal quality of Epimedium wushanense are improved by an isolate of dark septate fungus.

CONTEXT: Seven dark-septate endophytic (DSE) fungi have been isolated from the roots of Epimedium wushanense T. S. Ying (Berberidaceae), an important medicinal plant with various pharmacological activities. OBJECTIVE: The current study explores the effects of seven DSE fungi on the growth and accumulation of bioactive compounds in E. wushanense. MATERIALS AND METHODS: Each 1-month-old E. wushanense seedling was inoculated with one of the seven DSE fungi and was grown under greenhouse conditions for 90 d. The molecular identification of the fungi was based on the ITS1-5.8S-ITS2 nuclear ribosomal gene cluster. RESULTS: The results showed that the influence of DSE fungi inoculation varied between strains. Inoculation with DSE8 not only significantly enhanced plant height, root length, leaf area, leaf number, and shoot and root biomass but also improved the total flavonoid and icariin content, with an increase ranging from 20.24% to 237.97%. Three of the seven DSE fungi caused the inoculated plants to die, and the remaining three DSE strains showed neutral or negative effects on plant growth and the accumulation of bioactive compounds. According to the ITS sequence, DSE8 is congeneric to the genus Leptodontidium. DISCUSSION AND CONCLUSION: The findings indicate that application of DSE8 may be valuable to facilitate the cultivation of E. wushanense with a higher biomass and improved medicinal quality.

[Distribution and Community Characteristics or Epimedium myrianthum in Chongqing].

OBJECTIVE: To make clear the distribution area in Chongqing and community characteristics of Epimedium myrianthum, in order to provide evidence for sustainable utilization of resources. METHODS: Based on field investigation, recorded the GPS, and used the quadrat method to observe and analyze the abundance, frequentness and importance value of Epimedium myrianthum in the seven well-concentrated sample plots of different habitats. RESULTS: Epimedium myrianthum distributed in Changshou County, Dianjiang County, Zhongxian County and Fengdu County of Chongqing at present. The altitude of its distribution areas ranged from 351~663 m. The types of its habitats included coniferous forest, broad-leaved forest and bamboo forest. The plants in these communities were composed of 56 species belonging to 48 genera,33 families. Among associated plants, the dominant species of tree layer were Bambusa distegia, Quercus variabilis , Cupressusfunebris etc. The dominant species of shrub layer were Pittosporum illicioides, Loropetalum chinense, Myrsine africana, Ardisia japonica, Smilax china etc. The dominant species of field layer were Iris japonica, Miscanthus floridulus, Veronicastrum longispicatum, Carex lancifolia, Epimedium myrianthum etc. Species abundance index of D was between 1.1143 to 3.7781 in seven populations,the species in these communities were relatively less,and the numbers of individuals were relatively more. Compared with others, the population of the 3rd and 6th had relatively low index of H' (Shannon-wiener index) and E (Pielou index). The distribution of these species in the community was obviously uneven. The D value,H' value and E value of tree layer species richness were lower than those of shrub layer and field layer, the species diversity was worse. CONCLUSION: It is suggested to incorporate Epimedium myrianthum in the new edition of Chinese Pharmacopeia.

[Effect of suet oil on formation of self-assembled micelles of flavonoids in Epimedium].

In this paper, the action of suet oil in the preparation of self-assembled micelles of the active flavonoids in Epimedium in the simulated human environment was researched. Twelve suet oil samples were collected from different growing areas and different positions of sheep or goat to simulate the formation of micelles. Then the effects of the fatty acids in suet oil on the preparation of self-assembled micelles were studied furthermore. The results showed that the micelles had a dispersed state and spherical smooth surface. To compare the diameter, potential, encapsulation efficiency and drug loading of the 12 batches micelles, the micelles prepared by the suet oil from Qinghai were more stable and had a higher encapsulation efficiency. The fatty acids in suet oil could promote the formation of self-assembled micelles, but the whole suet oil had a better effect. Above all the study, we confirmed that the suet oil promoted the formation of self-assembled micelles of the flavonoids in Epimedium, it laid foundation for further research about increasing the efficacy of Epimedium and improved the absorption of the active flavonoids in Epimedium.

