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Micranthanosides I and II (1–2), two diterpenoid glucosides featuring a new 1,10-secograyanane skeleton, thirteen new diterpenoid glycosides (3–15), and 21 known analogues were obtained from the ethanol extract of the leaves and twigs of Rhododendron micranthum. Micranthanoside XII (12) represent the first example of 3,5-epoxy-4,5-seco-ent-kaurane diterpenoid. The structures of these compounds were determined by spectroscopic data analysis and quantum chemical calculations. To clarify the chemical basis and provide reference for rational use of this medicinal plant, the antinociceptive and the anti-inflammatory activities of the compounds were tested. In the acetic acid-induced writhing test, compounds 17 and 19 showed significant antinociceptive activity at a dose of 3 mg kg−1 and compounds 2, 6 and 32 showed significant antinociceptive activity at a dose of 10 mg kg−1. Toxic reactions such as nausea and convulsion were observed when 17, 19, 29, and 31 at a dose of 10 mg kg−1 or 30 and 33 at a dose of 1 mg kg−1 were administered. The anti-inflammatory activities of the isolated compounds were evaluated by measuring the inhibitory effects of LPS-induced NO production in BV2 cells. At 10 μM, micranthanoside IX (9) and rhodomicranoside F (26) showed moderate anti-inflammatory activities with inhibition rates of 56.31% and 72.43%, respectively.

Seventeen diterpenoids (1–17), classified into eight diverse carbon skeleton types, grayanane (1, 2, and 12), micranthane (3, 4, and 13), mollane (5–7 and 14), 1,5-seco-grayanane (8), kalmane (9–11), 1,5-seco-kalmane (15), A-homo-B-nor-ent-kaurane (16), and leucothane (17), respectively, were isolated from the leaves extract of Rhododendron micranthum. Among them, diterpenoids 1–9 are new compounds and their structures were elucidated via extensive spectroscopic methods, quantum chemical calculations including the 13C NMR-DP4+ analysis and electronic circular dichroism (ECD) calculations, and the single-crystal X-ray diffraction analysis. Micranthanol A (1) represents the first example of a 5αH,9αH-grayanane diterpenoid and a 6-hydroxy-6,10-epoxygrayanane diterpenoid, and micranthanone B (3) is the first 6,10-epoxymicranthane and the 5α-hydroxy-micranthane diterpenoids. 14-epi-Mollanol A (5) and mollanol B (6) represent the first examples of 14β-hydroxymollane diterpenoids. It is the first time to report mollane, 1,5-seco-kalmane, and A-homo-B-nor-ent-kaurane type diterpenoids from Rhododendron micranthum. All the seventeen diterpenoids showed significant antinociceptive activities at a dose of 5.0 mg/kg, and it is the first time to evaluate the antinociceptive activity of 1,5-seco-kalmane diterpenoid. Among them, compounds 3, 11, 14, and 15 exhibited significant antinociceptive activities even at a lower dose of 1.0 mg/kg. A preliminary structure–activity relationship for the antinociceptive effects of diterpenoids 1–17 is discussed, which provided a new basis to develop novel potent analgesics.

Rhododendron micranthum is an evergreen shrub species widely distributed in China that has high ornamental and medicinal value. However, there is a lack of molecular and genomic data for this plant, which severely restricts the development of its relevant research. The objective of the present study was to conduct a first genomic survey of R. micranthum and determine its whole-genome sequencing scheme. Next-generation sequencing (Illumina Hi-Seq Xten) was used to measure the genome size of R. micranthum, K-mer analysis were employed to investigate its genomic profile. Finally, we conducted bioinformatics methods to performed SSR (simple sequence repeat) prediction based on the genomic data. The genome size of R. micranthum was estimated to be 554.22 Mb. The heterozygosity ratio was 0.93%, and the sequence repeat ratio was calculated to be 49.17%. The clean reads of R. micranthum were assembled into 2281551 scaffolds with a N50 value of 916 bp. A total of 479724 SSR molecular markers were identified in the R. micranthum genome, and 871656 pairs of primers designed for application. Among of them, 100 primer pairs were validated, and 71 primer pairs were successfully amplified. In summary, the R. micranthum genome is complex with high heterozygosity and low repeated sequences. In future whole-genome research in R. micranthum, higher-depth ‘2+3’ (Illumina+PacBio) sequencing may yield better assembly results.

