[Effect of different fungicides on efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza].

Arbuscular mycorrhizal fungi provided is beneficial to Salvia miltiorrhiza for increasing yield, promoting the accumulation of active ingredients, and alleviating S. miltiorrhiza disease etc. However, the application of fungicides will affect the benefit of arbuscular mycorrhizal fungi and there is little research about it. This article study the effect of four different fungicides: carbendazim, polyoxin, methyl mopazine, and mancozeb on mycorrhiza benefit to S. miltiorrhiza by the infection intensity of arbuscular mycorrhizal fungi, the growth of S. miltiorrhiza, and the content of active ingredients. RESULTS:: showed that different fungicides had different effects. The application of mancozeb had the strongest inhibitory effect on the mycorrhizal benefit to S. miltiorrhiza. Mancozeb significantly reduced the mycorrhizal colonization and the beneficial effect of arbuscular mycorrhizal fungi on the growth and the accumulation of active components of S. miltiorrhiza. The application of polyoxin had no significant effect on mycorrhizal colonization. Instead, it had a synergistic effect with the mycorrhizal benefit to promoting the growth and accumulation of rosmarinic acid of S. miltiorrhiza. The inhibitory strengths of four fungicides are: mancozeb>thiophanate methyl, carbendazim>polyoxin. Therefore, we recommend applying biological fungicides polyoxin and avoid applying chemical fungicides mancozeb for disease control during mycorrhizal cultivation of S. miltiorrhiza.

Screening active ingredients of rosemary based on spectrum-effect relationships between UPLC fingerprint and vasorelaxant activity using three chemometrics.

Rosmarinus officinalis L., rosemary, is traditionally used to treat headache and improve cardiovascular disease partly due to its vasorelaxant activity, while the vasorelaxant ingredients remain unclear. In this study, chemical spectrum-pharmacological effect relationship (spectrum-effect relationship) was utilized for efficiently discovering the main vasorelaxant ingredients of rosemary. Ten kinds of rosemary extracts were prepared by different extracting solvents and macroporous resin purification, and their chemical components were analyzed by UPLC. At the same time, the vasorelaxant activities of the 10 kinds of rosemary extracts were estimated on isolated rat thoracic aorta, and three chemometrics named partial least squares regression (PLSR), grey correlation analysis (GRA), and the least absolute shrinkage and selection operator (LASSO) were applied to construct spectrum-effect relationship between the UPLC fingerprints and vasorelaxant activity of rosemary extracts. As a result, most rosemary extracts showed dose-dependent increase in vasorelaxant activity and five kinds of ingredients, including carnosol, carnosic acid, epirosmanol methyl ether, carnosol isomer, and augustic acid were screened as vasorelaxant ingredients. Further, the vasorelaxant activities of carnosic acid and carnosol were verified. Moreover, the increase of nitric oxide (NO) and the decrease of angiotensin-II (Ang-II) were thought to contribute to the vasorelaxant activity of rosemary.

[Effect of soil pH on efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza].

By comparing the effects of soil pH on the efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza， the study is aimed to provide guidance for the use of mycorrhiza in the cultivation of S. miltiorrhiza. In this experiment, the inoculant treated and the non-inoculant treated control were grown in different soil pH. The data was collected after 60 days of cultivation including rate of mycorrhizal infection, biomass, and three chemical constituents with known medicinal action. The results showed that Glomus versiforme was more apt to infect S. miltiorrhiza (F>94.00%; M>69.45%; m>73.66%) and promote the growth of S. miltiorrhiza under pH 5-9 soil. The mycorrhizal contribution to the growth of S. miltiorrhiza was the highest when grown in pH 8 soil. Plants grown with mycorrhiza in pH 8 soil had above-ground biomass more than 2 times and root biomass more than 5 times. The uninoculated plants grew better under acidic and neutral conditions, but the inoculated plants grew better under alkaline (pH 8) conditions. This result showed mycorrhiza can play a role in the adaptability of S. miltiorrhiza to the environment. Inoculation of mycorrhiza significantly increased the accumulation of rosmarinic acid, salvianolic acid B, and dihydrotanshinone by 6.59,5.03 and 2.20-folds. Based on our results alkaline soil (pH 8) is most suitable for the cultivation of S. miltiorrhiza by inoculation with the mycorrhiza G. versiforme.

[Study on characteristics and microscopic identification of fruits of Notopterygium franchetii and N. forrestii].

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.

[Quality assessment of commercial rosemary based on liquid/gas chromatography and mass spectrography with multivariate statistical analysis].

