Metabolite Chemical Composition of the Bletilla striata (Thunb.) Reichb. f. Endophyte Penicillium oxalicum.

Penicillium oxalicum strain can be isolated from the Bletilla striata (Thunb.) Reichb. f. tubers. Its solid-state fermentation products are concentrated by percolation extraction. Separation and purification have been conducted to the ethyl acetate extracts by preparative HPLC. Based on the use of spectrometry, we have determined 17 known compounds, 12,13-dihydroxy-fumitremorgin C (1), pseurotin A (2), tyrosol (3), cyclo-(L-Pro-L-Val) (4), cis-4-hydroxy-8-O-methylmellein (5), uracil (6), cyclo-(L-Pro-L-Ala) (7), 1,2,3,4-tetrahydro-4-hydroxy-4-quinolin carboxylic acid (8), cyclo-(Gly-L-Pro) (9), 2'-deoxyuridine (10), 1-(ß-D-ribofuranosyl)thymine (11), cyclo-(L-Val-Gly) (12), 2'-deoxythymidine (13), cyclo-(Gly-D-Phe) (14), cyclo-L-(4-hydroxyprolinyl)-D-leucine (15), cyclo-(L)-4-hydroxy-Pro-(L)-Phe (16), uridine (17). Here, we report compounds 1-3, 5, 7-8, 11-12, 14-17 are first found and isolated from this endophyte.

Lung protective effects of dietary malate esters derivatives from Bletilla striata against SiO2 nanoparticles through activation of Nrf2 pathway.

Objective: To study the protective activities of the dietary malate esters derivatives of Bletilla striata against SiO2 nanoparticles-induced A549 cell lines and its mechanism action. Methods: The components were isolated and elucidated by spectroscopic methods such as 1D NMR and 2D NMR. And MTT assays was used to tested these components on the A549 cell survival rates and ROS or proteins levels were detected by Western blotting. Results: A new glucosyloxybenzyl 2-isobutylmalate (a malate ester derivative), along with 31 known compounds were isolated and identified from n-BuOH extract of EtOH extract of B. striata. Among them, compounds 3, 4, 11, 12 and 13 possessed noteworthy proliferative effects for damaged cells, with ED50 of 14.0, 13.1, 3.7, 11.6 and 11.5 µmol/L, respectively, compared to positive control resveratrol (ED50, 14.7 µmol/L). Militarine (8) prominently inhibited the intracellular ROS level, and increased the expression of Nrf2 and its downstream genes (HO-1 and γ-GCSc). Furthermore, Nrf2 activation mediates the interventional effects of compound 8 against SiO2 nanoparticles (nm SiO2)-induced lung injury. Moreover, treatment with compound 8 significantly reduced lung inflammation and oxidative stress in nm SiO2-instilled mice. Molecular docking experiment suggested that 8 bound stably to the HO-1 protein by hydrogen bond interactions. Conclusion: The dietary malate esters derivatives of B. striata could significantly increase the viability of nm SiO2-induced A549 cells and decrease the finer particles-induced cell damages. Militarine is especially promising compound for chemoprevention of lung cancer induced by nm SiO2 through activation of Nrf2 pathway.

