Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application.

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4-5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell's resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

[Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome].

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1ß and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1ß and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

Aureimonas psammosilene sp. nov., isolated from the roots of Psammosilene tunicoides.

One motile strain designated, YIM DR1026T was isolated from the roots of Psammosilene tunicoides collected from Gejiu, Yunnan province, China. The cells of strain YIM DR1026T were Gram-negative and short-rod shaped. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM DR1026T was a member of the genus Aureimonas and closely related to Aureimonas rubiginis (96.7%). DNA-DNA relatedness values between strain YIM 1026T and Aureimonas rubiginis BCRC 80440T was 38.2 ± 1.5%. The ANI value between YIM DR1026T and other Aureimonas members were below the cut-off level (95-96%) recommended as the average nucleotide identity (ANI) criterion for interspecies identity. Strain YIM DR1026T grew at 4-30 °C (optimum 28 °C), pH 4.0-9.0 (optimum pH 6.0-7.0) and tolerated NaCl (w/v) up to 1% (optimum 0%). Q-10 was sole the respiratory ubiquinone present in YIM DR1026T. Polar lipids of strain YIM DR1026T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, sulfoquinovosyldiacylglycerol, unidentified aminolipid and unidentified polar lipid. The genomic G + C content was 64.6 mol%. The major fatty acids were C18:1ω7c, C16:0 and summed feature 3 (C16:1ω7c/C16:1ω6c). Based on phenotypic, phylogenetic, chemotaxonomic and genome comparison, strain YIM DR1026T represents a novel species of the genus Aureimonas, for which the name Aureimonas psammosilene sp. nov. is proposed. The type strain is YIM DR1026T (= KCTC 42691T = NBRC 112412T).

Nocardia zhihengii sp. nov., an actinobacterium isolated from rhizosphere soil of Psammosilene tunicoides.

A Nocardia-like actinobacterial strain, designated YIM TG2190T, was isolated from rhizosphere soil of Psammosilene tunicoides collected from Gejiu, Yunnan province, China. The cells of strain YIM TG2190T were observed to be Gram-stain positive and non-motile. The strain forms extensively branched substrate mycelia that fragments into rod-shaped elements. The 16S rRNA gene sequence analysis showed that strain YIM TG2190T is closely related to Nocardia nova (97.5%), Nocardia jiangxiensis (97.1%) and Nocardia miyunensis (96.8%). Growth occurs at 4-30 °C (optimum 28 °C), pH 6.0-8.0 (optimum pH 7.0) and the strain can tolerate NaCl (w/v) up to 3% (optimum 0-1%). The cell walls were found to contain meso-diaminopimelic acid. The whole-cell sugars were identified as glucose, mannose, ribose, galactose, arabinose and fucose. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and an unidentified phospholipid. The menaquinones detected were MK-9 (H2) and MK-8 (H4). The major fatty acids (> 5%) were found to be C16:0 (33.9%), summed feature 3 (21.7%), C18:0 10-methyl TBSA (13.7%) and C18:1ω9c (7.0%). The DNA G+C content was determined to be 61.1 mol%. DNA-DNA relatedness between the strain YIM TG2190T and N. nova CGMCC 4.1705T, N. jiangxiensis CGMCC 4.1905T and N. miyunensis CGMCC 4.1904T were 46.9 ± 2.6, 36.8 ± 1.3, and 35.7 ± 2.6%, respectively, values which are less than the threshold value (70%) for the delineation of prokaryotic genomic species. The phenotypic, chemotaxonomic and phylogenetic data indicates that strain YIM TG2190T represents a novel species of the genus Nocardia, for which the name Nocardia zhihengii sp. nov. is proposed. The type strain is YIM TG2190T (=KCTC 39596T = DSM 100515T).

[Construction of core collection of Psammosilene tunicoides based on polymorphism of ß-AS gene].

