Altitudinal Variation of Metabolites, Mineral Elements and Antioxidant Activities of Rhodiola crenulata (Hook.f. & Thomson) H.Ohba.

Rhodiolacrenulata (Hook.f. & Thomson) H.Ohba is an alpine medicinal plant that can survive in extreme high altitude environments. However, its changes to extreme high altitude are not yet clear. In this study, the response of Rhodiola crenulata to differences in altitude gradients was investigated through chemical, ICP-MS and metabolomic methods. A targeted study of Rhodiola crenulata growing at three vertical altitudes revealed that the contents of seven elements Ca, Sr, B, Mn, Ni, Cu, and Cd, the phenolic components, the ascorbic acid, the ascorbic acid/dehydroascorbate ratio, and the antioxidant capacity were positively correlated with altitude, while the opposite was true for total ascorbic acid content. Furthermore, 1165 metabolites were identified: flavonoids (200), gallic acids (30), phenylpropanoids (237), amino acids (100), free fatty acids and glycerides (56), nucleotides (60), as well as other metabolites (482). The differential metabolite and biomarker analyses suggested that, with an increasing altitude: (1) the shikimic acid-phenylalanine-phenylpropanoids-flavonoids pathway was enhanced, with phenylpropanoids upregulating biomarkers much more than flavonoids; phenylpropanes and phenylmethanes upregulated, and phenylethanes downregulated; the upregulation of quercetin was especially significant in flavonoids; upregulation of condensed tannins and downregulation of hydrolyzed tannins; upregulation of shikimic acids and amino acids including phenylalanine. (2) significant upregulation of free fatty acids and downregulation of glycerides; and (3) upregulation of adenosine phosphates. Our findings provide new insights on the responses of Rhodiola crenulata to extreme high altitude adversity.

Evaluation of Two Major Rhodiola Species and the Systemic Changing Characteristics of Metabolites of Rhodiola crenulata in Different Altitudes by Chemical Methods Combined with UPLC-QqQ-MS-Based Metabolomics.

Rhodiola species have a long history of use in traditional medicine in Asian and European countries and have been considered to possess resistance to the challenges presented by extreme altitudes. However, the influence of different Rhodiola species on quality is unclear, as well as the influence of altitude on phytochemicals. In this study, the phenolic components and antioxidant abilities of two major Rhodiola species are compared, namely Rhodiolacrenulata and Rhodiola rosea, and the metabolomes of Rhodiolacrenulata from two representative elevations of 2907 and 5116 m are analyzed using a UPLC-QqQ-MS-based metabolomics approach. The results show that the phenolic components and antioxidant activities of Rhodiolacrenulata are higher than those of Rhodiola rosea, and that these effects in the two species are positively correlated with elevation. Here, 408 metabolites are identified, of which 178 differential metabolites (128 upregulated versus 50 downregulated) and 19 biomarkers are determined in Rhodiola crenulata. Further analysis of these differential metabolites showed a significant upregulation of flavonoids, featuring glucosides, the enhancement of the phenylpropanoid pathway, and the downregulation of hydrolyzed tannins in Rhodiola crenulata as elevation increased. Besides, the amino acids of differential metabolites were all upregulated as the altitude increased. Our results contribute to further exploring the Rhodiola species and providing new insights into the Rhodiola crenulata phytochemical response to elevation.

Identification of a novel UDP-glycosyltransferase gene from Rhodiola rosea and its expression during biotransformation of upstream precursors in callus culture.

Roseroot (Rhodiola rosea L.) is a medicinal plant with adaptogenic properties and several pharmaceutically important metabolites. In this study, a full length cDNA encoding a UDPG gene of roseroot was identified, cloned and characterized. Its ORF (1425 bp) was transferred into E. coli, where the expression of the recombinant enzyme was confirmed. To monitor the enzyme activity, 3 precursors (tyramine, 4-hydroxyphenylpyruvate & tyrosol) of salidroside biosynthesis pathway were added to roseroot callus cultures and samples were harvested after 1, 6, 12, 24, 48 & 96 h. Along with the controls (without precursor feeding), each sample was subjected to HPLC and qRT-PCR for phytochemical and relative UDP-glycosyltransferase gene expression analysis, respectively. The HPLC analysis showed that the salidroside content significantly increased; reaching 0.5% of the callus dry weight (26-fold higher than the control) after 96 h when 2 mM tyrosol was given to the media. The expression of the UDP-glycosyltransferase increased significantly being the highest at 12 h after the feeding. The effect of tyramine and 4-hydroxyphenylpyruvate was not as pronounced as of tyrosol. Here, we introduce a R. rosea specific UDPG gene and its expression pattern after biotransformation of intermediate precursors in in vitro roseroot callus cultures.

