A Comparative Study on the Fingerprint of Paris polyphylla var. Polyphylla and Paris polyphylla var. yunnanensis from Different Origins in Dali and Seven Main Steroidal Saponins by HPLC / 中国药学杂志

OBJECTIVE: To study the fingerprints of rhizomes of Paris polyphylla Smith var. polyphylla and Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz from different origins in Dali and the differences of seven main steroidal saponins. METHODS: The fingerprints of rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali were established by HPLC. The similarity of fingerprints and seven main steroidal saponins were compared and analyzed. RESULTS: There were 14 common peaks in the fingerprints of rhizomes of Paris polyphylla var. polyphylla from different origins in Dali, and the similarity of the fingerprints of the samples from different habitats except Jinhua Weishan and Longjie Weishan was greater than 0.9. There were 13 common peaks in the fingerprints of rhizomes of Paris polyphylla var. yunnanensis from different origins in Dali, and the similarity of the samples from different origins was greater than 0.92. There were 11 common peaks in the fingerprints of rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali, and the similarity of the fingerprints was very low, between 0.057 and 0.225. Polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ are detected in the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali. The average peak areas of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ in Paris polyphylla var. yunnanensis from different origins in Dali were higher than those of Paris polyphylla var. polyphylla. Polyphyllin H was detected in the rhizomes of Paris polyphylla var. yunnanensis from Leqiu Nanjian, Jinhua Weishan, Fengyu Eryuan, Dengchuan Eryuan, Hongyan Midu, Xiyi Heqing and the rhizomes of Paris polyphylla var. polyphylla from Wanqiao Dali, Fengyi Dali, Xiyi Heqing, Hongyan Midu and Leqiu Nanjian. Polyphyllin Ⅲ was detected in the rhizomes of Paris polyphylla var. polyphylla from the origins except Longjie Weishan. Polyphyllin Ⅴ was also detected in the rhizomes of Paris polyphylla var. polyphylla in Fengyu Eryuan and Xiyi Heqing. CONCLUSION: Polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ are detected in the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis from different origins in Dali. The similarity of the HPLC fingerprints of the rhizomes of Paris polyphylla var. polyphylla and Paris polyphylla var. yunnanensis is very low, and there are great differences in seven main steroidal saponins. The contents of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅴ in Paris polyphylla var. yunnanensis are higher than those in Paris var. polyphylla.

Two new α-pyronoids from endophytic fungus Aspergillus oryzae derived from Paris polyphylla var. yunnanensis / 中草药

Objective: To study the chemical composition and structure of the secondary metabolites of the endophytic fungus Aspergillus oryzae from Paris polyphylla var. yunnanensis. Methods: A. oryzae was fermented by liquid fermentation. After extraction, it was separated and purified by various chromatography methods. The structure of the compounds was identified according to the physical and chemical properties and spectral data. Results: Four compounds were isolated and their structures were identified as 4-hydroxy-6-[(2S,3S)-3-hydroxybutan-2-yl]-3-methyl-2H-pyran-2-one (1), (R)-4-hydroxy-6-(1-hydroxy-2- methylpropyl)-3- methyl-2H-pyran-2-one (2), flufuran (3) and flufuran methyl ester (4). Conclusion: Compounds 1 and 2 are new α-pyronoids named asper-α-pyranone A and asper-α-pyranone B.

A new polyketide from endophytic fungus Aspergillus oryzae derived from Paris polyphylla var. yunnanensis / 中草药

Objective: To study the secondary metabolites of the endophytic fungus Aspergillus oryzae from Paris polyphylla var. yunnanensis in order to find new compounds. Methods: The endophytic fungus A. oryzae was fermented by liquid fermentation. After extraction, silica gel and macroporous adsorption resin were used to separate and purify the extract. The structures of the compounds were identified according to their physical and chemical properties and spectroscopic data. Results: Three compounds were isolated and their structures were identified as 3-amino-4,5-dihydroxy-4,6-dimethyl-2-(2-methylbutanoyl)cyclohex-2-enone (1), 12-N-methyl- cyclo-(L-tryptophyl-L-phenylalanyl) (2) and ditryptophenaline (3). Conclusion: Compound 1 is a new polyketide named asperpolyketide A.

