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The chemical constituents of Gentiana macrophylla Pall. under acidic condition were explored using gentiopicroside-rich secoiridoids extract treated with hydrochloric acid. 2 new secoiridoid-like structures gentiagenin A (1) and B (2), together with 4 known compounds, swerimilegenin G (3), 4-hydroxyindan-1-one (4), erythrocentaurin (5) and swerimilegenin E (6) were identified using 1D, 2D NMR and HR-ESI-MS spectral technology. In the activity evaluation, the 2 new compounds, such as gentiagenin A (1) and B (2), exhibited stronger inhibitory effect on NO production in RAW264.7 macrophage cells than that of gentiopicroside.

IntroductionProstaglandin E2 (PGE2) has been reported to cause cartilage degradation in the pathogenesis of osteoarthritis (OA). Matrix metallopeptidases (MMPs) play important roles in the pathogenesis of OA, while p-AKT and p-P39 signaling pathways were reported to be activated in the pathogenesis of OA. In this study, we aimed to investigate the effect of Gentiana macrophylla (GM) on the treatment of OA.Material and methodsPrimary rat chondrocytes were treated with PBS, IL-1β, and IL-1β+GM respectively to established in vitro models, and in vivo models were set up as a SHAM group, a monoiodoacetic acid (MIA) group, a MIA+GM (low dose) group and a MIA+GM (high dose) group.ResultsIn primary rat chondrocytes, the IL-1β treatment elevated the expression of PGE2 and COX2 mRNA. However, the GM treatment reduced the expression of PGE2 mRNA and COX2 mRNA. Also, the GM treatment reduced the expression of above MMPs in primary rat chondrocytes treated with IL-1β. Moreover, unlike P38 and AKT, GM treatment could reduce the expression of p-P38 and p-AKT in primary rat chondrocytes treated with IL-1β. Also, GM treatment reduced the up-regulated expression of COX2, MMPs including MMP-1, MMP-3 and MMP-13, and p-P38 and p-AKT in OA rat models, thus exhibiting a therapeutic effect on OA pathology.ConclusionsOur study demonstrated the inhibitory effect of GM on the up-regulated expression of PGE2, Cyclooxygenase-2 (COX-2), MMPs including MMP-1, MMP-3 and MMP-13, AKT and P38 in OA models, thus verifying the therapeutic effect of GM on the treatment of OA.

In September 2020, samples of galled roots with rhizosphere soil were collected from declining Gentiana macrophylla in Yulong County, China. The pathogenic nematodes were identified by observing morphological characteristics of females, second-stage juveniles and perineal pattern, sequence alignments, and specific amplification of sequence characterized amplified region (SCAR). The results showed that the perineal pattern of this nematode was round or oval, the dorsal arch was moderately high or low, one side or both of the lateral field extended to form a wing shape, the tail region had punctations, and the morphological characteristics and morphometric values of second-stage juveniles and females were similar to those of Meloidogyne hapla. The ITS region fragment of this nematode were highly similar to those of M. hapla in NCBI database, with a similarity of over 99.35%. Using the SCAR specific primers, a specific band with an expected size of approximately 440 bp was amplified from this nematode. Morphological and molecular identification supports the nematode species found on Gentiana macrophylla as M. hapla. This is the first report of this regulated root-knot nematode on Gentiana macrophylla in China.

The wild resources of the four original plants (Gentiana crasicaulis Duthie ex Burk, Gentiana daurica Fisch, Gentiana straminea Maxim, and Gentiana macrophylla Pall) of Gentianae Macrophyllae Radix are becoming exhausted. Predicting the distribution under current and future climate scenarios is of significance for the sustainable utilization of resources and ecological protection. In this study, we constructed four species distribution models (SDMs) combining species distribution informations, 19 bioclimatic variables, and the maximum entropy (MaxEnt) model. The results showed that these 4 plants prefer a cool and humid climate. Under the future climate scenarios, the areas of the highly suitable habitats for Gentiana crasicaulis Duthie ex Burk and Gentiana daurica Fisch were likely to decrease, while Gentiana straminea Maxim was likely to expand, and Gentiana macrophylla Pall was less affected. In addition, the centroids of the highly suitable habitats for the four species shifted north or west. Most notably, most of the highly suitable habitats for the four species remained unchanged, which would be the preferred area for semi-artificial cultivation. The above information in this study would contribute to the development of reasonable strategies to reduce the impact of climate change on the four original plants.

