Genetic diversity and population structure of a protected species: Polygala tenuifolia Willd.

Polygala tenuifolia Willd. is an important protected species used in traditional Chinese medicine. In the present study, amplified fragment length polymorphism (AFLP) markers were employed to characterize the genetic diversity in wild and cultivated P. tenuifolia populations. Twelve primer combinations of AFLP produced 310 unambiguous and repetitious bands. Among these bands, 261 (84.2%) were polymorphic. The genetic diversity was high at the species level: percentage of polymorphic loci (PPL)=84.2%, Nei's gene diversity (h)=0.3296 and Shannon's information index (I)=0.4822. Between the two populations, the genetic differentiation of 0.1250 was low and the gene flow was relatively high, at 3.4989. The wild population (PPL=81.9%, h=0.3154, I=0.4635) showed a higher genetic diversity level than the cultivated population (PPL=63.9%, h=0.2507, I=0.3688). The results suggest that the major factors threatening the persistence of P. tenuifolia resources are ecological and human factors rather than genetic. These results will assist with the design of conservation and management programs, such as in natural habitat conservation, setting the excavation time interval for resource regeneration and the substitution of cultivated for wild plants.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.

[Correlation analysis between effectiveness of element in rhizonsphere soil and quality of Danshen (Salvia miltiorrhiza)].

In order to investigate the content and distribution of available element in the rhizonsphere soil of the growing areas of Salvia miltiorrhiza Bunge, the contents of available element (N,P,K,B,Cu,Zn,Fe,Mn) in 26 soil samples were tested and evaluated. The results showed that the contents of available P and Fe were very plentiful, available K, Cu and Zn were rich, available N and Mn were deficient, available B was extremely deficient in all growing areas of S. miltiorrhiza of eight provinces in China. The correlation analysis showed that the contents of eight kinds of available elements were varying degree correlation. The stepwise regression analysis between the contents of available elements of rhizonsphere soil and ten kinds of active ingredients of Danshen (Salviae Miltiorrhizae Radix et Rhizoma) were researched. The results showed that the rates of contribution of available N,B,Mn and Fe to quality of Danshen were relatively large and they were the significant factors, and the other factors did not show statistical significance. The recommended fertilizing strategies is that the usage of N,B and Mn fertilizers should be controlled according to different stages of growth of S. miltiorrhiza, and P fertilizer should be reduced in all growing areas of S. miltiorrhiza.

[Study on quality assessment of Polygalae Radix based on HPLC-DAD fingerprint].

OBJECTIVE: To establish an HPLC fingerprint to evaluate the quality of Polygalae Radix, root xylem, and those collected in different growth ages or harvest time. METHOD: Separation was performed at 30 °C on a Kromasil C18 column (4.6 mm x 250 mm, 5 µm); the mobile phases was acetonitrile and 0.05% H3PO4 water in the gradient elution; the flow rate was set at 1.0 mL · min(-1) and the detection wavelength at 314 nm; the quality discriminant analyses were accomplished by means of similarity analysis, cluster analysis, principal component analysis and neural network model. RESULT: In 26 batches of Polygalae Radix, 24 batches fingerprint similarities were above 0.8. In 5 different growth or harvest time batches, 4 batches were above 0.8; in 8 batches root xylem samples, the similarities were all above 0.875. The similarity analysis was in accord with the quality discriminant analysis of cluster analysis, principal component analysis and neural network model. CONCLUSION: Fingerprint combined with chemical pattern recognition technique can effectively evaluate the quality of Polygalae Radix. The active substance species are all similar in cultivated, wild, different growth or harvest time Polygalae Radix and polygala root xylem, but the chromatography peak areas are different. The effective material contents are similar between wild and cultivated Polygalae Radix, but each chromatographic peak area of the root xylem is much smaller than that of Polygalae Radix. The chemical substance accumulation mainly depends on harvest month, but little growth time in Polygalae Radix.

