Genetic Diversity Assessment of a Chinese Medicinal Endemic Species, Aspidopterys obcordata var. obcordata, by Combined Molecular Marker Methods (ISSR & SRAP).

Aspidopterys obcordata var. obcordata, a medicinal plant endemic to China, is a narrowly distributed species and wild resources are extremely limited. To evaluate the genetic variability and degree of genetic divergence of A. obcordata var. obcordata, and to make rational scientific decisions on its harvest and germplasm conservation, we collected 122 samples from across nearly all of its distribution area and studied genetic diversity using inter-simple sequence repeats (ISSRs), sequence-related amplified polymorphisms (SRAPs), and a method combining the two techniques. The results revealed the high genetic diversity of A. obcordata var. obcordata, mainly due to its intra-population diversity, and the top two populations with the highest levels of intra-population diversity were ML and DH, individuals of which can serve as excellent germplasm candidates during the processing of germplasm screening and conservation. In general, the combining method was prior to the ISSR analyses and SRAP analyses results, except for a slight difference in the genetic structure of individual populations. Therefore, we suggest that a combination analysis of the two marker methods is ideal for evaluating the genetic diversity and genetic relationships of A. obcordata var. obcordata.

[DNA barcoding identification of original plants of a rare medicinal material Resina Draconis and related Dracaena species].

Resina Draconis, a rare and precious traditional medicine in China, is known as the "holy medicine for promoting blood circulation". According to the national drug standard, it's derived from the resin extracted from the wood of Dracaena cochinchinensis, a Liliaceae plant. In addition, a variety of Dracaena species all over the world can form red resins, and there is currently no molecular identification method that can efficiently identify the origin of Dracaena medicinal materials. In this study, seven species of Dracaena distributed in China were selected as the research objects. Four commonly used DNA barcodes(ITS2, matK, rbcL and psbA-trnH), and four highly variable regions(trnP-psaJ, psbK-psbI, trnT-trnL, clpP) in chloroplast genome were used to evaluate the identification efficiency of Dracaena species. The results showed that clpP sequence fragment could accurately identify seven species of Dracaena plants. However, due to the long sequence of clpP fragment, there were potential problems in the practical application process. We found that the combined fragment "psbK-psbI+ trnP-psaJ" can also be used for accurate molecular identification of the Resina Draconis origin plants and relative species of Dracaena, which were both relatively short sequences in the combined fragment, showing high success rates of amplification and sequencing. Therefore, the "psbK-psbI+ trnP-psaJ" combined fragment can be used as the DNA barcode fragments for molecular identification of Resina Dracon's origin plants and relative species of Dracaena. Research on the identification of Dracaena species, the results of this study can be used to accurately identify the original material of Resina Draconis, and providing effective means for identification, rational development and application of Resina Draconis base source.

[Research advance of functional plant pharmaceutical cycloartenol about pharmacological and physiological activity].

Cycloartenol, a phytosterol compound, also one of the key precusor substances for biosynthesis of numerous sterol compounds, has a variety of pharmacological activities such as anti-inflammatory, anti-tumor, antioxidant, antibiosis and anti-alzheimer's disease. Furthermore, cycloartenol also plays an important role in the process of plant growth and development. This article reviewed the research progress on cycloartenol pharmacological activity in domestic and foreign articles, and summarized the effect of cycloartenol and "cycloartenol pathway" on the plant growth and development, laying foundation for the its further study, development and utilization.

[Investigation, collation and research of traditional Dai medicine of China].