Radix Gentianae Macrophyllae (RGM) is a well-known traditional Chinese medicine with anti-inflammatory, analgesia and gastric motility strengthening effects. The iridoids are believed to be the RGM bioactive ingredients, however, an effective strategy for its total iridoids extraction and purification is lacking. In addition, a reliable method for the bioactive iridoid constituents digesting needs to be developed. In the present work, followed by process optimization via response surface modeling and Box-Behnken analysis, the maximum yield of total RGM iridoids was predicted to be 7.75 % from rhizomes of Gentiana macrophylla Pall. via three cycles of extraction in 69.16 % ethanol at liquid-solid ratio 12.27 (mL/g) for 110.91 min. The crude iridoid extract was further purified using HPD-300 macroporous resin to give the enriched product with the iridoid content up to 73.27 %. The anti-inflammatory effects of refined iridoid extracts were quantitatively measured to validate the purification procedure. The refined extract was detected to significantly inhibit the expression of NO through expression inhibition of COX-2, iNOS in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells with the IC50 of 10.49 μg/mL, which was stronger than that of crude extract with the IC50 of 51.11 μg/mL, indicating the iridoid compounds were enriched from crude extract through the purification process. Subsequently, an UPLC-QTOF-MS method was established for qualitative and quantitative analysis of the iridoid constituents in the crude and refined extracts. In addition, based on the UPLC-QTOF-MS data, 6 principle iridoids that give the most important support for the anti-inflammatory activities were digested by means of PCA and PLS analysis. The current study developed a new and effective strategy for optimal extraction, purification and identification of natural bioactive iridoids from Radix Gentianae Macrophyllae, which facilitates its further applications in pharmaceuticals and/or foods industry.

Adenosine deaminase (ADA) is an enzyme widely distributed from bacteria to humans. ADA is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Endophytes are endosymbionts, often bacteria or fungi, which live within plant tissues and internal organs or intercellular space. Endophytes have a broad variety of bioactive metabolites that are used for the identification of novel natural compounds. Here, 54 morphologically distinct endophyte strains were isolated from six plants such as Peganum harmala Linn., Rheum officinale Baill., Gentiana macrophylla Pall., Radix stephaniae tetrandrae, Myrrha, and Equisetum hyemale Linn. The isolated strains were used for the search of ADA inhibitors that resulted in the identification of the strain with the highest inhibition activity, Aspergillus niger sp. Four compounds were isolated from this strain using three-step chromatography procedure, and compound 2 was determined as the compound with the highest inhibition activity of ADA. Based on the results of 1H and 13C NMR spectroscopies, compound 2 was identified as 3-(4-nitrophenyl)-5-phenyl isoxazole. We showed that compound 2 was a new uncompetitive inhibitor of ADA with high cytotoxic effect on HepG2 and SMCC-7721 cells (the IC50 values were 0.347 and 0.380 mM, respectively). These results suggest that endophyte strains serve as promising sources for the identification of ADA inhibitors, and compound 2 could be an effective drug in the cancer treatment.