[Morphological character of growth cycle for Epimedium acuminatim and icariin content analysis].

OBJECTIVE: To study the relationship between morphological characteristics of growth cycle for Epimedium acuminatium and accumulation level of secondary metabolites. METHOD: After making habitat clear, methods of morphology observation, specimen preparation, organism charting were applied to record morphological characteristics of E. acuminatium at different stages. HPLC was employed to detect icariin content of different parts of the plant at the stage of vegetative growth and sexual reproduction. RESULT: The growth cycle of E. acuminatium was divided into vegetative growth stage and sexual and asexual reproduction coexistence stage. Seven to eight years were needed for the plants flowering and seeding of E. acuminatimcan in open land of forest edge, but within good nutrition conditions, growth cycle is shortened into three to four years. The difference of icariin content in two growth stages was not significant. CONCLUSION: Morphological change of lower and foliar organ can be utilized to estimate growth age. But after applying sexual propagation for both root and stem of grown plants, it's been difficult to determine their growth year. If individual biomass or yield is economical, plants can be harvested in both vegetative growth and sexual reproduction stages.

Characterization of a Crabs Claw Gene in basal eudicot species Epimedium sagittatum (Berberidaceae).

The Crabs Claw (CRC) YABBY gene is required for regulating carpel development in angiosperms and has played an important role in nectary evolution during core eudicot speciation. The function or expression of CRC-like genes has been explored in two basal eudicots, Eschscholzia californica and Aquilegia formosa. To further investigate the function of CRC orthologous genes related to evolution of carpel and nectary development in basal eudicots, a CRC ortholog, EsCRC, was isolated and characterized from Epimedium sagittatum (Sieb. and Zucc.) Maxim. A phylogenetic analysis of EsCRC and previously identified CRC-like genes placed EsCRC within the basal eudicot lineage. Gene expression results suggest that EsCRC is involved in the development of sepals and carpels, but not nectaries. Phenotypic complementation of the Arabidopsis mutant crc-1 was achieved by constitutive expression of EsCRC. In addition, over-expression of EsCRC in Arabidopsis and tobacco gave rise to abaxially curled leaves. Transgenic results together with the gene expression analysis suggest that EsCRC may maintain a conserved function in carpel development and also play a novel role related to sepal formation. Absence of EsCRC and ElCRC expression in nectaries further indicates that nectary development in non-core eudicots is unrelated to expression of CRC-like genes.

Variation of medicinal components in a unique geographical accession of horny goat weed Epimedium sagittatum Maxim. (Berberidaceae).

Herbal Epimedium species have been widely in Traditional Chinese Medicine for sexual enhancement, immunity improvement, anticancer and anti-aging treatment, with flavonoids and polysaccharides being the major active components. However, exhaustive depletion of wild sources warrants germplasm evaluation and quality resource exploration. A preliminarily analysis had previously indicated that a specific local geographic accession of Epimedium sagittatum found in Luotian (LT) county of Hubei Province (China) had a much higher content of total flavonoids and polysaccharides. In this study, we further investigated the medicinal component variation in the LT type under different light intensities and in different regions by the common-garden experiment. The results indicated a light intensity range of 40-160 µmol/m²/s was the most suitable for the synthesis and accumulation of total flavonoids, while polysaccharide accumulation was negatively correlated with the light intensity. Icariin was the component displaying the highest content among flavonoids, and the content of major flavonoid bioactive components was relatively stable in the third year after cultivation. There was significant correlation between the major flavonol glycoside constituents and the geographic location, and Central China followed by Northern China were the highly suitable regions for cultivation of LT type E. sagittatum. The results revealed that there was a functional balance between flavonoids and polysaccharides at different developmental stages, and the best harvesting stage should consider the primary contents of interest. This study provides important information on the exploration of quality resources, further breeding approaches and cultivation practices of E. sagittatum, and thus the important insights to enhance our understanding of quality control of traditional medicinal plants.