Psammosilene tunicoides is one of the main ingredients of the "Yunnan Baiyao". P. tunicoides is an endangered species included in the secondary protection list in China Plant Red Data Book as well as the endemic species in Southwest China. Its natural resources could not meet the needs of pharmaceutical production. Construction of core collection of P. tunicoides will lay the foundation for germplasm improvement and molecular breeding. The sequence variation of the key enzymes gene locus (ß-AS) were carried out to survey the population structure and population history of the species. Among the 11 populations across its geographical range, 36 haplotypes were identified. The levels of haplotype diversity (Hd=0.905) were high, while the levels of population differentiation (GST=0.280) were low. Analysisof molecular variance (AMOVA) indicated that a significantly greater proportion of total genetic variationpartitioned among populations thanwithin populations (values of 77.43% and 22.57%, respectively). These results in combination with the star-like phylogenetic network analysis indicate that Hap1 as an ancestral haplotypewas shared in four populations, Hap2, Hap4, Hap15 and Hap16 are occurred in two populations, the remains as private haplotype only distributed in single population. The strategy of core collection was constructed in order to maximumpreserve genetic diversity of P. tunicoides.

Streptomyces zhihengii sp. nov., isolated from rhizospheric soil of Psammosilene tunicoides.

An actinomycete strain, designated YIM T102(T), was isolated from the rhizospheric soil of Psammosilene tunicoides W. C. Wu et C. Y. Wu collected from Lijiang, Yunnan Province, China. The taxonomic position of the new isolate was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM T102(T) belongs to the genus Streptomyces. Strain YIM T102(T) was most closely related to Streptomyces eurocidicus NRRL B-1676(T) with a pairwise 16S rRNA gene sequence similarity of 98.9 %. However, DNA-DNA relatedness value between strain YIM T102(T) and S. eurocidicus NBRC 13491(T) was found to be 37.8 ± 1.8 %. The menaquinone composition detected for strain YIM T102(T) was MK-9 (H6) and MK-9 (H8), while the major fatty acids were summed feature 4 (38.0 %), anteiso-C15:0 (13.1 %), iso-C16:0 (10.1 %), summed feature 3 (9.8 %) and C16:0 (9.0 %) and iso-C15:0 (5.2 %). The whole-cell hydrolysates contained galactose, glucose, ribose and mannose, along with LL-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The DNA G+C content was 70.7 mol%. Strain YIM T102(T) also exhibited antagonistic activity against Alternaria alternata, Alternaria brassicae and Colletotrichum nicotianae Averna, based on the findings from the comparative analyses of phenotypic and genotypic characteristics; it is proposed that strain YIM T102 represents a novel species of the genus Streptomyces, for which the name Streptomyces zhihengii sp. nov. is proposed. The type strain is YIM T102(T) (=KCTC 39115(T) = DSM 42176(T) = CGMCC 4.7248(T)).

Triterpenoid saponins from Psammosilene tunicoides and their antinociceptive activities.

Herein, five undescribed oleanane-type triterpenoid saponins, namely, psammosaponins A-E, along with nine known compounds, were isolated from the roots of Psammosilene tunicoides. Moreover, part of the ethanolic extract of P. tunicoides was acid-hydrolyzed and three aglycones were isolated from the resulting hydrolysate. The structures of all compounds were established through extensive analysis involving 1D and 2D NMR experiments, HRESIMS measurements, chemical derivatization, and comparison of spectroscopic data with the values reported in the literature. In all, 10 of the isolated saponins and the three aglycones were evaluated in the acetic acid-induced writhing model for their antinociceptive activity. At a dose of 40 mg/kg, these compounds exhibited significant inhibitory effects on the mouse writhing response, with inhibitions ranging from 31.9% to 79.3%. In addition, the structure-activity relationships of the isolates were discussed. Among the isolates, quillaic acid 3-O-glucuronide and 16α-hydroxygypsogenic acid showed better antinociceptive activity with inhibitions of 79.3% and 73.7%, respectively. Both isolates also exhibited antinociceptive activities in hot plate and formalin tests on mice. Their antinociceptive mechanism was explored in lipopolysaccharide-stimulated RAW 264.7 cells. These isolates could significantly inhibit the production of nitric oxide and interleukin-6 and downregulate the expression levels of inducible NO synthase, COX-1, and COX-2.