A GC-MS fingerprint of rosemary oil and an UPLC fingerprint of non-volatile compounds of rosemary were established. Sixty-three kinds of volatile compounds and thirty-eight kinds of non-volatile compounds were identified tentatively using GC-MS and UPLC-Q-TOF-MS, respectively, and most of them are flavonoids, oxygenated monoterpenes, and diterpenoids, etc. According to multivariate data analysis, chemical differences existed among commercial rosemary samples, for example, the essential oil of imported rosemary sample belongs to Morocco/Tunisian type and the one of domestic sample belongs to Spanish type. The results of principal components analysis and partial least squares discriminant analysis showed that the imported and domestic rosemary samples were classified into two groups and fourteen components including eucalyptol, (+)-α-pinene, and carnosic acid, were found as the discrimination markers. In comparison with imported rosemary, it can be found that the contents of ten markers such as (+)-α-pinene, o-cymene, and carnosic acid were higher, nonetheless, lower contents for the other markers, in those domestic rosemary samples. Moreover, rosmarinic acid, an important bioactive component in rosemary extract, extensively varied among different samples, indicating that the quality evaluation of rosemary should be concerned.

[Discussion on related problems of model species of fungus： Paecilomyces hepialid].

Paecilomyces hepiali is a new species of fungus isolated from a field collection of Ophiocordyceps sinensis from Baima snow mountain, Diqing Tibetan Autonomous Prefecture, Yunnan Province by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The specimen was identified and named as Paecilomyces hepiali by Qing-Tao Chen, the professor of the Institute of Microbiology, Chinese Academy of Sciences (Paecilomyces hepiali) (2008), who identified a dried culture of living strain 82-2 as the holotype. Until now, the holotype (the voucher specimen) was deposited in the Herbarium of the Institute of Chinese Materia Medica (HICMM), China Academy of Chinese Medical Sciences, Beijing. The P. hepiali neotype designated by the paper "Neotypification of P. hepiali (Hypocreales)" published in TAXON 64 (1) by Yao Yi-Jian et al. in February 2015 is untenable.

[Study on microscopic identification of Astragalus complanatus and A. adsurgens seeds].

Astragalus adsurgens seed is one of the most common adulterants of Astragali Complanati Semen in the market, whose morphological characteristics are very similar with A. complanatus seeds (Astragali Complanati Semen). Many identification methods have been reported, such as morphological identification, fluorescence method, ultraviolet spectrophotometry, TLC, HPLC, protein electrophoresis and so on, but there's no much about microscopic identification. In the present study, the morphological characteristics and microscopic characteristics of these two seeds were investigated, which could provide scientific evidence for the identification and classification of Astragali Complanati Semen. Our results showed that these two seeds were slightly different in the color and the appearance, but significantly different in the microstructure of the seed coat and the hilum, and the distribution of fat droplets in the cotyledon cells. So these microscopic characteristics can be applied for the identification of Astragali Complanati Semen.

[Effect of five fungicides on growth of Glycyrrhiza uralensis and efficiency of mycorrhizal symbiosis].

In order to obtain the fungicides with minimal impact on efficiency of mycorrhizal symbiosis, the effect of five fungicides including polyoxins, jinggangmycins, thiophanate methylate, chlorothalonil and carbendazim on the growth of medicinal plant and efficiency of mycorrhizal symbiosis were studied. Pot cultured Glycyrrhiza uralensis was treated with different fungicides with the concentration that commonly used in the field. 60 d after treated with fungicides, infection rate, infection density, biomass indexes, photosyn- thetic index and the content of active component were measured. Experimental results showed that carbendazim had the strongest inhibition on mycorrhizal symbiosis effect. Carbendazim significantly inhibited the mycorrhizal infection rate, significantly suppressed the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. Polyoxins showed the lowest inhibiting affection. Polyoxins had no significant effect on mycorrhizal infection rate, the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. The other three fungicides also had an inhibitory effect on efficiency of mycorrhizal symbiosis, and the inhibition degrees were all between polyoxins's and carbendazim's. The author considered that fungicide's inhibition degree on mycorrhizal effect might be related with the species of fungicides, so the author suggested that the farmer should try to choose bio-fungicides like polyoxins.

[Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment].

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

[Exploration of property theory of Tibetan medicine].

Tibetan Herbal medicine has its own complete theory based on five sources doctrine. And the theories of "Liuwei", "Baxing" and "Shiqi Gongxiao" formed the basic core components of the property theory of Tibetan medicine. However, books and literature of Tibetan medicine have never been systematically expounded and discussed about it specially which thus will limit the further development of Tibetan medicine theory. In this thesis, we firstly introduced three basic core components of the property theory-the "Liu Wei", "Baxing", and "Shiqi Gongxiao" and their interactions as well. At the same time, the links and similarities between the theory of Tibetan medicine and Chinese medicine theory were compared. The job of the thesis done above is to lay the foundation for further systematic reveal and development of Tibetan medicine theory.