[Tissue cultivation of tiller buds of Epimedium wushanense].

OBJECTIVE: To established the rapid tissue propagation system of Epimedium wushanense, in order to provide theoretical basis for industrialized seed cultivation. METHOD: Tiller buds E. wushanense were used as explants, with MS, B5, WPM as basic media, and added with different concentrations of plant growth regulators such as 6-BA, NAA and GA3, in order to conduct a systematic study on induction and propagation conditions for tiller buds. RESULT: The suitable method for sterilizing bud was to disinfect with 75% ethanol for 30 s, and then treated with 0.1% HgCl2 for (4 + 2) min for consecutively twice, which could control the pollution rate below 5% and the survival rate above 75%. The optimal medium for bud induction was WPM + 6-BA 2.0 mg x L(-1) + NAA 0.1 mg x L(-1) + GA3 0.5 mg x L(-1), with the induction rate of 75.5%; meanwhile, the basic medium and 6-BA showed significant effect on the induction rate. The propagation medium suitable for buds was MS +6-BA 2.0 mg x L(-1) + NAA 0.5 mg x L(-1), with the propagation rate of 3.3. The optimal growth of rooting medium was 1/2 WPM + IBA 0.5 mg x L(-1) + activated carbon which (0.05%), with the rooting rate of 90%, three to six strong seedlings in each plant. CONCLUSION: The disinfection method suitable for tiller buds and the medium combination suitable for induction, propagation and rooting of adventitious buds are screened out to establish the rapid cultivation system for tiller buds of E. wushanense.

[Study on modules biomass structure of Epimedium acuminatum in different habitats].

OBJECTIVE: To study the biomass structure of Epimedium acuminatum Franch in the different ecological environments for the development and protection. METHOD: Through the scientific investigation in four typical habitats, the sampling spots were set up, the functional modules' biomass structure and relationship of E. acuminatum were researched. RESULT: The average of rhizome, as the largest biomass, and the average total biomass had the same pattern: the open areas of forest edge > shrub lumber > shrub-weed > stream drains. The ratio of the functional modules' biomass had different rates under different habitat conditions. By analyzing and combining investigation, the aerial part of E. acuminatum in the shrub-weed were comparative advantage, and the roots of nutrient accumulation of E. acuminatum in the open areas of forest edge were the highest efficient. Under different ecological condition, the distribution of water metabolism was different strategies. CONCLUSION: In the open areas of forest edge, E. acuminatum growing well, followed by shrub-weed. These two habitats are the ideal ecological environments while the rhizome or the whole plant used as medicine, but also the protection of E. acuminatum.

[Dormancy characteristics and breaking method of seeds from Epimedium wushanense].

OBJECTIVE: To research the mechanism of dormancy and find out the breaking method for the seeds of Epimedium wushanense. METHOD: The water permeability of seed coat was tested by weighing seeds. The germination inhibitor of the seeds were determined with biotic measurement. The development of embryos, germination rate and germination potential were determined after stratification. RESULT: The water permeability of seed coat was 41.86% after 5 h. The extracts of seeds had strong inhibition effects to the length growth of cabbage seedlings. The growth and development of embryos under the cold stratification (5 degrees C) were better than that under the other conditions. The embryo rate extended from 15.39% to 86.21% after 90 d. Germination rate and germination potential after stratification under 5 degrees C were significantly higher than that under other temperatures. CONCLUSION: The results showed that there was no obstacle of water permeability on the test of E. wushanense, after-ripening of embryogenesis and the germination inhibitor of the seed were the main reason for the seed dormancy. The cold stratification would be an effective way for breaking of the dormancy, which could significantly promote the seed embryogenesis and increase germination rate comparing to other methods.