[Preliminary study on correlation between diversity of soluble proteins and producing area of Cordyceps sinensis].

To analyze the content and type of soluble proteins in Cordyceps sinensis from different producing areas and processed with different methods with bradford method and 2-DE technology, in order to discover significant differences in soluble proteins in C. sinensis processed with different methods and from different producing areas. The preliminary study indicated that the content and diversity of soluble proteins were related to producing areas and processing methods to some extent.

[Corresponding relationship between Mantis and Mantidis Oötheca (Sangpiaoxiao)].

Mantidis Oötheca is commonly used Chinese medicine. Because of the used medicinal part is oötheca and many mantis species can yield ootheca, it is not possible to identify its original animal accurately. There is no unanimous conclusion about the corresponding relationship between Mantis and Mantidis Oötheca (Sangpiaoxiao). This relationship is the basis of the Mantidis Oötheca research. Our study combined the methods of artificial incubation oötheca and capture the living mantis to identify the species of Mantis and Mantidis Oötheca. The results showed that the origin insects of Mantidis Oötheca was Tenodera, Hierodula and Statilia genus insects. This has laid a foundation for further study of Mantidis Oötheca.

[Market survey and identification of hippocampus (Haima)].

OBJECTIVE: To clarify the commercial original species of hippocampus in the market. METHOD: Field survey and interview were applied to the investigation. RESULT: Present study identified the main commercial hippocampus of 13 species, including Pharmacopoeia contained four kinds of hippocampus (without Hippocampus japonicus) listed in Chinese Pharmacopoeia, and the identification method for the 13 kinds of commercially hippocampus was established. CONCLUSION: The further research on hippocampus should be strengthened for the establishment of hippocampal quality control standard.

[Determination of chlorogenic acid and total caffeoylquinic acid in 6 Folium Lonicerae].

OBJECTIVE: To determine the contents of chlorogenic acid and total caffeoylquinic acid in 6 Folium Lonicerae in Sichuan province and Chongqing city for evaluating the quality of them. METHODS: The HPLC analysis and spectrophotometry analysis were used. RESULTS: The contents of chlorogenic acid and total caffeoylquinic acid in 6 Folium Lonicerae had been determined. CONCLUSION: The method is accurate, simple and has a good reproducibility. The results can be used as a reference for the reasonable medication, quality control and further study of Folium Lonicerae.

[Isolation and identification of endophytic fungi from Ligusticum].

OBJECTIVE: To investigate the correlation between entophytic fungal community, habitations and varieties of Ligusticum. METHOD: The solidified potato dextrose agar (PDA) of plates was applied for the isolation of the endophytic fungi, and the identification was completed by spot-planting method. RESULT: Fifty strains of the entophytic fungi were isolated from the rhizome of L. chuanxiong collected from 6 habitations. They were morphologically identified as belonging to 13 genera, 4 families, 3 orders and 1 class. CONCLUSION: There were some differences at quantity, species and distributing of the entophytic fungi in different habitats and varieties of Ligusticum, which was suggested that entophytic fungal community is related with specific habitat.

Effects of tenuifolin extracted from radix polygalae on learning and memory: a behavioral and biochemical study on aged and amnesic mice.

Although normal cognitive changes take place when a person becomes older, aging in humans is generally associated with deterioration of cognitive performance and, in particular, of learning and memory. These cognitive deficits can cause debilitating consequences due to aging. There are a number of herbal medicines which are reported to improve brain function including intelligence. In the present study, improving effects of tenuifolin, extracted from Radix Polygalae (RP), on learning and memory in aged and dysmnesia mice were determined using step-down type passive avoidance test or Y type maze trial. Oral administration of tenuifolin (0.02, 0.04, 0.08 g/kgd(-1), for 15 d) evidently improved the latency and number of errors in aged and dysmnesia mice. The levels of cortical acetylcholine esterase (AChE) activity and hippocampal neurotransmitters in aged mice given tenuifolin (0.02, 0.04, 0.08 g/kgd(-1), for 15 d) were also investigated, and increased levels of norepinephrine (NE), dopamine (DA), decreased activity of AChE were found. However, serotonin (5-HT) had no significant difference from that of aged mice given distilled water. The evident improvement of learning and memory of aged mice is carried out by the effects of tenuifolin on the three stages of memory process, that is, acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the hippocampus and by decreasing the activity of AChE in the cortex.

