Study on fragmentation patterns of coumarins in Notopterygium inchum with ultrahigh performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry / 中国中药杂志

To demonstrate the fragmentation patterns of simple coumarins furanocourmarin(C_7-C_8), furanocourmarin(C_6-C_7) and dihydrofuran coumarin by mass spectrometry, with fraxin, scopoletin, isopsoralen, pimpinellin, isoimperatorin, notopterol and noda-kenin as study subjects, so as to provide a basis for rapid identification of compounds in different subtypes of coumarins. Ultrahigh performance liquid chromatography combined with quardrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was implemented in both positive and negative ion modes. Masslynx software was employed to provide the elemental constituents of each detected ion based on its accurate molecular weight. Chemdraw 2014 was used to cultivate mass number of each inferred structure. The fragment pattern of each compound was determined based on the structures inferred from all the relevant ions. And the patterns were drawn by Chemdraw 2014. The deviation between the calculated molecular weight of the inferred structure and the detected value of the ions was used to assess the correctness of the inferred structures in the fragmentation patterns. The results showed that with UPLC-Q-TOF, neutral loss of CO_2 and CO was reflected in lactone and furan skeletons from the courmarin structure. An even mass was attributed to the loss of an odd number of methyl radicals from compounds with a methoxy substituent. Furanocourmarin(C_7-C_8) produced a protonated molecular ion([M+H]~+), while the other courmarin subtypes produced either a sodium adduct of the molecular ion([M+Na]~+) or a sodium adduct of the molecular ion([M+Na]~+) with a protonated molecular ion([M+H]~+). The m/z 203.03 was a diagnostic ion for furanocourmarin(C_6-C_7), and the m/z 147.04 was supplementary evidence for furanocourmarin(C_6-C_7) identification. The characteristic ion of furanocourmarin(C_7-C_8) was m/z 131.05, while m/z 187.04 was the characteristic ion of dihydrofuran coumarin. The m/z 203.03 ion for furanocourmarin(C_7-C_8) was pretty weak. In negative ion mode, furanocourmarin(C_7-C_8) did not have any signals that were different from the other subtypes of courmarins. The fragmentation patterns in negative ion mode for the other subtypes of courmarins were similar to those in positive ion mode. Four types of fragmentation patterns were identified as forcourmarins from Notopterygium inchum. This study provides the basis for the rapid identification of courmarin subtypes by mass spectrometry.

Comparison of soil hydraulic properties in wild and cultivated areas of Notopterygium incisum / 中国中药杂志

To clarify the difference of soil moisture characteristics between mixed broad leaf-conifer forest soil and artificial cultivation of Notopterygium incisum, the HYPROP system and the dew point potential meter were used to determine soil water retention curves(SWRC) for samples of two horizons(i.e. 2-7 cm, 10-15 cm). The basic physical and chemical properties of soil and its water characteristic parameters were also determined. The result showed as fllows:①The bulk density of mixed coniferous-broad leaf forest soil was between 0.33 and 0.52 g·cm~(-3), significantly lower than the corresponding value of field soil(1.01-1.18 g·cm~(-3))(P<0.05), While the organic matter content was significantly higher than the corresponding value of field soil(P<0.05). ②The saturated water content(θ_s), field water holding capacity(θ_(FC)) and Water that can be effectively utilized by plants(θ_(PAC)) of mixed coniferous-broadleaved forest soil were significantly higher than the corresponding value of field soil(P<0.05), while the retained water content(θ_r) value that cannot be effectively utilized by plants was significantly lower than that of field soil(P<0.05). ③The values of structural porosity(0.13-0.24 cm~3·cm~(-3)) and Matrix porosity(0.34-0.44 cm~3·cm~(-3)) of mixed coniferous-broadleaved forest soil were higher than the corresponding values of field soil. Therefore, with low bulk density and high content of organic matter, mixed coniferous-broadleaved forest soil can store more water in soil in the form of effective water to meet the needs of plants for water, thus possibly forming high quality medicinal materials of Notopterygii Rhizoma et Radix. In conclusion, the results of this study can provide theoretical basis guidance for soil structure improvement and water management to form high quality medicinal materials in the artificial cultivation of N. incisum.