In order to find out the composition, characteristics and traditional utilization characteristics of Dai medicine and promote the rational protection, inheritance and utilization of the resources and traditional knowledge of Dai medicine in China, the resources of traditional Dai medicine have been investigated systematically and the traditional knowledge of Dai medicine have been analyzed in the article. We found out that there were altogether 1 077 kinds of traditional Dai medicine in China and among which 272 were the first time recorded in the condition of Dai folk medical uses. There were 1 053 plant medicines which belong to 169 family and 694 genus. These plant medicines mainly distributed in the southern, west southern and east southern area of Yunnan province, the southern area of Guangxi, Guangdong, Guizhou, Sichuan, Fujian province and tropical, subtropical district as Taiwan, and more than 94.49% plant medicines could be found in Yunnan province. From the point of plant life form, they were major herbaceous or shrubby plants; When it is used as medicinal part, root and rhizome of plants account for the highest proportion, the next were whole plant and leaves. From nature, flavor and channel tropism points of view, the largest proportion of Dai medicines were cool, bitter-tasted and possesses water element. In terms of treatment of disease types, most of the drugs can treat gastrointestinal diseases, next were drugs that could be used to treat upper respiratory infection, traumatological and rheumatic diseases, urinary infection, gynecological diseases, hepatopathy, puerperium fever and diseases caused by poisonous insects and beast of prey bite. The study revealed that the resources of traditional Dai medicine and traditional knowledge of application were abundant in China, but the resources of traditional Dai medicine and traditional knowledge of application were faced with the risk of gradually reduce and loss. The article suggested that we should take measures to strengthen the study of protection and utilization of important traditional Dai medicine and endangered resources along with the protection and transmission of traditional knowledge of Dai nationalistic medicine.

[Microscopic identification of dai medicine alpiniae kwangsiensis rhizoma and its confused species].

OBJECTIVE: To observe microscopic characteristics of rhizome of Alpinia kwangsiensis, Alpinia platychilus, Alpinia blepharocalyx, and to provide basis for their identification. METHODS: Microscopic identification of root transverse section by paraffin tissue section and free-hand section and powder were carried out to distinguish them. RESULTS: The microscopic identification can be made by cell structure of epidermis and endodermis, the existence of nonglandular hair, the lignification degree of vascular bundle fiber, the number and existence style of vascular bundle fiber, the number and size of tube, and so on. The powder identification can be made mainly according to the existence of nonglandular hair and spiral vessel, the shape of starch grain, and so on. CONCLUSION: The microscopic characteristics of rhizome can provide basis for the identification of three Alpinia species.

[Original plant identification of Dai nationality herb "Daibaijie"].

OBJECTIVE: To identify the original plant of "Daibaijie", commonly used Dai herb. METHOD: The literature review, morphology and anatomy, pharmacognosy, molecular biology, chemistry were used to analysis. RESULT: Daibaijie's historical scientific name, Dregea sinensis Hemsl., was mistakenly given "Daibaijie" and D. sinensis have significant differences from the distribution, morphology and anatomy, pharmacognosy, molecular biology and chemical composition. "Daibaijie" matches with the characteristics of Marsdenia tenacissima (Roxb.) Moon in Flora of China in English. CONCLUSION: Daibaijie's original plant is M. tenacissima (Roxb.) Moon. The description and illustration of M. tenacissima (Roxb.) Moon in Flora of China in China are wrong. The illustration of M. tenacissima in Flora of China in English is wrong too.

[Comparison of rattan microscopic characteristics of four Dai medicinal materials from Uncaria genus].

OBJECTIVE: To establish microscopic identification standards for four species from Uncaria genus. METHODS: The microscopic characteristics of transvers section and powder of these four species rattan were observed. RESULTS: As for these four species from Uncaria genus, the microscopic identification of rattan transvers section could be made by the existence of cork cambium and pericycle in the cortex, and the location of phloem fiber and stone cells, while the powder identification could be made according to the existence of scalariform vessel and calcium oxalate crystal, and the quantity of stone cells. CONCLUSION: The microscopic characteristics of rattan can provide information for identification of these four species from Uncaria genus.

[Optimal extraction technology for genistein in Flemingia macrophylla with response surface method].

OBJECTIVE: To optimize extraction techniques for genistein in Flemingia macrophylla by using response surface method. METHODS: Based on the single factor experiment, extraction temperature, extraction time and solvent ratio served as the response factor while the extraction rate of genistein served as the response value. Based on multiple linear regression and binomial expression, response surface method was employed to optimize the extracting process, and predicative analysis was conducted. RESULTS: The effects of factors in the descending order were as follows: extraction temperature, extraction time and solvent ratio. The optimal extraction technology was as follows: extraction temperature 80 degrees C, extraction time 3.5 h and solvent ratio 20 mL/g. Under these conditions, the extraction rate of genistein reached 0.161 mg/g. CONCLUSION: The results are agreed with model predictions. The technology can be used as the extraction process of genistein in Flemingia macrophylla.