[Resources of Chinese Herbs Sang (Morus sp.) in Sichuan and strategies for development and utilization].

A comprehensive analysis was conducted on resources composition of Chinese Herbs Sang (Morus sp.) in Sichuan using survey data and related literature. The original plants, germplasm collections, cultivation areas, main cultivated varieties and production sale of crude drugs of Sang in Sichuan were clearly expounded. Strategies for development and utilization of Sichuan mulberry resources were suggested.

[Principal component and cluster analysis of trace elements in Chinese herb Salvia miltiorrhiza and its relative species].

The contents of trace elements, including copper, zinc, iron, magnesium, calcium, chromium, lead, molybdenum, manganese, and cadmium in Chinese traditional herb S. miltiorrhiza and its relative species such as S. miltiorrhiza f. alba, S. evansiana, S. yunnanensis, S. przewarlskii, S. cavaleriei, S. cavaleriei var. simplicifolia, S. roborowskii, S. omeiana, S. tricuspis, S. brevilabra and S. cynica were determined by atomic absorption spectroscopy. The principal components analysis of SPSS was applied to the study of characteristic elements in S. miltiorrhiza and its relative species. Three principal components which accounted for 79.3% of total variance were extracted from the original data. The first factor accounted for 49.6% of the total variance, which means that iron, manganese, copper, zinc, cadmium and lead are the characteristic elements in S. miltiorrhiza and its relative species. The results of cluster analysis show that the samples could cluster reasonably into two groups. The samples of S. miltiorrhiza from different regions were classified into one group except S. miltiorrhiza from Zhejiang and S. miltiorrhiza f. alba. The other Salvia species were classified into another group except S. cavaleriei. The S. miltiorrhiza and other Salvia species can be distinguished by this method, whose accuracy of classification is 90%. The cluster analysis based on the contents of trace element in S. miltiorrhiza and its relative species provided a quick, accurate and simple method for authentication of herb Salvia miltiorrhiza.

[HPLC fingerprint of Rhizoma Polygoni Cuspidati].

OBJECTIVE: To establish the HPLC fingerprint of Rhizoma Polygoni Cuspidati (Polygonum cuspidatum). METHOD: The HPLC separation was carried with Diamonsil C18 column and eluted with a gradient from methanol and 0.1% phosphoric acid, the detection wavelength was at 230 nm and recording 70 min. The similarity of chromatograms was compared by mean of the software from Zhongnan University. RESULT: The constituents of P. cuspidatum were well separated by HPLC, and the similarity was above 0.80. CONCLUSION: The method can be used for the study of fingerprints of Rhizoma Polygoni Cuspidati.

[Study on relative bioavailability of ferulic acid of combination Radix Angelicae Sinensis and Cortex Cinnamomi in mice].

OBJECTIVE: To study the phamacokinetics and relative bioavailability of combination Radix Angelicae Sinensis and Cortex Cinnamomi. METHOD: The content of ferulic acid in plasma was determined directly by HPLC after oral administration of Cortex Cinnamomi in combination individually with Radix Angelicae Sinensis. The plasma concentration-time curve were plotted. The main pharmacokinetic parameters and relative bioavailability were obtained. RESULT: The plasma concentration-time curve of fenalic aclid conformed to one-compartment model. The relative bioavailability of Cortex Cinnamomi combined with Radix Angelicae Sinensis were 226.75%. CONCLUSION: By the statistical analysis, Cortex Cinnamomi combined with Radix Angelicae Sinensis can increase the relative bioavailability of ferulic acid.