Evaluation of whitening and antimicrobial activity of two strains of Bletilla striata WT and HL20.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata (Thunb.) Reixchb.f. is a perennial herb of the Orchid-aceae Bletilla and have various ethnopharmacological uses. As a traditional astringent hemostatic Chinese herbal medicine, B. striata has been widely used in the treatment of 127 different kinds of hemorrhagic diseases. Moreover, B. striata has been a beauty medicine since ancient times, with the first ancient records dating back to 2000 years ago, traditionally used to removing freckle and smooth the skin. Because of the high content of polysaccharides, which is considered the primary active substance of B. striata and having anti-aging, whitening, and anti-oxidation functions, this is also widely used in the cosmetics industry. AIM: We screened the germplasm resources of B. striata in the early stage and the superior HL20 strain was obtained. Our research aims to analyze and compare the whitening and antimicrobial activities of different extracts (aqueous extract, ethanol extract, and aqueous extract from ethanol extract filter residue) of the selected superior varieties (HL20) and the control (WT). MATERIALS AND METHODS: L-tyrosine and L-dopa were used as substrates to establish a tyrosinase inhibition system with arbutin as the positive control and the whitening activity was measured by the inhibition rate of TYR-M and TYR-D. Besides, an in vitro antimicrobial susceptibility test was performed to assess the antimicrobial activity of the B. striata extracts. In a nutshell, the method of punching diffusion was used to thoroughly examine the effects of three extracts from two strains on the antimicrobial activity of five types of microorganism in cosmetics microbiological testing products. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of different extracts were also assessed. RESULTS: Results showed that the whitening and antimicrobial properties of the HL20 strain were found to be more potent than those of the WT strain. Compared with the other two extraction methods, the aqueous extract from ethanol extract filter residue of B. striata exhibited better inhibition of tyrosinase activity. The antimicrobial assay manifested that only the ethanol extract of B. striata had an inhibitory effect and had a potent antimicrobial impact on E. faecalis. CONCLUSIONS: In summary, we evaluated the pharmacological activity of the pre-selected excellent variety (HL20) in terms of whitening and antimicrobial activity. Our results reveal that the selected strain (HL20) has certain advantages over the control (WT). These characteristics make it a candidate additive for whitening cosmetics. Our study also provides a further contribution to the product application of B. striata in cosmetics and antimicrobial agents and the selected HL20 also lays a foundation for the breeding of superior B. striata varieties.

Complete chloroplast genome sequence of Bletilla striata (Thunb.) Reichb. f., a Chinese folk medicinal plant.

Bletilla striata (Thunb.) Reichb. f. is one of the commonly used traditional Chinese medicine with tuber as medicine. We report herein the complete chloroplast genome sequence of Bletilla striata (Thunb.) Reichb. f. It has a length of 159,491 bp, which contained a small single-copy (SSC) region of 18,778 bp and a large single-copy (LSC) region of 87,139 bp, separated by two copies of an inverted repeat (IR) of 26,787 bp. The chloroplast genome contains 114 unique genes, including 80 PCG, 30 tRNA, and 4 rRNA genes. In addition, 18 genes contained one or two introns, which of those including 10 PCG genes possess a single intron, and 2 PCG genes harbor two introns; and 6 tRNA genes harbor a single intron. In this study, Bletilla stariata is sister to Bletilla formosana and clustered within the group consisting of the species that belong to Orchiidaceae.

Cloning, sequence characterization, expression profiling of hormones response of phenylalanine ammonia-lyase gene in Bletilla striata / 中草药

Objective: Phenylalanine ammonia-lyase gene (PAL) is one of the key enzymes associated with stress resistance in secondary metabolism pathway of plants. Exploring its sequence information and expression profiling information in stress response could comprehensively peep at the protein structure, functions and signal network of plant stress resistance. Methods: The full cDNA length of PAL from Bletilla striata was cloned by RT-PCR and RACE approaches. Physicochemical properties and conserved domain of BsPAL protein were determined by a series of bioinformatics tools as Protparam, SOPMA, SWISS-MODEL, etc. Multiple alignment and phylogenetic tree were achieved by DNAMAN and MEGA Software, respectively. The qPCR was employed to examine the expression profiles of BsPAL under exogenous hormone stress. Results: The full cDNA of BsPAL was 2 708 bp, encoding a 797 amino-acid protein with a molecular weight of 86 216.94 and an isoelectric point (pI) of 6.24. The BsPAL protein included the typical structural domain and active site of PALs in other plants, and without transmembrane region, which was more homologous with PALs of Dendrobium officinale and Phalaenopsis aphrodita. The qPCR Results: revealed the expression level of BsPAL in roots was much higher than that in leaves and stems. Under MeJA treatment, the expression trend of BsPAL was first gradually ascending and then descending, while SA treatment had the reverse effect. Conclusion: The BsPAL’s sequence characterizing, expression profiling and responding patterns against SA and MEJA provided a research basis for elucidating the metabolic pathways of phenylpropanoid and hormone signaling research in B. striata.