Pharmacodynamics assessment of ß-carboline from the roots of Psammosilene tunicoides as analgesic compound.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Psammosilene tunicoides (W. C. Wu et C. Y. Wu) is a well-known medicinal herb for the treatment of pain, hemostasia and rheumatoid arthritis among Chinese people. AIM OF THE STUDY: The present study aimed to investigate the antinociceptive activity and mechanism of ß-carboline alkaloids 1-4 which were extracted from the roots of P. tunicoides. MATERIALS AND METHODS: The analgesic effects were evaluated using peripheral and central pain mouse models of nociception, including the formalin test and the tail flick test. The levels of glutamic acid (Glu) and nitric oxide (NO) in cerebellar cortexes and spinal cords (L4-6) were determined. The anti-inflammatory of all compounds were then assessed on RAW264.7 cells. RESULTS: Our results showed that compounds 1-4 had significant analgesic effects on both phases of formalin test of mice. Furthermore, all compounds showed suppressive effects on Glu in the brain and NO levels in the brain cortex and the spinal cord. Except for compound 1, the others could extend the pain threshold of hot water tail-flick in mice. In addition, compounds 2 and 3 (60 µmol/kg) could decrease GABAAα1 protein levels in spinal cord. All compounds exhibited anti-inflammatory effects by inhibiting lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells with half-maximal inhibitory concentration (IC50) 1.1-34.9 µM. CONCLUSION: ß-carboline alkaloids from the roots of P. tunicoides had significant analgesic activity by both central and peripheral mechanisms. Our findings suggested that regulating the release of NO or Glu or GABAα1 are some of the mechanisms of analgesic activity of ß-carboline alkaloids.

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid.

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

Chitosan elicitation of saponin accumulation in Psammosilene tunicoides hairy roots by modulating antioxidant activity, nitric oxide production and differential gene expression.

Elicitation is one of the most effective strategies for enhancing plant bioactive compounds, such as triterpenoid saponins. Chitosan gained worldwide attention for biostimulant activity, but little is known about its roles in the elicitation of triterpenoid saponin in medicinal plants. Here, we explored the regulatory network of chitosan on saponin accumulation in hairy root cultures of Psammosilene tunicoides, a valuable medicinal herb known for its pain-relieving properties endemic to China. Compared with control, the highest total saponin accumulation exhibited a 4.55-fold enhancement in hairy roots elicited by 200 mg L-1 chitosan for nine days. High-performance liquid chromatography (HPLC) revealed the yields of quillaic acid, gypsogenin and gypsogenin-3-O-ß-D-glucuronopyranoside were significantly increased after chitosan treatments. Moreover, exogenous chitosan application dramatically triggered the reactive oxygen species (ROS) scavenging enzyme activities and nitric oxide (NO) content in hairy roots. Comparative transcriptome analysis from chitosan-treated (1 and 9 d) or control groups revealed that differentially expressed genes (DEGs) were greatly enriched in plant-pathogen interaction and metabolic processes. The transcriptions of candidate DEGs involved in chitosan-elicited saponin metabolism were increased, especially genes encoding antioxidant enzymes (SOD, POD and GR), stress-responsive transcription factors (WRKYs and NACs) and terpenoid biosynthetic enzymes (DXS, GPPS and SE). Taken together, these results indicate that chitosan elicitor promotes triterpenoid saponin biosynthesis by enhancing antioxidant activities, NO production and differential gene expression in P. tunicoides hairy roots.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Tunicyclin L, a cyclic peptide from Psammosilene tunicoides: Isolation, characterization, conformational studies and biological activity.

Tunicyclin L (1), cyclo (L-Pro1-Gly-L-Phe1-L-Ile-L-Pro2-L-Phe2 -L-Thr-L-Val), and 11 known compounds, including one cyclic peptide (2), eight carboline alkaloids (3 -10), one lignan (11) and one flavone (12) were isolated from the roots of Psammosilene tunicoides. Their structures were elucidated on the basis of extensive UV, IR, MS, NMR spectroscopic data and comparison with literature. Single-crystal X-ray diffraction results revealed the stereochemistry of the 24-membered ring cyclic peptide (1). Among these known compounds, compound 6 was found to be a new natural product, and compounds 3, 4, and 11 were isolated from this plant for the first time. Five compounds (1, 3, 4, 7, and 9) showed moderate anti-acetylcholinesterase (AChE) activity.

Triterpenoid saponins from the roots of Psammosilene tunicoides.

Seven oleanane-type triterpenoid saponins, tunicosaponins B-D (1-3), F-I (4-7), along with eight known triterpenoid saponins (8-15), were isolated from the roots of Psammosilene tunicoides. The structures of compounds 1-7 were determined by comprehensive spectroscopic analysis, including 1D and 2D NMR techniques, mass spectrometry and chemical methods. Triterpene glycosides have been considered as major active constituents of P. tunicoides. This work provides a more complete insight into the saponin constituents of P. tunicoides.

Complete plastid genome sequence of the rare and endangered medicinal herb Psammosilene tunicoides, endemic to China.