Enhanced Production of Phenolic Compounds in Compact Callus Aggregate Suspension Cultures of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal plant of the trans-Himalayan region of Ladakh. It is used for the treatment of numerous health ailments. Compact callus aggregate (CCA) suspension cultures of Rhodiola imbricata were established to counter extinction threats and for production of therapeutically valuable phenolic compounds to meet their increasing industrial demands. The present study also investigated the effect of jasmonic acid (JA) on production of phenolic compounds and bioactivities in CCA suspension cultures. CCA suspension cultures established in an optimized Murashige and Skoog medium supplemented with 30 g/l sucrose, 3 mg/l NAA, and 3 mg/l BAP showed maximum biomass accumulation (8.43 g/l DW) and highest salidroside production (3.37 mg/g DW). Upon 100 µM JA treatment, salidroside production (5.25 mg/g DW), total phenolic content (14.69 mg CHA/g DW), total flavonoid content (4.95 mg RE/g DW), and ascorbic acid content (17.93 mg/g DW) were significantly increased in cultures. In addition, DPPH-scavenging activity (56.32%) and total antioxidant capacity (60.45 mg QE/g DW) were significantly enhanced upon JA treatment, and this was positively correlated with increased accumulation of phenolic compounds. JA-elicited cultures exhibited highest antimicrobial activity against Escherichia coli. This is the first report describing the enhanced production of phenolic compounds and bioactivities from JA-elicited CCA suspension cultures of Rhodiola imbricata.

Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew.

Rhodiola imbricata is a rare medicinal herb well-known for its adaptogenic and antioxidant properties due to the presence of a diverse array of secondary metabolites, including phenylethanoids and phenylpropanoids. These secondary metabolites are generating considerable interest due to their potential applications in pharmaceutical and nutraceutical industries. The present study investigated the influence of light quality on growth, production of industrially important secondary metabolites and antioxidant activity in callus cultures of Rhodiola imbricata. Callus cultures of Rhodiola imbricata were established under different light conditions: 100% red, 100% blue, 100% green, RGB (40% red: 40% green: 20% blue) and 100% white (control). The results showed that the callus cultures grown under red light accumulated maximum amount of biomass (7.43 g/l) on day 21 of culture, as compared to other light conditions. Maximum specific growth rate (0.126 days-1) and doubling time (132.66 h) was observed in callus cultures grown under red light. Reverse phase-high performance liquid chromatographic (RP-HPLC) analysis revealed that the callus cultures exposed to blue light accumulated maximum amount of Salidroside (3.12 mg/g DW) on day 21 of culture, as compared to other light conditions. UV-Vis spectrophotometric analysis showed that the callus cultures exposed to blue light accumulated maximum amount of total phenolics (11.84 mg CHA/g DW) and total flavonoids (5.53 mg RE/g DW), as compared to other light conditions. Additionally, callus cultures grown under blue light displayed enhanced DPPH free radical scavenging activity (53.50%). Callus cultures grown under different light conditions showed no significant difference in ascorbic acid content (11.05-13.90 mg/g DW) and total antioxidant capacity (27.37-30.17 mg QE/g DW). The correlation analysis showed a positive correlation between total phenolic content and DPPH free radical scavenging activity in callus cultures (r = 0.85). Taken together, these results demonstrate the remarkable potential of light quality on biomass accumulation and production of industrially important secondary metabolites in callus cultures of Rhodiola imbricata. This study will open new avenues and perspectives towards abiotic elicitation strategies for sustainable growth and enhanced production of bioactive compounds in in-vitro cultures of Rhodiola imbricata.

Response to climate change of montane herbaceous plants in the genus Rhodiola predicted by ecological niche modelling.