Effect of inoculation time of arbuscular mycorrhizal fungi on endogenous hormone content in Paris polyphylla var. yunnanensis seedlings / 中草药

Objective: To study the effect of the inoculation time of arbuscular mycorrhizal (AM) fungi on endogenous hormone content in Paris polyphylla var. yunnanensis seedlings. Methods: The indoor pot test was applied. The seeds, one-year-old seedlings, and two-year-old seedlings of P. polyphylla var. yunnanensis were inoculated by different mixed AM fungi and cultivated in the sterilized soil matrix respectively. The contents of endogenous hormone zeatin riboside (ZR), gibberellin (GA), indole acetic acid (IAA) and abscisic acid (ABA) were determined by HPLC. Results: The results showed that the endogenous hormone (ZR, GA, IAA and ABA) contents in rhizome and fibrous roots of P. polyphylla var. yunnanensis from different regions or different time inoculated by mixed AM fungi were various. As a whole, the contents of endogenous hormone ZR and GA increased obviously in the rhizome and fibrous roots of the wild and cultivated P. polyphylla var. yunnanensis compared with the control group (CK), while the content of ABA reduced. The IAA/ABA value, GA/ABA value and ZR/ABA value increased obviously in the rhizome and fibrous roots of P. polyphylla var. yunnanensis. Conclusion: The one-year-old of P. polyphylla var. yunnanensis inoculated with S2 and S6 mixed AM fungi showed the best comprehensive effect for promoting growth of the rhizome and fibrous roots of P. polyphylla var. yunnanensis seedlings.

Rapid identification of Paris polyphylla var. yunnanensis and its closely relative species by infrared spectroscopy combined with morphological characteristics / 中草药

Objective: To explore morphology and infrared spectrum identification evidence of Paris polyphylla var. yunnanensis and its closely relative species, and further analyze their genetic relationship to provide basis for the development and utilization of medicinal plant resources of genus Paris. Methods: The morphology and infrared spectrum of P. polyphylla var. yunnanensis and its closely relative species were studied systematically and compared with each other. The original infrared spectra data were pretreated by automatic baseline correction, automatic smoothing, ordinate normalization, and second derivative, and analyzed by principal component analysis (PCA), partial least squares discrimination analysis (PLS-DA), and hierarchical cluster analysis (HCA). Results: There were typical characteristics of P. polyphylla var. yunnanensis and its closely relative species, such as plant size, leaves, veins, sepals, petals, flower pavilions and stamens, which could provide morphological identification evidence. There were obviously differences of the second derivative in 3 000-2 000 cm-1 and the fingerprint in 1 800-500 cm-1 in the six types of genus Paris. Both HCA and PLS-DA could better distinguish Paris polyphylla var. yunnanensis and its closely relative species, which could provide an infrared spectral identified evidence. The Results: of HCA showed that P. polyphylla var. yunnanensis, P. polyphylla var. chinensis, P. polyphylla, P. polyphylla var. stenophylla and P. polyphylla var. nana were relatively close. Conclusion: Fourier transform infrared spectroscopy (FTIR) combined with original plant morphological identification can quickly identify P. polyphylla var. yunnanensis and its closely relative species, which will provide a scientific basis for the cultivation, clinical application, and resource development of genus Paris.

Effects of Paris polyphylla ethanol extract on human osteosarcoma and its mechanism / 中草药

Objective To evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and investigate its underlying mechanisms. Methods The anti-proliferation activity of PPEE was tested on 143B, MG-63, U-2 OS, and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The anti-vasculogenic mimicry effects of PPEE were investigated by Matrigel culture assays. The related proteins expression was evaluated by Western blottig. Results PPEE evidently suppressed cell proliferation of 143B, MG-63, and U-2 OS cells with IC50 values of 10-60 μg/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1 expression. It also promoted apoptosis in 143B cells by up-regulating the expression of cleaved Caspase-3, Caspase-8, and Caspase-9, and Bax/Bcl-2 ratio. Additionally, PPEE inhibited 143B cells vasculogenic mimicry formation at non-cytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP-2, and MMP-9. Finally, four weeks’ daily oral administration of PPEE exhibited potent antitumor and anti-vasculogenic mimicry activity in 143B xenograft model with low toxicity. Conclusion These findings demonstrated that PPEE possesses anti-osteosarcoma and anti-vasculogenic mimicry activity in vitro and in vivo, and its underlying mechanisms may be related to inducing apoptosis, blocking cell cycle, and destroying vasculogenic mimicry formation of osteosarcoma cells. Therefore, PPEE is a potential candidate for osteosarcoma treatment.