Coumarins from Notopterygium incisum and their inhibitory effect against lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells / 中草药

Objective: To study coumarins from Notopterygium incisum and their anti-inflammatory effect. Methods: Coumarins were separated and purified by repeated column chromatography on silica gel and HPLC, and their chemical structures were determined by spectral data analyses of MS and NMR. Lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells accompanying overproduction of pro-inflammatory mediator nitric oxide (NO) were applied to investigate the anti-inflammatory effect of these purified coumarins. Results: Twenty-four coumarins were obtained and identified as isoimperatorin (1), angenomalin (2), psoralen (3), bergapten (4), capillarin (5), osthenol (6), 5-dehydronotopterol (7), anhydronotopoloxide (8), 7'-O-methylnotoptol (9), bergamottin (10), 7-isopentenyloxy-6-methoxy-coumarin (11), pabulenol (12), notopterol (13), demethylfuropinarine (14), notoptol (15), cnidilin (16), 6-isopentenyloxyumbelliferone (17), nodakenitin (18), isopimpinellin (19), nodakenin (20), decuroside V (21), decuroside I (22), marmesin-11-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (23), and forbesoside (24), respectively. Coumarins 7-10, 13, and 15 exhibited potent inhibitory activity against LPS-induced NO production in RAW 264.7 cells with half maximal inhibitory concentration values from 8.50 to 35.12 μmol/L. Conclusion: Compound 7 is a new natural product; compound 17 is obtained from the roots and rhizomes of N. incisum for the first time; Unsaturated double bond on C-5 in the aromatic ring significantly improved the anti-inflammatory activity of coumarins.

GC-MS Analysis of Volatile Oil of Notopterygium inchum and Notopterygium franchetii / 中国实验方剂学杂志

Objective:To study the volatile oil of Notopterygium inchum and N. franchetii by using gas chromatography-mass spectrometry （GC-MS）. Method:Ten batches of N. inchum and N. franchetii were harvested from Sichuan and Gansu，including eight batches of wild samples， one batch of cultivated N. inchum and one batch of cultivated N. franchetii. Volatile oil was extracted according to the methods in Chinese Pharmacopeia， and the chemical constituents were analyzed with GC-MS technology. Result:Ninety-seven chemical compounds were identified，including monoterpenes，sesquiterpenes and non-terpenes. The calculation results of the relative content of chemical components showed that α-pinene（25.39%-41.68%），β-pinene（19.34%-40.95%） and limonene（4.59%-11.99%） were main compounds in volatile oil of N. inchum and N. franchetii. Relative content of α-pinene was higher than that of β-pinene in wild N. inchum and cultivated N. franchetii， while relative content of β-pinene was higher than that of α-pinene in wild N. franchetii. Relative content of β-pinene in samples stored for one year was lower than that of newly harvested ones. The partial least squares discriminant analysis with SIMCA-P+11.5 software showed that the samples from Gansu and Sichuan provinces had no significant differences， and additionally，one-year-stored samples and newly-harvested samples were clustered into two groups. Conclusion:This study showed that the storage had influence on the chemical constituents of volatile oil in N. inchum， and it also revealed the differences in volatile oil components of wild and cultivated Notopterygium. The above mentioned work provided chemical basis for the pharmacology comparison study of volatile oil from different kinds of N. inchum and N. franchetii.

Study on Anti-inflammatory Mechanism of Couplet Medicine of Notopterygium incisum -Angelica pubescens Based on Network Pharmacology / 中国药房

OBJECTIVE： To predict the anti-inflammatory active components and mechanism of couplet medicine of Notopterygium incisum-Angelica pubescens. METHODS： According to the principle of oral bioavailability≥30％ and drug- likeness≥0.18， active components of N. incisum and A. pubescens were screened； TCMSP was used to predict and screen the potential target of them. Using “Anti-inflammatory” as keyword， inflammatory related target genes were retrieved from human gene database Genecards. Common target was screened by mapping the target genes of active ingredients from couplet medicine of N. incisum-A. pubescens. The active ingredient-target network was established by using Cytoscape 3.5.1 software. The screened targets were used to construct the target protein interaction （PPI） network on the STRING V 10.5 platform. Its anti-inflammatory mechanism was studied by KEGG signaling pathway and GO biological enrichment analysis. RESULTS： Totally 15 active components such as coumarin， beta-sitosterol， ammidin， nodakenin were selected from couplet medicine of N. incisum-A. pubescens. Acting on 49 targets such as transcription factor AP-1， PI3-kinase subunit gamma， estrogen receptor， they mainly involved 19 signaling pathways such as hepatitis B and cell apoptosis， and were involved in 47 biological processes such as regulating inflammatory response and prostaglandin biosynthesis. CONCLUSIONS： The anti-inflammatory mechanism of active components of couplet medicine of N. incisum-A. pubescens on multi-target， multi-channel and multi-biological processes is predicted， and it points out the direction for further anti-inflammatory mechanism study.