[A accurate identification method for Chinese materia medica--systematic identification of Chinese materia medica].

This paper put forward a more accurate identification method for identification of Chinese materia medica (CMM), the systematic identification of Chinese materia medica (SICMM) , which might solve difficulties in CMM identification used the ordinary traditional ways. Concepts, mechanisms and methods of SICMM were systematically introduced and possibility was proved by experiments. The establishment of SICMM will solve problems in identification of Chinese materia medica not only in phenotypic characters like the mnorphous, microstructure, chemical constituents, but also further discovery evolution and classification of species, subspecies and population in medical plants. The establishment of SICMM will improve the development of identification of CMM and create a more extensive study space.

[Study on digitization of difference in drug color and odor of Magnoliae Officinalis Cortex before and after perspiration].

OBJECTIVE: To digitalize the changes in characters of Magnoliae Officinalis Cortex after perspiration with colorimeter and electronic nose. METHOD: With perspired and non-perspired Magnoliae Officinalis Cortex as objective, colorimeter and electronic nose were used to detect their color characteristic parameter and odor characteristic parameter. Finally, an identification model was established. RESULT: In terms of drug color, the color characteristic parameter model was established for perspired and non-perspired Magnoliae Officinalis Cortex on the basis of L*, a*, b* color spaces. The range of 90% of reference values of perspired Magnoliae Officinalis Cortex: L* (52.22-59.42), a* (5.36-7.68), b* (22.04-27.05). The range of 90% of reference values of non-perspired Magnoliae Officinalis Cortex: L* (38.42-47.31), a* (9.63-11.85), b* (18.48-25.53). In terms of drug odor, the principal component analysis (PCA) and the partial least squares method (PLS) showed significant difference between perspired and non-perspired Magnoliae Officinalis Cortex. CONCLUSION: The difference in drug color and odor of Magnoliae Officinalis Cortex before and after perspiration can be digitalized according to color and odor characteristic parameters tested with colorimeter and electronic nose.

[Research wilt disease of Salvia miltiorrhiza and its pathogen].

Salvia miltiorrhiza is a highly valued traditional chinese medicine for the treatment of atherosclerosis-related disorders in china, such as cardiovascular and cerebrovascular diseases in China. The wilt disease is serious in the culture of S. miltiorrhiza. Wilt disease cause biomass of plant shoots and roots is lessened, active components are decreased. To solve these problems, we research the pathogen causing wilt disease of S. miltiorrhiza. The suspected pathogen is identified by morphology and etiological test. The identification was further confirmed by alignment the sequences of internal transcribed spacer (ITS) amplified by PCR. Our result show the wilt disease of S. miltiorrhiza mostly occurred in July and August, which is hot and wetter. The wilt disease rate of S. miltiorrhiza continuous cropping for one year in S. miltiorrhiz stubble is 10%, but the wilt disease rate of S. miltiorrhiza continuous cropping for three years in S. miltiorrhiz stubble is 60%-70%. The root rot of S. miltiorrhiz caused by the wilt disease, so the wilt disease was mistaken for the rot root in production. Morphological characteristics show the pathogen is Fusarium oxysporum. The sequence of ITS wes determined and found by BLAST shared 99% identity to that of F. oxysporum f. sp. cucumerinum. So it comes to the conclusion that the causing agent of wilt disease on S. miltiorrhiza belongs to F. oxysporum.

[Hypothesis and establishment of 2-dimensional molecular marking method inidentification of Chinese material medica].

This paper introduced a new identification method, the 2-dimensional molecular marking method (2-DM), for Chinese materia medica identification. It can be used in genuine/false discriminating and quality evaluating for the Chinese materia medica. Concept, principle and process of 2-DM method were introduced in this paper. The technical advantages and contributions of 2-DM method in the study of Chinese materia medica were also discussed. Generally speaking, the occurring of 2-DM method would not only expand connotation of identification of Chinese materia medica but provide another effective way for quality evaluating.

[Construction of medicinal plant core collection based on molecular phylogeography].

The paper introduces the current situation and the characteristics of the medicinal plant core collection. It expounds the significance and research methods for the medicinal plant core collection based on molecular phylogeography. Guided by molecular phylogeography, the essay explores the feasibility and methods of medicinal plant core collection for the medicinal plants with rich wild resources and without wild resources. It further forecasts the application of medicinal plant core collection methods on the basis of molecular phylogegraphy.

[Research of mechanism of secondary metabolites of phenolic acids in Salvia miltiorrhiza hairy root induced by jasmonate].