Gentiopicroside isolated from gentiana macrophylla Pall. belongs to iridoid glycosides. This study aimed to evaluate the protective effect of gentiopicroside against ethanol-induced gastric mucosal injury in mice. Mice were proactively administrated with gentiopicroside by intragastric administration once a day for 3 consecutive days. On the 3rd day, gastric ulcer in mice was induced with 70% ethanol after the last intragastric administration. The stomach tissues were submitted for evaluation of the severity of gastric mucosal alterations. Gentiopicroside administrated orally ameliorated the severity of gastric mucosal alterations. Oral administration of gentiopicroside significantly increased heat shock protein-70 and glutathione levels and superoxide dismutase activity, normalized epidermal growth factor and vascular endothelial growth factor levels, and decreased the levels of tumour necrosis factor-α, interleukin-6 and malondialdehyde, and myeloperoxidase activity in gastric tissue. These findings demonstrated that gentiopicroside has protective effect against ethanol-induced gastric mucosal injury in mice through the improvements of antioxidative and anti-inflammatory effects, as well as up-regulation of heat shock protein-70 level and normalization of epidermal growth factor and vascular endothelial growth factor levels. The results presented in this study provide some evidence for the development of a novel antigastric ulcer agent.

Background Quality evaluation of multi-species resourced herb medicine (MSRHM) is a main problem for quality control of herb medicine. Current quality evaluation methodology lost consideration of species discrepancy. New quality evaluation strategy for MSRHM is in urgent need. Qinjiao, a representative MSRHM, originated from Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crassicaulis Duthie ex Burk. or Gentiana dahurica Fisch., has been used as an important herb medicine over 2000 years for expelling wind-dampness and relieving impediment pain. However, quality evaluation among species has never been revealed. The current work proposes an integrated quality evaluation strategy for MSRHM of Qinjiao, which may promote innovation of quality control of MSRHM. Methods In this work, 58 batches of Qinjiao covering 4 species were collected. Genetic comparative analysis based on ITS2 sequence was conducted. Metabolomics analysis based on TOF–MS and NMR spectrum were carried out. Compounds underlying species differences were identified and their discrepancies among species were investigated by ANOVA analysis and multivariate analysis. Results Four species of Qinjiao can be authenticated by ITS2 sequence comparation. Metabolomics analysis by TOF/MS and NMR revealed chemical discrepancies among species of Qinjiao. Maximum discrepancy was present between Gentiana crassicaulis Duthie ex Burk. and Gentiana dahurica Fisch. Chemical difference among species were tentative explored. For TOF–MS profiling, 28 constituents were tentative identified, 17 of which were further confirmed by standards. For 1H-NMR profiling, signals from 5 compounds were assigned. Contents discrepancies were investigated by ANOVA analysis. It seems that (seco)iridoids like loganic acid, gentiopicroside or swertiamarin were richer in specie of Gentiana crassicaulis Duthie ex Burk., while flavonoid (morroniside) and triterpenoids (roburic aicd, ursolic acid, oleanolic acid, β-sitosterone) were richer in specie of Gentiana dahurica Fisch. The current research demonstrates that metabolite profiling based on both UPLC/Q-TOF MS and 1H-NMR coupled with ITS2 sequence comparation can be a powerful tool for quality investigation of MSRHM of Qinjiao. Conclusions A comprehensive quality evaluation strategy for MSRHM was proposed by integrating UPLC-Q-TOF–MS, NMR based metabolic analysis and ITS2 sequence genetic comparation. The proposed quality evaluation strategy shall promote innovation of quality control of traditional Chinese medicine.

Gentimilegenins A, B (1, 2), (6R, 8R)-6-hydroxy swerimuslactone A (3), (6R, 8S)-6-hydroxy swerimuslactone A (4), 4-hydroxy roburic acid methyl ester (5), (±) 3'-hydroxy gentioxepine (6), N-heptacosanoyl anthranilic acid (7a), N-nonacosanoyl anthranilic acid (7b), together with 40 known compounds were isolated from the roots of Gentiana macrophylla Pall. Their structures were elucidated on the basis of comprehensive analysis of HRESIMS, IR, 1D-, 2D-NMR and X-ray diffraction. The anti-inflammatory effects of selected compounds were also evaluated through the detection of their inhibitory effects on NO production in LPS-induced RAW264.7 macrophage cells.