Chemical constituents of coumarins compounds from Notopterygium incisum and their anti-oxidant activity / 中草药

Objective: To investigate the coumarin compounds from Notopterygium incisum and their anti-oxidant activities. Methods The coumarin compounds and their analogues were separated and purified by recrystallization, silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. The structure was identified by modern spectroscopy. The isolated compound was tested for anti-oxidant activity by DPPH and ABTS assay. Results Ten compounds were isolated from the dichloromethane extraction layer of 70% ethanol extract, which were identified as bergaptol (1), d-laserpitin (2), falcarindiol (3), phenethyl ferulate (4), selinidin (5), archangelicin (6), notoptol (7), nodakenetin (8), (+)-cis-khellactone (9), and oxypeucedanin hydrate (10). The anti-oxidant activity of these compounds was tested by DPPH and ABTS methods. Conclusion Compounds 2 and 6 are isolated from this plant for the first time. Compounds 1 and 7 have shown the strongest anti-oxidant activity. The structure-activity relationship (SAR) studies revealed the -OH and unsaturated double bond on C-5′ in the aromatic ring significantly improved the anti-oxidant activity than other coumarin compounds.

Geographic distribution of genetic variation of natural populations in an endangered and rarely herb Notopterygium forbesii var. oviforme / 中草药

Objective: In order to determine the geographic distribution of genetic variation and structure of natural populations in an endangered and rarely medicinal herb Notopterygium forbesii var. oviforme. Methods The primers of polymorphic microsatellite molecular markers of N. forbesii var. oviforme were firstly developed using the next generation reduced-representation sequencing technology. Then, the genetic polymorphisms of the whole geographical distributional population samples of N. forbesii var. oviforme were investigated based on the polymorphic SSRs. Results:A total of 780 SSR-containing DNA sequences were obtained by genome De novo assembly. Ten pairs of polymorphic SSR primers were developed and used to analyze the genetic variability of 105 individuals from six natural populations that covered the entire geographical distributions of N. forbesii var. oviforme. The results showed that the number of observed alleles (No) per locus varied between 1 and 6 (mean = 3.530). The mean observed heterozygosity (Ho) per population ranged from 0.305 to 0.457. These finds suggested that N. forbesii var. oviforme had the moderate to high level of genetic variability. Bayesian clustering analyses demonstrated that the six natural populations of N. forbesii var. oviforme have formed two clear genetic lineages. The gene flow and/or genetic introgression have occurred between these two groups. Conclusion:The next generation reduced-representation sequencing technology largely enriched SSRs database of N. forbesii var. oviforme. The geographic distribution patterns of genetic variations of N. forbesii var. oviforme may be related to the long evolutionary history of species and pollen dispersal of long distance among different natural populations.

Anti-rheumatoid arthritic effect of volatile components in notopterygium incisum in rats via anti-inflammatory and anti-angiogenic activities / 中国天然药物

Notopterygium incisum (QH) has been used for the treatment of rheumatoid arthritis (RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis (AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner (2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils (5, 15, and 45 μg·mL) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.

Effects of soil water content on growth and accumulation of active ingredients of Notopterygium incisum / 中国中药杂志

To investigate the adaptive mechanism of Notopterygium incisum to water changes, one-year-old seedlings were used as test materials. The characteristics of growth, physiology, and active ingredients of N. incisum were studied at different soil water content by pot experiments. The result showed that: ①The water content at 100% field capacity is conducive to the growth and accumulation of biomass of N. incisum. The leaf length, petiole length, petiole diameter, root diameter, root length and biomass of underground dry of N. incisum were all significantly increased at the 100% field capacity, and they slightly declined at 70% field capacity. 20% field capacity could not provide the necessary water to growth of N. incisum, all N. incisum withered and died. ②With the extension of moisture treatment time, the relative content of chlorophyll (SPAD) in leaves and the activity of peroxidase (POD) in rhizome showed a trend of increasing firstly, decreasing then; the tendency of activity of superoxide dismutase (SOD) in rhizome increased; the content of malondialdehyde (MDA) in rhizome decreased firstly and increased then;. For the late test, the SPAD value, the activity of POD and SOD in N. incisum at the 40% field capacity were lower than 70% field capacity and 100 field capacity, while the content of MDA in rhizome were higher than 70% field capacity and 100% field capacity. ③ At 70% field capacity, the active ingredients accumulated most in the N. incisum. At the 70% field capacity, the notopterol and isoimperatorinwerehigher than100 field capacity and 40 field capacity. In summary, the soil water content at 70%-100% field capacity would contribute to the growth and accumulation of active ingredients of N. incisum.