Climate change profoundly influences species distributions. These effects are evident in poleward latitudinal range shifts for many taxa, and upward altitudinal range shifts for alpine species, that resulted from increased annual global temperatures since the Last Glacial Maximum (LGM, ca. 22,000 BP). For the latter, the ultimate consequence of upward shifts may be extinction as species in the highest alpine ecosystems can migrate no further, a phenomenon often characterized as "nowhere to go". To predict responses to climate change of the alpine plants on the Qinghai-Tibetan Plateau (QTP), we used ecological niche modelling (ENM) to estimate the range shifts of 14 Rhodiola species, beginning with the Last Interglacial (ca. 120,000-140,000 BP) through to 2050. Distributions of Rhodiola species appear to be shaped by temperature-related variables. The southeastern QTP, and especially the Hengduan Mountains, were the origin and center of distribution for Rhodiola, and also served as refugia during the LGM. Under future climate scenario in 2050, Rhodiola species might have to migrate upward and northward, but many species would expand their ranges contra the prediction of the "nowhere to go" hypothesis, caused by the appearance of additional potential habitat concomitant with the reduction of permafrost with climate warming.

[Resource investigation about Tibetan medicine Rhodiola kirilowii].

The investigation aims to better understand the resource status of Rhodiola kirilowii, analysis the suitable habitat of wild Rh. kirilowii and protect the wild resources of Rh. Kirilowii, promoting the sustainable utilization of Rh. kirilowii resources. In this paper, we investigated the wild resources of Rh. kirilowii in 16 counties of Sichuan, Qinghai, Gansu and Yunnan by means of investigation and sampling investigation combined with interview. The results showed that the population densities of wild Rh. kirilowii in 4 provinces were very different and the reserve of wild resources decreased gradually in many areas. According to the survey results, the current total reserve of Rh. kirilowii in four provinces was about 1 100 t. The reserve of wild Rh. kirilowii in Sichuan province was the largest. Simultaneously, the Rh. kirilowii had a certain ecological value. We found that a sand control base with planting Rh. kirilowii was set up in Hongyuan County of Sichuan Aba Tibetan and Qiang Autonomous Prefecture. The investigation provides a scientific basis for the development and sustainable utilization of Rh. kirilowii resources.

[Potential distribution of Rhodiola crenulata in Tibetan Plateau based on Maxent model].

Wildlife tending and artificial cultivation is an important way to protect the wild resources of Rhodiola crenulata. It is a study hotspot at present. The distribution information of R. crenulata was collected by query data and field survey, the ecological suitability regionalization was conducted based on maximum entropy model combine with ecological factors, including climate, soil and altitude. To provide the reference for production layout, suitable planting area and the selection of artificial planting base by studying the ecological suitability regionalization of R. crenulata. The potential distribution areas mainly concentrated in the easen Tibet, western Sichuan, southern Qinghai, and Gansu Gannan Tibetan Autonomous Prefecture, Yunnan Diqing Tibetan Autonomous Prefecture. There were 5 major environmental factors to have obvious influence on ecology suitability distributions of R. crenulata, including altitude (contribution rate of 61.8%), precipitation of warmest quarter (contribution rate of 19%), the coefficient of variation of precipitation seasonality (contribution rate of 4.7%), the SD of temperature seasonality (contribution rate of 4%), mean temperature of driest quarter (contribution rate of 2.5%). The AUCs of ROC curve were both above 0.9, indicating that the predictive results with the Maxent model were highly precise. The study of the ecological suitability regionalization of R. crenulata based on Maxent can provide a scientific basis for the selection of artificial planting base.

Changes in the Content of the Glycosides, Aglycons and their Possible Precursors of Rhodiola rosea during the Vegetation Period.