OBJECTIVE: To study the mechanism of secondary metabolites of some phenolic acids in the hairy roots of Salvia miltiorrhiza induced by methyl jasmonate. METHOD: The hairy roots of S. miltiorrhiza were induced with methyl jasmonate (100 micromol x L(-1)) and collected at 0, 12, 24, 36 h after treatment. Real-time quantitative PCR was used for detecting the mRNA expression level of the key enzyme genes on the secondary metabolites pathway of rosmarinic acid, while a LC-MS method was developed to determine the content of rosmarinic acid, caffeic acid and salvianolic acid B. RESULT AND CONCLUSION: The concentration of phenolic acids grew up and accumulated quickly in the hairy roots with exogenous signal molecule MJ induced, and it was showed that the content of CA and RA reached the maximum after 24 h and the content of LAB reached the maximum in 36 h by MJ induced. The induction mechanism may be activated with different levels of RA synthesis in PAL, 4CL, C4H genes on the key enzyme phenylalanine pathway and TAT, HPPR genes on tyrosine pathway. The time of gene expression was different, among them, 4CL and PAL genes were more important. In a word, the result can provide some basis data about the mechanism of secondary metabolites of phenolic acids for further research.

[Discussion on applications and mechanisms of biocontrol microoganisms used for controlling medicinal plant soil-borne diseases].

In recent years, the soil borne disease of medicinal plants becomes severely during the process of cultivation and directly endangered the production and quality of raw materials used in traditional Chinese medicine (TCM). The chemical pesticides have been constantly used to prevent and control the soil borne disease, but only a few are effective. Meanwhile, the excessive uses of chemical pesticides also lead pesticide residues in TCM, which often exceed limit of the standard, and harm the human health and cause environmental pollution. Therefore, biological control has become a hot research point for its environmental advantages. This paper mainly discussed the mechanisms of different species of microorganisms, which could control the soil borne disease of medicinal plants, from the following aspects: improving host plants' nutrient absorption, the nutrient and space competition with the pathogenic bacteria, changing the morphology and anatomical structure of roots, adjusting the host plants' endogenous hormones, restoring the balance of host rhizosphere soil microecology and activating the host plants' defense system etc. Then put forward the prospect of biocontrol agents in the future.

[Effect of elicitors on induction and manipulation of secondary metabolic effective ingredients in Salvia miltiorrhiza].

The application of elicitors, which is currently the focus of research, has been considered as one of the most effective methods to improve the synthesis of secondary metabolites in medicinal plants. Biotic and abiotic elicitors can regulate the secondary metabolic pathways of effective ingredients in Salvia miltiorrhiza. This paper has introduced the research progress about the induction and the regulation mechanism of S. miltiorrhiza by elicitors.

[Study on modules biomass structure of Epimedium acuminatum in different habitats].

OBJECTIVE: To study the biomass structure of Epimedium acuminatum Franch in the different ecological environments for the development and protection. METHOD: Through the scientific investigation in four typical habitats, the sampling spots were set up, the functional modules' biomass structure and relationship of E. acuminatum were researched. RESULT: The average of rhizome, as the largest biomass, and the average total biomass had the same pattern: the open areas of forest edge > shrub lumber > shrub-weed > stream drains. The ratio of the functional modules' biomass had different rates under different habitat conditions. By analyzing and combining investigation, the aerial part of E. acuminatum in the shrub-weed were comparative advantage, and the roots of nutrient accumulation of E. acuminatum in the open areas of forest edge were the highest efficient. Under different ecological condition, the distribution of water metabolism was different strategies. CONCLUSION: In the open areas of forest edge, E. acuminatum growing well, followed by shrub-weed. These two habitats are the ideal ecological environments while the rhizome or the whole plant used as medicine, but also the protection of E. acuminatum.

[Study on electrophoretogram of Magnolia officinalis seeds from different locations].

OBJECTIVE: To find the identification method of seeds from different locations, we analyzed the SDS-PAGE electrophoretogram of Magnolia officinalis seeds from 5 different locations of Enshi district, Hubei province, the genuine producing area of M. officinalis, and compared band numbers, band intensity and protein molecular weight of different locations. The result showed that there were significant differences among seeds from different locations. 9 bands with the molecular weight about 80.34, 63.07, 56.48, 51.32, 40.64, 25.18, 22.63, 21.70, 18.14 kDa were involved in all seeds electrophoretograms and could be as standard bands of M. officinalis seeds. Seeds from Badong county, Jianshi county and Enshi city had their own special bands which were very different from other locations. So this electrophresis method can be applied to identify different locations of M. officinalis seeds.