Anti-rheumatoid arthritic effect of volatile components in notopterygium incisum in rats via anti-inflammatory and anti-angiogenic activities / 中国天然药物

Notopterygium incisum (QH) has been used for the treatment of rheumatoid arthritis (RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis (AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner (2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils (5, 15, and 45 μg·mL) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.

Study on characteristics and microscopic identification of fruits of Notopterygium franchetii and N. forrestii / 中国中药杂志

Rhizoma et Radix Notopterygii is a rare and endangered Chinese medicine. In the collection of Notopterygium franchetii fruits, we collected a sample of N. forrestii , which is a spurious breed. Fruits of N. franchetii and N. forrestii are very similar in morphology and can be easily confused. Until now the morphological identification of the fruits of Notopterygium has not been reported. To provide a scientific basis for the identification of N. franchetii and N. forrestii fruits, the morphology and microscopic identification were studied in this paper. In this study, stereomicroscope and paraffin sections were used to compare the morphological characteristics and microscopic characteristics of these two fruits. Our results showed that these two fruits were different in size, surface texture and the number of vertical edges on the back. These traits can be used as diagnostic characteristic of these two fruits. The difference between the number of tubing and the endosperm cell contents can be used as microscopic identification features. The above discriminative characteristics can distinguish the two fruits and provide scientific basis for the identification and germplasm evaluation of Notopterygium fruits.

Industrialization condition and development strategy of Notopterygii Rhizoma et Radix / 中国中药杂志

Notopterygii Rhizoma et Radix, the underground part of Notopterygium incisum and N. franchetii, is used as a classical traditional Chinese medicine, and as raw materials for 262 Chinese patent drugs production in 694 pharmaceutical factories currently. It plays an important role in the whole Chinese medicine industry with irreplaceable important economic and social values. However, wild resource of was abruptly depleted, and large-scale artificial cultivation has been inapplicable. In this study, Utilization history and the industrialization status of Notopterygii Rhizoma et Radix were summarized. Resource distribution, ecological suitability of Notopterygii Rhizoma et Radix and core technologies for seeds production, seedling breeding, large-scale cultivation has been reported by current studies, and basic conditions are already available for industrialization production of Notopterygii Rhizoma et Radix. However, there still some key technical problems need to be solved in the further research, and some policy dimensions need to be focused on in the coming industrialization cultivation of Notopterygii Rhizoma et Radix.

Cultural regionalization for Notopterygium incisum based on 3S technology platform Ⅱ. Evaluation for quality suitability for N. incisum based on collocated Cokriging / 中国中药杂志

Quality characteristics based on active substance are focused for in the cultivation of traditional Chinese medicine(TCM) plants due to their economic values. However, ecological and quality suitability are not always a coincidence, which makes traditional cultural regionalization for TCM cultivation based on ecological suitability have great limitations in practical applications. A regionalization method integrated GIS and medicinal plants quality have been analyzed by using field quality data of Notopterygii Rhizoma et Radix as a case study. Spatial interpolation based on medicine quality by Cokriging method is reasonable, and the predicted values of interpolation are correlated with measured values significantly, which shows that cultural regionalization for TCM cultivation based on spatial quality suitability is possible theoretically. The results indicate that the most suitable areas for quality suitability were mainly distributed in Sichuan province (29.42%), while the spatial distribution of quality suitability and ecological suitability was not coincidence. The cultivation regionalization of TCM plants based on quality suitability is helpful to high quality and quantity cultivation of those TCM plants which spatial distribution of geoherbalism and ecological suitability is separated.

Cultural regionalization for Notopterygium incisum based on 3S technology platform Ⅲ. Functional regionalization for cultivation based on suitability evaluation of growth and production quality / 中国中药杂志

Based on the research results of suitability evaluation of growth and production quality, a functional production cultivation regionalization with high feasibility and operability to solve the problem of the separation of high quality and high yield were formatted for protection, wild monitoring, and cultivation of this plant by weighted sum of spatial suitability data of ecology and quality, as well as integrated with land use and cover data. The results of the study revealed that good quality and high yield area were mainly distributed in the original production areas in Sichuan province and where could carry out monitoring and commercialization cultivation. This method is expected to overcome shortage of traditional regionalization methods difficult to distinguish the contribution of ecological factors and quality factors, which provide an innovative theory and methodology for regionalization, and is helpful to practical application of wild resource protection, nature research, monitoring, and commercialization cultivation for Chinese material medica.