Isolation, purification and structural characterization of Bletilla striata polysaccharides and its antitumor activity / 中草药

Objective: Bletilla striata polysaccharide (BSP-1) extracted from the stem tubers of B. striata was purified to study the structural properties and antitumor activity. Methods: The crude polysaccharide was fractionated by DEAE-cellulose column chromatography, and three fractions were obtained including BP-1, BP-2, and BP-3, respectively, BP-1 was further purified by SephadexG-200 column chromatography, and a sub-fraction named BSP-1 was obtained. Subsequently, HPGPC, IR spectroscopy, gas chromatography (GC), GC coupled to mass spectrometry (GC-MS) and methylation analysis, and 1H- and 13C-NMR were employed to characterize the structural properties of the polysaccharide fractions. Moreover, the anticancer activity was investigated in vivo. Results: The results showed that the molecular weight of BSP-1 were 4.72 × 105. BSP-1 was consisted of mannose (Man) and glucose (Glc) residues. The backbone of BSP-1 was composed of β-1,4-linked Man and Glc residues at the end with a molar ratio of 8:1. BSP-1 could inhibit the tumor proliferation of HepG2-bearing mice. The tumor weight of mice was significantly inhibited by BSP-1 with inhibitory rate of 66.42%. Conclusion: Structural characterization of BSP-1 provides the significant reference for the further development and elucidation of polysaccharides from B. striata as new drugs.

Glycoside constituents from Bletilla striata / 中草药

Objective: To study the glycoside chemical constituents of Bletilla striata grown in Guizhou Province. Methods: The glycoside chemical constituents were separated and purified by silica gel column chromatography, MCI, gel column chromatography, medium pressure preparation, highperformance liquid chromatography and other modern separation methods. The structures were identified based on the spectral data. Results: Thirteen glycosides were isolated from the 95% ethanol extracts: 1-methyl-3-phenylpropyl-β-D-glucopyranoside (1), 1-(4-β-D-glucopyranosyl oxybenzyl) 4-ethyl (2R)-2-isobutylmalate (2), 3’,5- dimethoxy-bibenzyl-3-O-β-D-glucopyranoside (3), syringaresinol mono-β-D-glucoside (4), 4-O-(6’-O-glucosyl-p-coumaroyl)-4- hydroxybenzyl alcohol (5), (4-hydroxyphenyl) methyl-β-D-glucopyranoside (6), 4-methylphenyl-β-D-glucopyranoside (7), benzyl-β-D- glucopyranoside (8), phenyl-β-D-glucopyranoside (9), 4-[(acetyloxy) methyl] phenyl-β-D-glucopyranoside (10), batatasin III-3-O- glucoside (11), gastrodin (12) and 4-(4-β-D-glucopyranosyl-oxybenzyl)-(2R)-2-isobutylmalate (13). Conclusion: Compound 1 is a new natural product. Compounds 3-6 are isolated from this plant for the first time; Compounds 2, 7-13 are obtained from this genus for the first time.

Progress on research of tissue culture of Bletilla striata / 中草药·英文版

Rhizoma Bletillae is in high demand as a traditional Chinese medicine, and the natural resources have been severely damaged due to excessive exploitation. Because Bletilla striata seeds are small and have no endosperm, the seed germination rate is low in natural conditions. Traditional division propagation could not meet the demands of large-scale cultivation. Tissue culture can provide many seedlings in a short time and is more effective and convenient than other methods. Most studies on tissue culture of B. striata selected seeds as explants. This review summarized the processes of the aseptic seed germination pathway. It included such stages as seed germination, proliferation of clusters of buds, induction of rooting and transplanting of seedlings. Influential factors as well as optimum combination and concentration of the plant growth regulators of each stage were also summarized. The further research on tissue culture of B. striata was also prospected.

Cytotoxic, anti-inflammatory and hemostatic spirostane-steroidal saponins from the ethanol extract of the roots of Bletilla striata.