Two new grayanane-type (1 and 2) and one new kalmane-type diterpenoids (3), together with 16 known compounds, were isolated from the roots of Rhododendron micranthum. The structures of new compounds were fully determined on the basis of spectroscopic analysis, including HRESIMS, 1 D and 2 D NMR data. An acetic acid-induced writhing test in mice was proceeded to evaluate the antinociceptive activities of compounds 1–3, 5–6, 9–14 and 16. Compared to vehicle-injected mice, compounds 1, 6, 14 and 16 showed significant antinociceptive effects with writhe inhibition rates of 45.8%-64.2% at a dose of 0.1 mg/kg, and compounds 10, 12 and 13 showed significant antinociceptive effects with writhe inhibition rates of 33.9%-64.8% at a dose of 5 mg/kg. Compound 2 showed potent antinociceptive effects with writhe inhibition rates of 86.1% and 54.7% at doses of 8 mg/kg and 0.8 mg/kg, respectively.

This research was performed for the purpose of providing useful and base data and preservation strategy of growth characteristics of Rhododendron micranthum. From the result of research, wild growth area of Rhododendron micranthum was discovered near steep forest road on 695∼901m in altitude above sea level of Baekdu-daegan National Arboretum. Vegetation colony structure of SiteⅠwas resulted in Pinus densiflora(tree stratum M.I.P.=45.3%) is dominants, Betula schmidtii(arborescent M.I.P.=9.8%) is second dominant, and SiteⅢ resulted in Pinus densiflora(tree stratum M.I.P.=30.5%) is dominants, Betula schmidtii(arborescent M.I.P.=10.3%) is second dominant. SiteⅡ resulted in Betula schmidtii(arborescent M.I.P.=30.4%) is dominants and Pinus densiflora tree(tree stratum M.I.P.=8.3%) is second dominants. For herbs, it was researched that the rate of one year herb of chrysanthemum family(10), rice family(3), sedge(2) was higher than near forest area. Among Species diversity index, that of Shannon was in range of 0.7348∼1.1090. After comparing this with similar area, this value was relatively low. Dominance analysis result was analyzed that various groups are evenly distributed. Monthly highest average temperature is 23.9° in Site A and Site B, and monthly lowest average temperature is -4.5℃ in SiteⅠand -4.7℃ in SiteⅡ. Monthly maximum average humidity is 85.6% in SiteⅠand 83.2% in SiteⅡ. Monthly minimum average humidity is 60.9% in SiteⅠand 60.8% in SiteⅡand it showed that these 2 area are located far away but have similar temperature and humidity characteristic and climate environment is similar. It was analyzed that humidity is high with the result of annual average humidity is over 60%. In growth characteristic, it showed that doubt damage of Stephanitis pyrioides, exposure of roots, peeled bark was discovered and supplement on this is urgent.

Thirteen new grayanane diterpenoid glucosides, 3- epi-grayanoside B (1), micranthanosides A-E (2-6), 7α-hydroxygrayanoside C (7), micranthanoside F (8), 14β-acetyoxymicranthanoside F (9), micranthanoside G (10), 14- O-acetylmicranthanoside G (11), 14β-hydroxypieroside A (12), and micranthanoside H (13), and six known analogues (14-19) were isolated from the leaves of Rhododendron micranthum. The structures of 1-19 were elucidated based on spectroscopic analysis, comparison with literature, and chemical methods. The absolute configurations of 3- epi-grayanoside B (1) and micranthanosides A (2) and C (4) were defined by single-crystal X-ray diffraction analysis. This is the first report of the crystal structures of grayanane diterpenoid glucosides. 3- epi-Grayanoside B (1) represents the first example of a 3α-oxygrayanane diterpenoid glucoside, and micranthanosides A-D (2-5) are the first examples of 5α-hydroxy-1-β H-grayanane diterpenoids. In addition, micranthanosides C-F (4-6 and 8) and 14β-acetyoxymicranthanoside F (9) represent the first examples of grayanane glucosides with the glucosylation at C-16. All the grayanane diterpenoid glucosides 1-19 were assayed for their anti-inflammatory, antitumor, and PTP1B inhibitory activities, but did not show significant activities at 40 μM. Grayanane diterpenoid glucosides 1-18 were evaluated for their antinociceptive activity, and compounds 2, 3, 7-10, 12, 13, and 16 showed significant antinociceptive effects with percentage inhibitions in excess of 50%.