The nonstructural protein NS1 of human parvovirus B19 (B19) is known to exacerbate disease activity in systemic lupus erythematosus (SLE). However, no specific medicine for B19 infection is available. The roots of Gentiana macrophylla Pall. (GM), the traditional Chinese medicine "Qinjiao," have been used for centuries to treat rheumatic disease, including SLE. Herein, we aimed to investigate the effects of GM root extract (100 and 300 mg/kg body weight) on B19-NS1-exacerbated liver injury in NZB/W F1 mice; liver tissues were assessed by hematoxylin-eosin staining and immunoblotting. The GM root extract significantly decreased B19-NS1-exacerbated liver inflammation by suppressing the expressions of hepatic inducible nitric oxide synthase, cyclooxygenase type 2 (COX-2), interleukin (IL)-1β proteins, values of serum asparate transaminase (AST) and alanine transaminase (ALT), and lymphocyte infiltration (P .05). It also significantly reduced the B19-NS1-exacerbated hepatic matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) expressions by downregulating tumor necrosis factor (TNF)-α/NF-κB (p65) signaling. These findings suggest a therapeutic potential of GM root extract against B19-NS1-exacerbated liver inflammation in SLE.

Gentiana crassicaulis Duthie ex Burk. and Gentiana macrophylla Pall. are two main sources of Radix Gentianae Macrophyllae (Qinjiao) available in markets, which has a wide range of anti-inflammatory effects and has been extensively used for fighting rheumatoid arthritis. However, they vary in terms of chemical compositions, pharmacological activities, and biomass. In this study, a combined chemical and genetic (HPLC and DNA barcoding) approach was used to compare these two plants. Four predominant bioactive compounds, namely, gentiopicroside, loganic acid, swertiamarin, and sweroside, were used to assess the chemical variations. Based on chemical variations, 15 samples were clustered into two groups through PCA analyses. DNA barcoding utilizing the variable nuclear ITS2 regions were sequenced, aligned, and compared. Together with 61 sequences collected from GenBank, 76 batches of Qinjiao were clustered in two groups according to species origin. The genetic relationships indicated by the ITS2-based NJ tree were consistent with the chemical variations. Thus, the chemical profiles determined by HPLC and DNA profiles obtained from ITS2 region could be applied for the quality control of Qinjiao.

To obtain an alternative source for the production of gentiopicroside, here genomic DNA segments of the medicinal plant Gentiana macrophylla were randomly transferred into Hansenula polymorpha by 25 KeV nitrogen ions (N+) at a dose of 2.5 × 1016 ions/cm2 under vacuum pressure of 1 × 10−3 Pa. To screen for potential gentiopicroside-producing recombinant yeast strains, geraniol 10-hydroxylase (G10H) and secologanin synthase (SLS) involved in the gentiopicroside biosynthesis pathway were used as molecular markers. Based on the conserved protein sequences of G10H and SLS, degenerate primers were designed and used for colony polymerase chain reaction (PCR). PCR-positive results for both the G10H and SLS genes were obtained in 79 out of 653 transformants by low-energy ion beam-mediated transformation. These 79 potential gentiopicroside-producing strains were further analysed by Fehling's test, thin-layer chromatography, high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. The results showed that the retention time and ion peaks of the sample from one stable recombinant strain designated as DL67 were consistent with those of the gentiopicroside standard. The corresponding gentiopicroside yield was 8.41 mg/g dry cell weight after strain DL67 was cultured for 96 h. This could offer a new starting point for the construction of recombinant yeasts for production of medicinal plant compounds.

A simple, reliable and rapid ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of four secoiridoid (gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid), the bio-active ingredients in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim-pack XR-ODS column with a mobile phase consisting of methanol and 0.1% formic acid in water. A triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple-reaction monitoring scanning. The lower limits of quantitation were 0.25–30 ng/mL for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard (amygdalin) from rat plasma were all >71.4%. The validated method was successfully applied to a comparative pharmacokinetic study of four analytes in rat plasma between normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan and Gentiana macrophylla extract, respectively. Results showed significant differences in pharmacokinetic properties of the analytes among the different groups. Copyright © 2015 John Wiley & Sons, Ltd.