Dynamics of plant biomass and nutrients accumulation of Notopterygium incisum in artificial cultivation / 中国中药杂志

In this study,field cultivation experiments of Notopterygium incisum had been carried out for three years, and samples had been collected monthly during growth seasons, and biomass and nutrient elements of aerial and underground part of sampled plants had been determined to assess their seasonal and interannual dynamics respectively. The results showed that biomass of underground part (dry weight) increased mainly in the second year after seedling transplanting, i.e., biomass increased about 32 times in the second year whilst less than 6 times and 2 times in the first year and in the third year, respectively. Therefore, efforts for yield improvement should be focused on the first two year in artificial cultivation of N. incisum. Accumulation of nutrient elements increased steady in the underground part during the first and second year, then showed a sharp decline in the first phase of growth season in the third year, while its accumulation in July to August of third year was higher than the value of second year. Ca, Fe, B and Zn were larger demand nutrient elements to meet growth demands whether for the underground part or aerial part during the second year and third year for N. incisum cultivation. This result provided instructional guidance and scientific basis for artificial cultivation and specific fertilizer of N. incisum.

Ecological suitability assessment for Notopterygium plants transplanting along altitudinal gradients / 中国中药杂志

A comprehensive field research had been focused on growing status, underground biomass and active constituents of Notopterygium incisum and N. franchetii to evaluate the ecological suitability and appropriate cultivation zones by growing the two species seedlings along different elevation gradient. The results showed that compared to the survival rate and underground biomass, the beneficial altitude region to N. incisum was ranged from 2 600 m to 4 100 m, while N. franchetii required a lower altitude which ranges from 1 700 m to 3 600 m. For the active constituent contents, the values were higher in the range of 2 600 to 3 600 m for N. incisum, but for N. franchetii, the range was form 1 700 to 3 600 m. This result provides instructional guidance and scientific basis for artificial cultivation of N. incisum and N. franchetii.

Absorption Characteristics of Isoimperatorin from Notopterygium in Different Parts of Rat's Intestines / 医药导报

Objective To study the absorption features of isoimperatorin in intestine of rat.Methods Establish a single-pass intestinal perfusion model of rat,take phenolsulfonphthalein as a marker for the detection of isoimperatorin concentration from crude extracts of notopterygium,and observe the absorption features of isoimperatorin and its reference substance in intestine of rat.Results The content of isoimperatorin in crude extracts was (0.43±0.02)% (n=5).The absorption parameters of duodenum,jejunum,ileum,colon were (5.28±0.82),(4.47±0.56),(4.17±0.94),(4.32±0.68)×10-5 cm·s-1,respectively.There were no significant differences among them (P>0.05).Compared with the reference substance of isoimperatorin,crude extracts showed better absorption features.Conclusion Isoimperatorin from crude extracts have better characteristics of absorption.This study can provide theoretical basis for design of notopterygium oral formulation.

Identification of Notopterygium seeds using DNA barcoding method / 中国中药杂志

In order to guarantee the species correction of Notopterygium seeds, a molecular identification method with ITS2 as DNA barcode has been verified. In this study, 27 samples of Notopterygium seeds were collected from the main producing area of Notopterygium. The morphological characteristics of the Notopterygium seeds were firstly surveyed. Then the DNA extraction, PCR amplification, DNA sequencing and DNA assembly were carried out. The species identification for a Notopterygium seed was implemented through distance method, NJ-tree method and the DNA barcoding system for traditional Chinese medicine (www.tcmbarcode.cn). The results showed that the seeds of N. incisum and N. franchetii had similar morphological characteristics and were difficult to distinguish clearly based on morphological descriptions. With the results of molecular identification, 24 samples were genuine including 13 N. incisum seeds samples and 11 N. franchetii genuine seeds samples. In conclusion, DNA barcode technology can accurately and efficiently identify the species of Notopterygium seeds. Furthermore, this study will provide a new method for germplasm resources identification of medicinal materials and supplies some guidelines for establishing Chinese herbal seeds and seedlings quality standards.