[Total flavonoids and icariin contents of Epimedium pubescens in different types of communities and their relationships with soil factors].

Epimedium pubescens and soil samples were collected from the Betula albosinensis community (community I ), Cyclobalanopsis glauca var. gracilis community (community II), and Cinnamomum longepaniculatum community (community III) in Tangjiahe Nature Reserve in August 2009, with the total flavonoids and icariin contents in E. pubescens samples measured by high performance liquid chromatography and UV-spectrophotometer, and the relationships between these contents and soil parameters analyzed. Among the plant organs of E. pubescens, leaf had the highest contents of total flavonoids and icariin, while stem had the lowest one. The total flavonoids and icariin contents of E. pubescens were significantly higher in community I [(5.32 +/- 0.23)%, (0.47 +/- 0.05)%] than in communities II [(4.06 +/- 0.03)%, (0.32 +/- 0.01)%] and III [(4.15 +/- 0.07)%, (0.28 +/- 0.09)%] (P<0.05), and negatively correlated with soil total nitrogen and alkali-hydrolyzable nitrogen (P<0.05) but positively correlated with soil pH (P<0.01), suggesting that the soil with lower level of nitrogen and higher level of acidity in community I could enhance the total flavonoids and icariin contents of E. pubescens.

[The contents changes of polysaccharides and total flavonoids during flower bud differentiation of Epimedium sagittatum].

OBJECTIVE: To study the dynamic changes of the contents of polysaccharides and total flavonoids Daring flower bud differentiation of Epimedium sagittatum. METHODS: The polysaccharides and total flavonoids from Epimedium sagittatum were extracted by Microwave-assisted method. The content of polysaccharides was determined by anthrone sulphuric acid colorimetry and that of total flavonoids was determined at 270 nm by ultraviolet spectrophotometry. RESULTS: The polysaccharides content in leaf decreased during the initial stages of floral bud differentiation and then increased, while the content in stem and fibrous root decreased steadily until floral bud differentiation completed. The content in rhizome kept invariant. In the flower bud physiological differentiation period,the content of total flavonoids in leaf and stem decreased and then kept invariant, while the content in rhizome increased and kept invariant. The content of total flavonoids in fibrous root increased during the initial stages of floral bud differentiation and then decreased. CONCLUSION: The contents of polysaccharide and total flavonoids have relation with the flower bud differentiation of Epimedium sagittatum.

[Research on quality of Epimedium extract in market].

OBJECTIVE: To review the quality of Epimedium extract in the market. METHOD: The contents of icariin, epimedin C, sagittatoside B and total flavonoids in Epimedium extracts sold in the market were assayed by the methods of HPLC and UV respectively. HPLC fingerprintings were obtained at the same time. RESULT: The contents of icariin in most of the extracts are closely similar with the ones labeled by the companies. 3 type chromatograms were classified in all the HPLC fingerprintings, and were corresponded with their raw materials. The contents of epimedin C, sagittatoside B and total flavonoids were different in the samples with the same content of icariin. CONCLUSION: We can primarily confirm the origin of raw materials by comparing the HPLC fingerprinting of extracts with the ones of materials. The difference of extracts quality mainly comes from the difference of materials. So we suggest that Epimedium extract product should be labeled the origin of materials, and assayed with more compound contents, to ensure the quality stabilization.

[A review on research of raw material and cut crude drug of Herba epimedii in last ten years].

Researches on taxonomy, resources, cultivation, quality, chemicals and processing of Herba Epimedii in last ten years were reviewed. The taxonomy of Chinese species in "Flora of China (Vo1.29)" (published in 2001) and "The genus Epimedium and other herbaceous Berberidaceae" (published in 2002) were mainly introduced and commented. The quality evaluation of raw material of Herba Epimedii, and effects of distribution, growing time and medicinal part on the quality were summarized.