An efficient protocol for plant regeneration was developed from protoplasts of Gentiana macrophylla Pall. through somatic embryogenesis. Viable protoplasts were isolated from cell suspensions derived from young seedling leaves in an enzyme solution containing 2 % Cellulase Onozuka R-10, 0.5 % Macerozyme R-10, 0.5 % Hemicellulase, and 0.4 M sorbitol with a yield of 6.2 × 106 protoplasts g−1 fresh weight. Liquid, solid–liquid double layer (sLD) and agar-pool (aPL) culture systems were used for protoplast culture. The frequency of protoplast cell divisions and colony formations in aPL culture were significant (p < 0.05) higher than those in liquid and sLD cultures. The highest division frequency and plating efficiency were 37.7 % and 16.5 %, respectively, in aPL culture supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.5 mg l−1 6-benzylaminopurine (BA). Protoplast-derived microcalli obtained from aPL culture system were transferred to solid MS medium with a reduced concentration of 2,4-d (0.5 mg l−1) to promote the formation of embryogenic calli. Somatic embryos developed into plantlets on MS medium supplemented with 2 mg l−1 BA at a rate of 51.3 %. RAPD analysis of G. macrophylla revealed a low variation among regenerants.

Background Radix Gentianae Macrophyllae, commonly known as Qinjiao (秦艽in Chinese), is dried roots of medicinal plants that belong to Gentianaceae family and Gentiana genus. It has medically been used for the treatment of various diseases such as arthritis, stroke, facial paralysis, and scapulohumeral periarthritis in China since ancient times. Purpose The aim of this paper is to provide a comprehensive and constructive overview of Qinjiao mainly containing Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crasicaulis Duthie ex Burk., and Gentiana daurica Fisch. in botany, traditional use, phytochemicals, pharmacology (biological activities and pharmacokinetics), quality control, and authentication according to the up-to-date data of available scientific literatures. Materials and methods All information regarding these four plants was collected from various academic search engines for example Google, Google Scholar, Web of Science, SciFinder, Pubmed, CNKI, and Wanfang. Additional information was obtained from botanical books, Chinese classic texts, and medical monographs. Results So far 166 compounds have been isolated and identified from Qinjiao plants together with Gentiana tibetica King ex Hook. f., Gentiana siphonantha Maxim., Gentiana officinalis H. Smith, and Gentiana waltonii Burk. Their constituents are mainly classified into iridoid glycosides, triterpenes, flavones, sterols, benzene derivatives, etc. The pharmacological studies demonstrate that Qinjiao plants display a wide range of bioactivities e.g. anti-inflammatory, anti-oxidant, hepato-protective, cardio- and neuro-protective, insecticidal, and anti-influenza effects. The conventional quality control is performed by determination of the concentration of some compounds, e.g., gentiopicroside, or establishment of the fingerprint. In addition to seed propagation, tissue culture technology has been used to address the limited supplies and guarantee the sustainable development of Qinjiao in the experimental scale. Conclusions Although the identification of compounds from Qinjiao and demonstration of medicinal uses in vitro and in vivo have been carried out, various other studies on these plants should deserve our more attention. More efforts should be concentrated on the underlying mechanisms of their beneficial bioactivities. The proper toxic evaluation is indispensable to guarantee the safety, efficacy, and eligibility for medical use. To sum up, the summarized achievements could highlight the importance of Qinjiao and provide a solid foundation for scientists not only to further exploit the therapeutic potentials, but also possibly develop novel drugs in the subsequent research.