Phytochemical participants in the biosynthetic pathway of salidroside and cinnamyl alcohol glycosides were studied from seven Rhodiola rosea L. individuals originating from a wild population. Plants were grown in a phytotron and samples were taken at 3 weekly intervals during the vegetation period. Based on HPLC analysis, all the key compounds to which roseroot medicinal property is attributed were detected, with salidrosde being the most dominant, followed by its aglycone, tyrosol. The contents of all compounds were 2-3 times more in the rhizomes than in roots. The highest content of salidroside, tyrosol, rosarin, rosavin and cinnamyl alcohol was recorded in rhizomes and at the beginning of shoot elongation. The seven roseroot individuals showed a very high deviation in their chemical content at each sampling time. Our statistical analysis showed that the trend of salidroside accumulation in the rhizome was the most similar in all studied plants. These results have important implications for choosing a reasonable harvest time to obtain the maximum phytochemical content and a better understanding of active compounds formation in R. rosea L.

The role of biotechnology for conservation and biologically active substances production of Rhodiola rosea: endangered medicinal species.

At present, more than 50,000 plant species are used in phytotherapy and medicine. About 2/3 of them are harvested from nature leading to local extinction of many species or degradation of their habitats. Biotechnological methods offer possibilities not only for faster cloning and conservation of the genotype of the plants but for modification of their gene information, regulation, and expression for production of valuable substances in higher amounts or with better properties. Rhodiola rosea is an endangered medicinal species with limited distribution. It has outstanding importance for pharmaceutical industry for prevention and cure of cancer, heart and nervous system diseases, and so forth. Despite the great interest in golden root and the wide investigations in the area of phytochemistry, plant biotechnology remained less endeavoured and exploited. The paper presents research on initiation of in vitro cultures in Rhodiola rosea and some other Rhodiola species. Achievements in induction of organogenic and callus cultures, regeneration, and micropropagation varied but were a good basis for alternative in vitro synthesis of the desired metabolites and for the development of efficient systems for micropropagation for conservation of the species.

Improved mass multiplication of Rhodiola crenulata shoots using temporary immersion bioreactor with forced ventilation.

A temporary immersion bioreactor system was found to be suitable for mass shoot proliferation of Rhodiola crenulata. The shoot multiplication ratio and hyperhydration rate reached 46.8 and 35.4%, respectively, at a temporary immersion cycle of 3-min immersion every 300 min. Forced ventilation was employed in the temporary immersion bioreactor culture in order to decrease the hyperhydration rate, improve shoot quality and enhance the multiplication ratio. The highest multiplication ratio of 55.7 was obtained under a temporary immersion cycle of 3-min immersion every 180 min with the forced ventilation at an air flow rate of 40 l/h, and the hyperhydration rate was reduced to 26.1%. Forced ventilation also improved the subsequent elongation and rooting rate of these proliferated shoots, and the shoot cultures from the temporary immersion bioreactor formed complete plantlets when subcultured onto a rooting medium containing 5 µmol/l indole-3-acetic acid.

[Effects of different light and GA3 on the germination and seedling growth of Rhodiola sachalinensis].

OBJECTIVE: To improve germination rate and research the seed germination character of Rhodiola sachalinensis. METHODS: Made seed germinated under water or with moisture, at light or dark condition, effects of 6 different light qualities (black, white, blue, green, red and far red light) illumination on seed germination were studied. The seedling growth at different temperature were studied as well. RESULTS: The results indicated that light was required for Rhodiola sachalinensis seeds germinate, 83.3% germination rate at light and soaking in watercondition; Seed germination were improved to 84%, 70.7% and 52.0% respectively in white, red and green light quality illumination, the germination rate reached 84% after 9 h of white light illumination. Germination rate were 80% after 100 mg/L GA3 treatment for 3 h under dark condition. 20 degrees C was the optimal temperature for Rhodiola sachalinensis seedling growth, under with the plant height, fresh weight and dry weight of plant were the best. Seedling growth rate and the dry weight increased obviously at 35 d and 50 d. CONCLUSION: The experiment definited the best condition for Rhodiola sachalinensis seed germination and seedling growth, supplied basic data for large scale planting Rhodiola sachalinensis using seed propagation technology.

Salidroside production by hairy roots of Rhodiola sachalinensis obtained after transformation with Agrobacterium rhizogenes.