Nine previously undescribed diterpene glucosides, rhodomicranosides A−I, comprising leucothane, 4,5-seco-ent-kaurane, and grayanane types, respectively, were isolated from the leaves of Rhododendron micranthum, along with seven known diterpenoids. Their structures were elucidated based on extensive spectroscopic analyses such as HRESIMS, 1D and 2D NMR, UV, and IR, and their absolute configurations were determined by various methods including X-ray diffraction analysis, electronic circular dichroism spectroscopy (ECD), calculated ECD, and Mo2(OAc)4-induced ECD, as well as chemical methods. This is the first time to report the crystal structures of leucothane diterpene glycosides. Rhodomicranosides A−C represent the first examples of 15α-hydroxy-leucothane diterpenoids, leucothane diterpene diglucosides, and 9β-hydroxy-leucothane diterpenoids, respectively. Rhodomicranosides D and E are the second and third examples of 4,5-seco-ent-kaurane diterpenoids, and this is the first time to report 4,5-seco-ent-kaurane-type diterpenoids from the genus of Rhododendron. Rhodomicranosides F and G are the first examples of 5α-H-grayan-1(10),9(11)-diene-6-one diterpenoids. Some isolated diterpenoids were evaluated for their analgesic activity in an acetic acid-induced writhing test, and rhodomicranosides A−E and H, pierisformoside F, iso-grayanotoxin II, and grayanotoxins I, III, and IV showed significant analgesic effects with the percentage inhibitions over 50% at the dose of 1.0 mg/kg. In particular, grayanotoxins I and III exhibited more potent analgesic activity than morphine at a dose of 0.2 mg/kg, and they showed significant analgesic activity even at a lower dose of 0.04 mg/kg with the inhibition rates of 71.5% and 69.3%, respectively. Their preliminary structure-activity relationships were discussed.

The habitats characteristics of Rhododendron micranthum Turcz. were investigated to compile basic data for conservation and restoration. Natural habitats were located at altitudes of 100-500 m with inclinations of 10-35 o . Rhododendron micranthum population was classified into Pinus densiflora dominant population. In the study sites, soil organic matter, total nitrogen, available phosphate, exchangeable potassium, exchangeable sodium, exchangeable calcium, exchangeable magnesium, cation exchange capacity and soil pH were 4.10-8.64%, 0.18-0.46%, 8.69-26.70 mgkg -1 , 0.10-0.23 cmol + kg -1 , 0.06-0.10 cmol + kg -1 , 0.85-4.10 cmol + kg -1 , 0.24-0.64 cmol + kg -1 , 12.76-20.90 cmol + kg -1 , 4.34-5.15. Rooting rate is too low, cutting propagation, breeding methods are not good. Also, this study was investigated seed germination of R. micranthum depends on soaking treatment. R. micranthum was soaked with tap water for four days, the average values of germination day were represented of 70%/1 week.Key Words: Rhododendron micranthum, Seed germination rate, rooting rate, rare plant, propagation

Selection of reliable reference genes is a prerequisite for accurate normalization of gene expression in quantitative real-time polymerase chain reaction (qRT-PCR). The present study is the first report of a systematic evaluation of potential reference genes in Rhododendron micranthum Turcz. The expression stabilities of 6 candidate reference genes (18S, ACT, CYP, UBQ, TUA, and EF1α) in different tissues and the same tissue at different developmental stages were assessed in Rh. micranthum Turcz, using two analysis software packages, geNorm and NormFinder. The results indicated that EF1α and UBQ were the most stable reference genes for different tissues (leaf, root, stem and flower) at the same developmental stage, whereas 18S should be avoided. During leaf development, EF1α and 18S can be used as the most reliable reference genes, but CYP was the most variable one. During flower development, the most stable genes were CYP and EF1α, while 18S was the least stable one. In order to demonstrate the importance of using suitable reference genes for obtaining an accurate and reliable results in gene expression studies, the expression pattern of an independent target gene, RhMDH gene was analyzed in parallel, the RhMDH showed an expression difference of 58-fold in stem when normalized with the most stable gene (EF1α) or the least stable gene (18S) act as reference, respectively. Our results support that the selection of suitable reference genes is necessary to achieve accurate results in qRT-PCR.

The proanthocyanidins of the leaves of 16 taxa of the Rhododendron genus (Ericaceae) [Rhododendron 'Catawbiense Grandiflorum', Rhododendron 'Cunningham's White', Rhododendron smirnowii Trautv., Rhododendron calophytum Franch., Rhododendron dichroanthum ssp. scyphocalyx (Balf. f. & Forrest ) Cowan, Rhododendron micranthum Turcz., Rhododendron praevernum Hutch., Rhododendron ungernii Trautv., Rhododendron kaempferi Planch., Rhododendron degronianum ssp. heptamerum var. hondoense (Nakai ) H. Hara, Rhododendron fortunei Lindl., Rhododendron ponticum L., Rhododendron galactinum Balf. f. ex Tagg., Rhododendron oreotrephes W. W. Sm., Rhododendron brachycarpum ssp. brachycarpum D. Don ex G. Don, and Rhododendron insigne Hemsl. & E. H. Wilson ] were investigated qualitatively by liquid chromatography-mass spectrometry in series. Twenty-nine dimeric proanthocyanidins based on (epi)catechin and (epi)gallocatechin were detected and characterized on the basis of their unique fragmentation pattern in the negative ion mode tandem mass spectrometry spectra. All of them were extracted for the first time from these sources, and ten of them were not reported previously in nature. The position of the galloyl residue was assigned on the basis of the retro-Diels-Alder fragmentation and the dehydrated retro-Diels-Alder fragmentation; it resulted from the loss of gallic acid as a neutral loss in the negative ion mode. Furthermore, four caffeoylquinic acids, six p-coumaroylquinic acids, epigallocatechin, gallocatechin, catechin, epicatechin, epigallocatechin gallate, catechin gallate, epicatechin gallate, gallocatechin gallate, two quercetin-O-hexosides, quercetin-O-galloyl-hexoside, quercetin-O-pentoside, quercetin-O-rhamnoside, quercetin-O-pentoside-O-hexoside, quercetin-O-rhamnoside-O-hexoside, quercetin-O-feruloyl-hexoside, quercetin-O-(p-hydroxy)benzoyl-hexoside, taxifolin-O-pentoside, myricetin-O-rhamnoside, two myricetin-O-pentosides, three myricetin-O-hexosides, and two myricetin-O-galloyl-hexosides were detected and shown to possess characteristic tandem mass spectrometry spectra and were tentatively assigned on the basis of their retention time.