Four new spirostane steroidal saponins, (1α,3α)-1-O-[(ß-d-xylopyranosyl-(1→2)-α-l-rhamnopyranosyl)]-3-O-d-glucopyranosyl-5α-spirostan (1), (1α,3α)-1-O-[(ß-d-xylopyranosyl-(1→2)-α-l-rhamnopyranosyl)oxy]-3-O-d-glucopyranosyl-25(27)-ene-5α-spirostan (2), (1α,3α)-1-O-[(ß-d-xylopyranosyl-(1→2)-α-l-rhamnopyranosyl)oxy]-epiruscogenin (3), and (1α,3α)-1-O-[(ß-d-xylopyranosyl-(1→2)-α-l-rhamnopyranosyl)oxy]-epineoruscogenin (4) together with two known compounds, bletilnoside A (5) and 3-O-ß-d-glucopyranosyl-3-epi-neoruscogenin (6), were isolated from the ethanol extract of the roots of Bletilla striata (Thunb.) Reichb. f. The structures of the isolated compounds were established based on 1D and 2D ((1)H-(1)H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxicities against seven tumor cell lines, anti-inflammatory activities against Cox-1 and Cox-2, and hemostatic activities. As a result, compounds 1-4 and 6 exhibited significant cytotoxicities against all the tested tumor cell lines with IC50 value less than 30µM and selective inhibition of Cox-2 comparable with the standard drug NS-398 (>90%). Additionally, compounds 1-6 showed potent hemostatic activities.

Effect of fertilizer application of nitrogen,phosphorus,and potassium on output and polysaccharides in Bletilla striata / 中草药

N. By contraries,the effect of N,P_2O_5,and K_2O fertilizer on output of B.striata could be in the order of K_2O

Determination of relative molecular weight of polysaccharides in Bletilla striata by MALDI-TOF-MS / 中草药

Objective To investigate the relative molecular weight of polysaccharides in Bletilla striata by matrix-assisted laser desorption ionizatior/time-of-flight mass (MALDI-TOF-MS). Methods 2, 5-Dihydroxybenzoic acid (DHB) as a matrix was used. By drop drying at room temperature the proportion of the matrix and polysaccharide sample was 1.5∶1. Baseline mode and laser intensity at 1 500-2 500 units were carried out. Results The relative molecular weights of polysaccharides in B. striata were 6?104-3?105, whose distribution rang was wide. Conclusion The relative molecular weights of polysaccharides in B. striata are obtained by MALDI-TOF-MS technique in the proportion of DHB-polysaccharides in B. striata 1.5∶1, which is valuable for the preparation and good quality by the direct drop drying method.

Effect of ultra-fine powder of Bletilla striata on experimental gastric ulcer in rats / 中草药

Objective To compare the pharmacodynamics of common Bletilla striata powder(80 and 200 meshes) with ultra-fine powder of B.striata on experimental gastric ulcer in rats and to clarify how much the dose of B.striata was decreased after superfine comminution.Methods The models of gastric ulcer were set up by anhydrous alcohol and acetic acid to measure the gastric ulcer area and count out the inhibitory rate;The tissue slice of gastric ulcer cicatrix was got far HE staining.Based on the viewpoint of pathology the morbid state was observed.Results B.striata powder could decrease the gastric ulcer area significantly,and the groups of ultra-fine powder were advantaged to the others.Conclusion After superfine comminution,the potency of B.striata can be improved with its dose reducing.It is good for mo-dernization of making dosage forms.

Sulfation techniques of Bletilla striata polysaccharide by orthogonal design / 中草药

Objective To optimize the sulfation-conditions of Bletilla striata polysaccharide (BSPS). Methods BSPS was sulfated with chlorsulfonic acid-pyridine. Reagent proportion, reaction temperature, and reaction time were selected with substitution degree and yield as index by L_9(3~4) orthogonal design. Results Results showed that the condition with 6∶1 volume ratio of pyridine and chlorine sulfonic acid, reaction temperature of 85 ℃, reaction time of 2 h was the optimum. Conclusion The optimal method for sulfation of BSPS is feasible, and it provides valuable parameters for further enlarged test.