Psammosilene tunicoides W.C. Wu et C.Y. Wu, a monotypic species and endemic to Southwest China, is a rare and endangered traditional medicinal herb with satisfactory effects on multifold pathology. In order to provide crucial data for protection, we reported and analyzed the complete plastid genome of P. tunicoides as the foundation of germplasm conservation. The complete plastid genome is a typical quadripartite circular molecule of 153,957 bp in length, including a large single copy (LSC) region of 83,972 bp and a small single copy (SSC) region of 17,495 bp separated by two inverted repeat (IR) regions of 26,245 bp. In total of 111 unique genes were predicted, including 78 protein-coding genes (PCGs), 29 transfer RNA genes, and four ribosomal RNA genes. The overall GC content of P. tunicoides is 36.5%. The phylogenetic analysis revealed that P. tunicoides and Dianthus caryophyllus formed an independent clade with a 100% bootstrap support.

Development, characterization, and cross-amplification of microsatellite markers for Psammosilene tunicoides (Caryophyllaceae).

PREMISE OF THE STUDY: Psammosilene tunicoides (Caryophyllaceae) is a narrowly distributed and endemic plant species in southwestern China. The overexploitation of natural P. tunicoides has led to the destruction of many populations. Population and genetic studies will provide crucial data for the protection and management of P. tunicoides. In this study, we develop simple sequence repeat markers of P. tunicoides to analyze population diversity. METHODS AND RESULTS: Microsatellite loci of P. tunicoides were isolated with FIASCO. Eleven polymorphic and 10 monomorphic primers were developed. The 11 polymorphic primers were tested in three P. tunicoides populations, yielding two to nine alleles per locus. Levels of observed heterozygosity varied from 0.000 to 1.000, and levels of expected heterozygosity ranged from 0.000 to 0.615. In addition, three of these loci were successfully amplified, and showed polymorphism, in three Silene species. CONCLUSIONS: These microsatellite markers can be valuable tools to investigate the genetic diversity and population structure of P. tunicoides.

Acute and sub-acute oral toxicity studies of the aqueous extract from radix, radix with cortex and cortex of Psammosilene tunicoides in mice and rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Psammosilene tunicoides is one of the important ingredients of a famous Chinese traditional medicine formulation "Yunnan Baiyao". Also, this plant is commonly used as an anodyne and hemostatic agent in southwest China. Currently, little toxicological information is available on its safety following prolonged use. AIM OF THE STUDY: In this study, we sought to evaluate the toxicity of the three different parts of Psammosilene tunicoides: Psammosilenes Radix (PR), Psammosilenes Radix with Cortex (PRC) and Psammosilenes Cortex (PC) by acute and sub-acute toxicity studies. MATERIALS AND METHODS: In the acute toxicity study, mice were orally administrated with different doses of PR, PRC and PC. General behavior and mortality were observed up to 14 days. In sub-acute toxicity study, these aqueous extracts were given orally as a single administration to rats at doses of 0.3, 0.6 and 1.2g/kg/day, respectively, for 28 days. General behavior, body weight, biochemical, hematological, organ coefficients and pathological morphology parameters were detected. RESULTS: In acute study, single oral administration of the aqueous extract of PR, PRC and PC caused dose-dependent general behavior adverse effects and mortality. The LD50 values of PR, PRC and PC were 4.64g/kg, 4.85g/kg and 6.40g/kg, respectively. In sub-acute study, the administration of the extract of PR, PRC and PC during 28 days at all doses reduced spontaneous activities with both genders. Occasional nasal secretion with blood at high doses (1.2g/kg) of PR, PRC and PC were observed. Daily single oral administration provoked varying degrees of growth retardation in female rats. The relative heart and spleen weight in the female rats were reduced after the administration. On the hematological and biochemical analyses, the administration of the extract of PR, PRC and PC during 28 days mainly caused variation of indexes in female rats. Histopathological analysis has shown vascular congestion in heart, thickened alveolar wall and emphysema in lung, and vascular congestion in kidney of rats after sub-acute oral administrations. CONCLUSIONS: As shown in the results, Psammosilene tunicoides has a toxic potential in acute and sub-acute oral administrations. However, there is no direct relationship between toxicity and the cortex. Daily oral administration of three different parts from Psammosilene tunicoides (PR, PRC and PC) may cause damages to heart, lung and kidney in rats. Thus these extracts should be used with caution.