We surveyed that vascular plants can be classified into 90 families and 240 genus, 336 species, 69 variants, 22 forms, 3 subspecies, total 430 taxa. Dicotyledon plant is 80.9%, monocotyledon plant is 9.8%, Pteridophyta is 8.1%, Gymnosermae is 1.2% among the whole plant family. Rare and endangered plants are Crypsinus hastatus, Lilium distichum, Viola albida, Rhododendron micranthum, totalling four species. Endemic plants are Carex okamotoi, Salix koriyanagi for. koriyanagi, Clematis trichotoma, Thalictrum actaefolium var. brevistylum, Galium trachyspermum, Asperula lasiantha, Weigela subsessilis, Adenophora verticillata var. hirsuta, Aster koraiensis, Cirsium chanroenicum and Saussurea seoulensis total 11 taxa. Specialized plants are 20 classification for I class, 7 classifications for the II class, 7 classifications for the III class, 2 classification for the IV class, and 1 classification for the V class, total 84 taxa. Naturalized plants specified in this study are 10 types but Naturalization rate is not high compared to the area of BaekDu-DaeGan. This survey area is focused on the center of BaekDu-DaeGan, and it has been affected by excessive investigations and this area has been preserved as Buddhist temples’ woods. Therefore, we need health care provisions and monitoring for this.

Eleven grayanane diterpenoids, 1-epi-grayanotoxin IV, 1-epi-grayanotoxin II, 6-deoxy-1-epi-grayanotoxin XVII, 6-deoxygrayanotoxin XVII, 16-acetylgrayanotoxin II, 3-oxograyanotoxin IX, 14-deoxygrayanotoxin VIII, 14-acetylisograyanotoxin II, rhodomicranols C–E, and a leucothane diterpenoid, rhodomicranol F, together with eleven known diterpenoids were isolated from leaves of Rhododendron micranthum. Their structures were elucidated by spectroscopic analyses, with the absolute configurations of 1-epi-grayanotoxin IV and rhodomicranol C determined by single-crystal X-ray diffraction with Cu Kα radiation, and the structures of 14-acetylisograyanotoxin II and known grayanotoxins IX and X confirmed by single-crystal X-ray diffraction. All twenty-three diterpenoids were evaluated for their in vitro immunomodulatory activities, and none showed significant immunomodulatory activities in a dose-dependent manner. In addition, they are non-toxic to the murine lymphocytes in the general cytotoxicity assay.

A new diterpene with an unprecedented carbon skeleton, micranthanone A (1), two new grayanane diterpenoids bearing an unusual 5,6-(3,4-dihydroxylbenzylidene acetal) motif, rhodomicranols A (2) and B (3), and three known grayanane diterpenoids (4–6) were isolated from Rhododendron micranthum. Their structures were elucidated by spectroscopic analyses, calculated ECD, and single-crystal X-ray diffraction. The in vitro immunomodulatory activities of 1–6 were evaluated, and a plausible biogenetic pathway for 1 is proposed.

A reversed-phase high-performance liquid chromatography method was established for the simultaneous determination of four major constituents, namely myricitrin, hyperin, quercitroside, and quercetin in Folium Rhododendri Micranthi. The optimal conditions of separation and detection were achieved on a C18 analytical column with a gradient mobile phase consisting of acetonitrile and 1% acetic acid at the flow rate of 1.0 mL/min, and detection wavelength was set at 355 nm. All calibration curves showed good linear regression (r > 0.9993) within test ranges. The reproducibility of relative standard deviations was less than 2.3%, and recovery of analytes was greater than 99.4%, respectively. The method was successfully applied to determine the contents of four compounds in Folium Rhododendri Micranthi. The results indicated that the contents of myricitrin, hyperin, quercitroside and quercetin in Folium Rhododendri Micranthi were 0.166%, 0.303%, 0.299%, and 0.053%, respectively.