The Research of Effects of Applying Cataplasm of Paris Polyphylla Smith Var. Yunnanensis (Franch.) Hand.-Mazz on Acute Gouty Arthritis and Inflammation / 昆明医科大学学报

Objective This study is designed to improve topical medication in the Department of Orthopedics in Kunming Municipal Hospital of Traditional Chinese Medicine by applying cataplasm made from the active ingredient (polyphyllin) of Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz ("Dian Chonglou"in Chinese) and to study the mechanism of anti-inflammation. Methods SD male rats were randomly divided into two groups: control group and treatment group. The rats in two groups were all injected 50ul 5% sodium urate suspension into tibiotarsal joint cavity to duplicate the model of acute gouty arthritis.The rats in the control group were applied with cataplasm without polyphyllin, whereas those in the treatment group applied with cataplasm of polyphyllin. After 4,24,48,72 hours,the ankle diameter and circumference were measured. ELISA was used to test the interleukin-1β (IL-1β) andtumor necrosis factor (TNF) in peripheral blood. Western blotting was applied to detect the changes in TLR4 expression of monocyte.Results Compared with the control group, the added value of the diameter and circumference of ankle decreased significantly in the treatment group 4, 24,48 and 72 h after the treatment.IL-1β in the treatment group was lowered at 4,24 and 72 h. TLR4in the treatment group decreased at 48 h. Conclusion Cataplasm with polyphyllin can alleviate the acute inflammatory reaction in gouty joint of rat by reducing the expression of IL-1βand TLR4.

Effect of arbuscular mycorrhizal fungi on endogenous hormones in Paris polyphylla var. yunnanensis / 中草药

Objective In order to study the dynamic variation of endogenous hormones in Paris polyphylla var. yunnanensis inoculated by different foreign arbuscular mycorrhizal fungi species. Methods With sterilized soil as growth substrate, the fresh seeds of P. polyphylla var. yunnanensis were co-cultured with 28 arbuscular mycorrhizal (AM) fungi species under the condition of pot culture at room temperature. The rhizomes and fibrils of P. polyphylla var. yunnanensis were collected, and the contents of endogenous hormone zygotic nucleotides (ZR), gibberellin (GA), indole acetic acid (IAA) and abscisic acid (ABA) were determined by HPLC, then content and proportion of endogenous hormones were analyzed, respectively. Results The results showed that it could increase the mycorrhizal colonization rate and seedling rate of P. polyphylla var. yunnanensis inoculated by different foreign AM fungi species. The content change of ZR, GA, IAA and ABA appears different in the rhizomes and seedling fibrils of P. polyphylla var. yunnanensis after the inoculation of AM fungi species, but it has preferences. Compared with the control group (CK), the content of ZR and GA rose obviously in the rhizomes and seedling fibrils of P. polyphylla var. yunnanensis after the inoculation of AM fungi species, the content of ABA reduced and the content of IAA did not obvious change. The ABA/ZR proportion, ABA/GA proportion and ABA/IAA proportion reduced obviously in the rhizomes of P. polyphylla var. yunnanensis after the inoculation of AM fungi species but there was different change in seedling fibrils of P. polyphylla var. yunnanensis. Conclusion In conclusion, it is inferred that the AM fungi can promote the survival rate of the P. polyphylla var. yunnanensis seedlings, and the different AM fungi strains affect the content of endogenous hormones of P. polyphylla var. yunnanensis.