Objective To search for the potential medicinal plant part of Gentiana macrophylla based on changes of secondary metabolites and trace elements in the flowers of G. macrophylla. Methods HPLC was used to detect the changes of the active constituents (longanic acid, sweroside, gentiopicroside, and swertiamarin) and ICP-AES was used for mineral nutrients in G. macrophylla during flower development. And soluble sugar, starch, crude protein, hemicelluloses, cellulose, and lignin were determined. Results Biomass of flower in full bloom (D2) phase was considerable during flower development, in which the contents of longanic acid and gentiopicroside were at the highest levels with 2.65 and 2.88 times higher than those recorded in Chinese Pharmacopoeia 2010, sweroside and swertiamarin in the flowers were reaching 6.06 and 1.25 times higher than those in roots. Florescence is the most valuable stage during flower development. The concentration of Fe, Mg, K, P, and B was higher in the flowers than that in roots. The accumulation of active constituents in the plant was influenced by the contents of metabolically linked carbon and nitrogen compounds. Conclusion The secondary metabolites, mineral nutrients, and physicochemical indicators are tightly regulated by flower organ development, D2 is an important stage for both biomass and extraction of active constituents such as longanic acid. The flowers of G. macrophylla could be used as a potential medicinal plant part for longanic acid at a high level.

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates - five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) - using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔC t, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions.

In this paper, a combined high performance counter-current chromatography (HPCCC) and preparative high-performance liquid chromatography (HPLC) method was employed for rapid separation and enrichment of bioactive constituents from a water extract of Gentiana macrophylla Pall. With a two phase solvent system composed of ethyl acetate-n-butanol-water-acetic acid (2: 3: 5: 0.6, v/v), the water extract of G. macrophylla Pall was fractionated into six fractions with three targets isolated and four others highly concentrated, which were then further purified by preparative-HPLC. As a result, 37 mg deglucoserrulatoside, 22.4 mg loganic acid, 3.9 mg isoorientin, 22.4 mg swertiamarin, 52.3 mg gentiopicroside, 27.5 mg sweroside, and 7.9 mg macrophylloside D with the purity of 95.3%, 90.2%, 98%, 98%, 99.2%, 98.8%, and 98.4%, respectively, were isolated from the water extract of Gentiana macrophylla Pall. The structures were confirmed by UV spectra, MS, as well as NMR measurements.

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.

The leaf explants from the eight-week-old Gentiana macrophylla Pall sterile seedlings were precultured for three days on Murashige and Skoog (MS) agar medium with 1 mg/l of 6-benzyladenine, and then incubated in MS liquid medium containing Agrobacterium rhizogenes R1000 for 2 h with shaking. After 10 days of culture on hormone-free MS agar medium with 500 mg/l cefotaxime under illumination of 300 μmol/(m2 s) with a 16-h photoperiod at 24°C, up to 18.3% of the infected explants produced typical hairy roots (HRs). Finally, three stable HR lines with no morphologically visible differences were obtained. The HR lines could vigorously grow and propagate on/in hormone-free half-strength MS solid/liquid medium. Moreover, they changed from white to green when moved from darkness to the light and spontaneously produced calli, which were able later to produce adventitious buds. PCR analysis revealed that three HR lines, which were undergone to continuous subculturing over two years, still contained the rolC gene from the A. rhizogenes Ri plasmid. HPLC revealed the HR line possessing a certain capacity of synthesizing gentiopicroside (0.11 mg/g dry wt).

Gentiana macrophylla is a traditional Chinese medicinal plant. Its dominant active constituents are secoiridoids, mainly gentiopicrin. The objective of this study was to determine whether endophytic fungi isolated from this plant produce the bioactive ingredient gentiopicrin. Primary screening was done by Dragendorff's reaction and the strain re-selection was done with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. In this study, 20 strains of endophytic fungi were isolated from G. macrophylla, and the extracts from five strains had a positive Dragendorff's reaction. Two strains (QJ16 and QJ18) had a component with the same R(f) value in TLC as that of authentic gentiopicrin and one ingredient of the QJ18 extract had a retention time identical with that of authentic gentiopicrin in HPLC. Therefore, the fungus appears to produce the bioactive ingredient gentiopicrin, as does its host plant, and could be used for the production of gentiopicrin by fermentation.