Hairy roots induced by Agrobacterium rhizogenes grow faster, and are considered as genetically stable. These hairy roots can be used as an interesting material for the production of secondary metabolites of pharmaceutical value. Salidroside has been identified as the major compounds from the roots of Rhodiola sachalinensis A. BOR. Here, we provide an update that adds new perspectives on the prospects and challenges of producing Salidroside from hairy roots induced by Agrobacterium rhizogene in Rhodiola sachalinensis A. BOR. For high salidroside production, the optimal concentration for precursor (Tyrosol, Tyrosine, and Phenylalanine) and elicitor (Aspergillus niger, Coriolus versicolor, and Ganoderma lucidum) was added in the LB liquid medium, respectively. The addition of elicitor in the liquid MS medium and the utilization of precursor from chemical feeding enhanced biomass accumulation and salidroside production. The optimal concentration for elicitor and precursor in the liquid medium was 0.05 mg/l and 1 mmol/l, respectively.

[Effects of plant growth substances on induction and culture of callus from Rhodiola quadrifida].

OBJECTIVE: To investigate the effect of plant growth substances on induction and culture of callus from Rhodiola quadrifida and also to analyze salidroside contents in the callus. METHOD: The optimum combination of plant growth substances in MS solid medium for induction and culture of callus was established using orthogonal design. The contents of salidroside was analyzed by HPLC. RESULT: MS medium containing 2,4-D 1 mg x L(-1), NAA 2 mg x L(-1), 6-BA 0.5 mg x L(-1) and KT 0.1 mg x L(-1) could induce the callus from R. quadrifida most effectively;the induction rate was 83.3%. The optimized combination of plant growth substances for callus subculture was 2,4-D 1 mg x L(-1), 6-BA 0.1 mg x L(-1) and KT 0.5 mg x L(-1). The dry weight could reach 11.77 g x L(-1) when the callus was cultured in the optimum medium for 30 d and salidroside content was 0.28%. CONCLUSION: The quantities of plant growth substances required for induction and culture of callus are different in R. quadrifida. The callus could produce salidroside.

[Seasonal variations in biomass and salidroside content in roots of Rhodiola sachalinensis as affected by gauze and red film shading].

Rhodiola sachalinensis A. Bor, a perennial herb, belonging to the family Crassulaceae, is mainly distributed in mountains at the altitudes of 1,700-2,500 m. It is a typical alpine plant and a very important medicinal plant with high activities of anti-fatigue, anti-senescence, and anti-radiation, due to the secondary metabolite salidroside in its root. Our previous findings have proven that red light promotes salidroside synthesis remarkably but decreases biomass insignificantly, resulting in a higher yield of salidroside in roots of Rh. sachalinensis in a greenhouse. In order to investigate the influences of shading and red light on seasonal variations in biomass and salidroside content in Rh. sachalinensis roots, the effects on 3 or 4 years old Rh. sachalinensis plants in a nursery in Daxinganling Mountain (124 degrees 02' E, 50 degrees 30' N) were studied in 2001. Compared to the control (CK) of full sunlight, 6 treatments with neutral transparent film and gauze, or red film alone had been conducted for 131 days. In treatment I, Rh. sachalinensis was shaded with neutral transparent film and gauze to achieve an irradiance 51.8% of full sunlight. In treatment II, the plants were shaded by red film alone, but the irradiance was as that in treatment I. In treatments III, IV, V and VI, neutral transparent film and gauze were originally used on May 8, then shifted to red film on Jun 3, July 4, August 4 and September 2, respectively and all experiments stopped on September 16, 2001. Rh. sachalinensis roots were harvested on 2-4th from June to September and finally on September 16, and root-biomass and salidroside content were measured. Root-biomass in plants decreased significantly under shading with neutral transparent film and gauze compared to the control with full sunlight, but little variations in salidroside content and yield. In comparison with shading by neutral transparent film and gauze, root-biomass reduced lightly and salidroside content and yield in roots were increased remarkably under red-film shading. At the end of the season, salidroside content under red light was 163% in 3-year-old and 155% in 4-year-old Rh. sachalinensis roots; whereas salidroside yields were 144% in 3-year-old and 145% in 4-year-old Rh. sachalinensis roots to those in plants under shading. The results also showed that the enhancement in the salidroside content and yield were little related to the duration of red film shading, which implied that in order to increase salidroside content and get higher salidroside yield, but less affect root-biomass, Rh. sachalinensis may be shaded with red film just several days before harvest.