Transcriptome profiling reveals candidate flavonoid-related genes during formation of dragon's blood from Dracaena cochinchinensis (Lour.) S.C.Chen under conditions of wounding stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Dragon's Blood (Resina Draconis) is a red resin that has been used in traditional medicine to promote blood circulation, regenerate muscles, reduce swelling and pain, stop bleeding, etc., and its main chemical constituents are flavonoids. Dracaena cochinchinensis (Lour.) S.C.Chen is the only plant defined by the Pharmacopoeia of the People's Republic of China as a source of dragon's blood. AIM OF THE STUDY: We aimed to reveal genes involved in the biosynthesis and accumulation of flavonoids of D. cochinchinensis which is under wounding stress by performing a de novo transcriptome analysis. MATERIALS AND METHODS: D. cochinchinensis samples were collected for transcriptome sequencing and bioinformatics analysis at 0 days (0 d), 3 days (3 d), 6 days (6 d), and 10 days (10 d) after induction wounding stress, and tissues were microscopically observed after wounding stress. RESULTS: A total of 63,244 unigenes were obtained through bioinformatics analysis, and genes associated with the biosynthesis of flavonoids were identified. Through the analysis of DEGs after wounding stress in D. cochinchinensis, based on gene expression consistent with flavonoid accumulation levels, 20 genes in connection with the flavonoid synthesis pathway and 56 genes that may be responsible for flavonoid modification and transport, and also revealed TFs (MYB, bHLH) that may be responsible for flavonoid biosynthesis. Analysis of DEGs between the four periods revealed that after wounding stress, the greatest number of significant DEGs were enriched during the first 3 days, while fewer DEGs were enriched after day 3, which corresponding to only about 1/10 (353/3883) the number of DEGs during the first 3 days. In addition, putative unigenes involved in lignin biosynthesis, such as CSE, HCT, CCR, F5H, and CAD, were significantly down-regulation after D. cochinchinensis wounding stress, but the putative unigenes responsible for flavonoid biosynthesis, such as CHS, CHI, DFR, F3'5'H, F3H, ANR, FLS, and ANS were significantly up-regulation. CONCLUSION: We performed de novo transcriptome analysis of D.cochinchinensis under wounding stress, candidate genes and TFs involved in the biosynthesis and accumulation of flavonoids were identified, which is the first report on the transcript variants in flavonoid form accumulation in D. cochinchinensis under wounding stress. According to the results of DEGs analysis, wounding stress attenuated lignin biosynthesis meanwhile promoted flavonoid biosynthesis. In addition, we also compared the transcriptomics of the two different original plants (D.cochinchinensis and D.cambodiana) that form dragon's blood in order to provide further understanding of the formation of dragon's blood.

Complete Chloroplast Genome Analysis of Two Important Medicinal Alpinia Species: Alpinia galanga and Alpinia kwangsiensis.

Most Alpinia species are valued as foods, ornamental plants, or plants with medicinal properties. However, morphological characteristics and commonly used DNA barcode fragments are not sufficient for accurately identifying Alpinia species. Difficulties in species identification have led to confusion in the sale and use of Alpinia for medicinal use. To mine resources and improve the molecular methods for distinguishing among Alpinia species, we report the complete chloroplast (CP) genomes of Alpinia galanga and Alpinia kwangsiensis species, obtained via high-throughput Illumina sequencing. The CP genomes of A. galanga and A. kwangsiensis exhibited a typical circular tetramerous structure, including a large single-copy region (87,565 and 87,732 bp, respectively), a small single-copy region (17,909 and 15,181 bp, respectively), and a pair of inverted repeats (27,313 and 29,705 bp, respectively). The guanine-cytosine content of the CP genomes is 36.26 and 36.15%, respectively. Furthermore, each CP genome contained 133 genes, including 87 protein-coding genes, 38 distinct tRNA genes, and 8 distinct rRNA genes. We identified 110 and 125 simple sequence repeats in the CP genomes of A. galanga and A. kwangsiensis, respectively. We then combined these data with publicly available CP genome data from four other Alpinia species (A. hainanensis, A. oxyphylla, A. pumila, and A. zerumbet) and analyzed their sequence characteristics. Nucleotide diversity was analyzed based on the alignment of the complete CP genome sequences, and five candidate highly variable site markers (trnS-trnG, trnC-petN, rpl32-trnL, psaC-ndhE, and ndhC-trnV) were found. Twenty-eight complete CP genome sequences belonging to Alpinieae species were used to construct phylogenetic trees. The results fully demonstrated the phylogenetic relationship among the genera of the Alpinieae, and further proved that Alpinia is a non-monophyletic group. The complete CP genomes of the two medicinal Alpinia species provides lays the foundation for the use of CP genomes in species identification and phylogenetic analyses of Alpinia species.

Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl.

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.

Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes.

To ensure the safety of medications, it is vital to accurately authenticate species of the Apocynaceae family, which is rich in poisonous medicinal plants. We identified Apocynaceae species by using nuclear internal transcribed spacer 2 (ITS2) and psbA-trnH based on experimental data. The identification ability of ITS2 and psbA-trnH was assessed using specific genetic divergence, BLAST1, and neighbor-joining trees. For DNA barcoding, ITS2 and psbA-trnH regions of 122 plant samples of 31 species from 19 genera in the Apocynaceae family were amplified. The PCR amplification for ITS2 and psbA-trnH sequences was 100%. The sequencing success rates for ITS2 and psbA-trnH sequences were 81% and 61%, respectively. Additional data involved 53 sequences of the ITS2 region and 38 sequences of the psbA-trnH region were downloaded from GenBank. Moreover, the analysis showed that the inter-specific divergence of Apocynaceae species was greater than its intra-specific variations. The results indicated that, using the BLAST1 method, ITS2 showed a high identification efficiency of 97% and 100% of the samples at the species and genus levels, respectively, via BLAST1, and psbA-trnH successfully identified 95% and 100% of the samples at the species and genus levels, respectively. The barcode combination of ITS2/psbA-trnH successfully identified 98% and 100% of samples at the species and genus levels, respectively. Subsequently, the neighbor joining tree method also showed that barcode ITS2 and psbA-trnH could distinguish among the species within the Apocynaceae family. ITS2 is a core barcode and psbA-trnH is a supplementary barcode for identifying species in the Apocynaceae family. These results will help to improve DNA barcoding reference databases for herbal drugs and other herbal raw materials.

The effects of genetic variation and environmental factors on rhynchophylline and isorhynchophylline in Uncaria macrophylla Wall. from different populations in China.

Uncaria macrophylla Wall. is an important Chinese medicinal herb. Rhynchophylline (RIN) and isorhynchophylline (IRN) are its major active compounds. We investigated the influence of genetic differentiation and environmental factors on the RIN and IRN to find the main influencing factors of their contents and lay the foundation for the following cultivation and breeding. We used inter-simple sequence repeat (ISSR) markers to investigate the genetic diversity, and high-performance liquid chromatography (HPLC) to measure the contents of RIN and IRN in 200 samples of U. macrophylla obtained from nine natural populations, and then to analyze the correlation between genetic differentiation, environmental factors of sampling sites and the contents of RIN and IRN. We found that High intra-population (80.05%) and low inter-population (19.95%) genetic diversity existed in the samples of U. macrophylla. To some extent, genetic differentiation and the contents of RIN and IRN had correlation in individual populations (such as JH, MH, XM, and ML). The RIN and IRN contents were significant negatively correlated with the precipitation in May (RIRN = -0.771, p = 0.015) and June (RRIN = -0.814, p = 0.008; RIRN = -0.921, p = 0.000), indicating that precipitation was the main affecting factor of their contents. Interestingly, the analysis results showed that the RIN content had a significant positive correlation (r = 0.585, p = 0.000) with the IRN content (they are isomers); the proportion of RIN had a significant negative correlation with the sum of the two (r = -0.390, p<0.0001), while the proportion of IRN had a significant positive correlation (r = 0.390, p<0.0001). It meant that, with the total quantity of the two compounds increased, the proportion of RIN decreased and the proportion of IRN increased, illustrating that their conversion exist some regularity. Moreover, the content ratio of RIN and IRN was significant positively correlated with the January precipitation (r = 0.716, p = 0.030), implying that January may be the key period for the mutual transformation of RIN and IRN.