Cloning and expression of squalene epoxidase from Paris polyphylla var. yunnanensis / 中草药

Objective: To clone and express a full-length cDNA encoding squalene epoxidase which is key to the biosynthetic pathway of triterpenoid saponins in Paris polyphylla var. yunnanensis. Using real-time PCR method to detect the relative content of SE1 gene and SE2 gene from the cDNA sample. Methods: Total RNA was extracted from the roots of P. polyphylla var. yunnanensis and transcription was reversed. Using the reversed transcription of cDNA as template, specific primer was designed according to the transcriptome data about two groups of SE gene sequence from the HiSeq2500 sequencing platform, and then SE gene was cloned. The production was inserted to pEASY-T1 Simple Cloning Vector, and pEASY-E1-SE expression vector was built after sequencing appraisal right. The ArtMedia protein Expression/Amp+ medium was used to induce expression automatically. Using real-time PCR method to detect the relative contents of SE1 gene and SE2 gene from the cDNA sample. Results: Two SE genes of P. polyphylla var. yunnanensis were obtained, which were named as ppSE1 and ppSE2, repectively. cDNA was 1 598 bp of ppSE1 and 1 509 bp of ppSE2, respectively. The full length for ppSE1 was 1 932 bp, length of ORF was 1 578 bp, coding 525 AA; The full length for ppSE2 was 1 828 bp, length of ORF was 1 548 bp, coding 515 AA. Fluorescence quantitative PCR results showed that the expression of ppSE1 and ppSE2 genes had significant differences in stem and leaf, and the expression of ppSE1 was most pronounced in the leaf. Enzyme digestion and sequencing results showed that the prokaryotic expression vector pEASY-E1-SE was built successfully. SDS-PAGE analysis showed that two fusion proteins of SE genes were induced expression successfully in BL21 (DE3) express competent cells. Conclusion: The SE gene of P. polyphylla var. yunnanensis has been cloned, and the SE protein with biological activity in vitro has been obtained. The ppSE1 and ppSE2 genes have different expression patterns in P. polyphylla var. yunnanensis, and play a different role in the synthesis of secondary metabolites.

Accumulation differences of four polyphyllins in Paris polyphylla and Paris polyphylla var.yunnanensis / 中成药

AIM To investigate the accumulation differences of polyphyllins Ⅰ,Ⅱ,Ⅵ and Ⅶ in Paris polyphylla Smith var.polyphylla and Paris polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz..METHODS The contents of four polyphyllins in the roots,leaves and stems of P.polyphylla were determined by HPLC.The fingerprints of twenty batches of samples were established.Cluster analysis was adopted in the evaluation of accumulation differences of various polyphyllins in different parts of two kinds of plants.RESULTS There were significant differences in various polyphyllins' contents and total contents in different populations of P.polyphylla (P ＜ 0.05,P ＜ 0.01).Polyphyllin Ⅵ was not detected in the roots,and the average contents of polyphyllins Ⅰ and Ⅱ were the highest.The polyphyllin Ⅶ' s average content and total content were the highest in the leaves.Only polyphyllin Ⅶ was detected in the stems,and the total content was the lowest.The similarities of roots of P.polyphylla and P.polyphylla var.yunnanensis were high,while those of leaves and stems were low.According to the total contents of polyphyllins in different parts,the samples could be divided into three types.CONCLUSION P.polyphylla can be considered as the substitute of P.polyphylla var.yunnanensis.

Study on activity of VEGF-binding factors in Paris polyphylla / 中草药

Objective: To screen and isolate vascular endothelial growth factor (VEGF)-binding factors from Paris polyphylla var. yunnanensis, and explore their property for stimulating VEGF activity. Methods: The VEGF-binding factors from water extract of P. polyphylla var. yunnanensis were screened by VEGF-affinity chromatography and further purified by HPLC method. Their activity on proliferation of VEGF-dependent cells was determined by MTT analysis with sensitive cell line HepG2 as model. We also predicted the possible components according to RRLC-QTOF and UV results. Results: The VEGF-binding factors screened from the water extract of P. polyphylla var. yunnanensis were named as factor B3 which included three compounds and could induce the proliferation of VEGF-dependent cell line HepG2 but not the VEGF-independent cell line HEK293. Further studies indicated that factor B3 had an additive effect with VEGF to induce the proliferation of HepG2, and the additive effect could be attenuated by VEGF antibody. In addition, the proliferation of HepG2 cells induced by factor B3 alone could also be attenuated by VEGF antibody. Furthermore, based on the results of RRLC-QTOF and UV analysis, we predicted that factor B3 are probably members of saponin family. Conclusion: The factor B3 isolated from P. polyphylla var. yunnanensis has the property to stimulate VEGF activity.