Integrated multi-omics analysis reveals genes involved in flavonoid biosynthesis and trichome development of Artemisia argyi.

Artemisia argyi is an herbaceous plant of the genus Artemisia. Its young and mature leaves are used as food and medicine, respectively. Glandular trichomes (GTs) are distributed on the leaf surface in A. argyi and are generally considered the location of flavonoid biosynthesis and accumulation. However, the mechanism of flavonoid biosynthesis and accumulation in A. argyi remains unclear. In this study, the coregulatory genes involved in flavonoid biosynthesis and trichome development in this species were screened and evaluated, and the biosynthetic pathways for key flavonoids in A. argyi were uncovered. AaMYB1 and AaYABBY1 were screened using weighted gene co-expression network analysis, and both genes were then genetically transformed into Nicotiana tabacum L. cv. K326 (tobacco). Simultaneously, AaYABBY1 was also genetically transformed into Arabidopsis thaliana. The total flavonoid and rutin contents were increased in tobacco plants overexpressing AaMYB1 and AaYABBY1, and the expression levels of genes participating in the flavonoid synthesis pathway, such as PAL, FLS, and F3H, were significantly up-regulated in plants overexpressing these genes. These results indicated that AaMYB1 and AaYABBY1 promote flavonoid biosynthesis in tobacco. Furthermore, compared to that in the wild-type, the trichome density was significantly increased in tobacco and A. thaliana plants overexpressing AaYABBY1. These results confirm that AaYABBY1 might be involved in regulating trichome formation in A. argyi. This indicates the potential genes involved in and provides new insights into the development of trichome cellular factories based on the "development-metabolism" interaction network and the cultivation of high-quality A. argyi.

Morphology and mass spectrometry-based chemical profiling of peltate glandular trichomes on Mentha haplocalyx Briq leaves.

Mentha haplocalyx Briq (M. haplocalyx) is a herbaceous plant that has long been used as a food, medicinal spice, and flavoring agent in traditional Chinese medicine. Its secondary metabolites, having high commercial values, are mainly produced in tiny specialized structures called glandular trichomes (GTs). The primary purpose of this study was to examine the morphology and metabolites of peltate GTs in M. haplocalyx.Peltate GTs possessed globular dome shapes and intense auto-fluorescence on the surfaces of M. haplocalyx leaves. Structure subsidence and cuticle rupture were found throughout the aging stage of peltate GTs. According to histochemical staining results, the secretion of peltate GTs contained anthraquinone, flavonoids, phenolic acid and terpenoids. In M. haplocalyx peltate GTs and leaf tissues without peltate glandular trichomes, ten and two volatile compounds were identified respectively. Peltate GTs contained 42 non-volatile chemicals with a variety of structural types, including 20 flavonoids, 17 phenolic acids,1 diterpene, 3 anthraquinone and 1 alkane. Meanwhile, 15 non-volatile compounds were discovered in leaf tissues without peltate glandular trichomes, and they were all included in the list of peltate GTs' 41 components. Therefore, Peltate GTs were shown to be the primary site of not just volatile compounds but also non-volatile chemicals in M. haplocalyx. This study provides an important theoretical basis and technical approach for clarifying the bio-active metabolite biosynthesis in M. haplocalyx.

The pharmacophylogenetic relationships of two edible medicinal plants in the genus Artemisia.

Artemisia argyi and Artemisia indica are edible medicinal plants belonging to the genus Artemisia in the Asteraceae. There are many similarities in their morphology, traditional curative effect, and modern pharmacological treatment. In this study, we built distribution maps of A. argyi and A. indica in China and a phylogenetic tree of common medicinal plants in Asteraceae. Then, we verified the chemical composition changes of A. argyi and A. indica via their metabolome. Traditional efficacy and modern pharmacological action were verified by network pharmacology and in vitro using RAW264.7 cells. The results showed that A. argyi and A. indica are widely distributed in China, and they shared pharmaphylogeny, which provides theoretical support for the mixed use of A. argyi and A. indica in most regions of China. Furthermore, there were both similarities and differences in volatile oil and flavonoid composition between A. argyi and A. indica. The network pharmacology results showed that A. argyi and A. indica had 23 common active compounds and that both had pharmacological effects on chronic gastritis (CG). Molecular docking analyses showed that quercetin, luteolin, and kaempferol have strong binding affinities with the target proteins JUN, TP53, AKT1, MAPK3, TNF, MAPK, and IL6. The cell experiment results further demonstrated that A. argyi and A. indica treat CG via the NOD-like receptor pathway. Based on the theory of pharmaphylogeny, this study explored the pharmaphylogeny between A. argyi and A. indica from various perspectives to provide a basis for the substitution of A. argyi and A. indica.

Morphogenesis, ultrastructure, and chemical profiling of trichomes in Artemisia argyi H. Lév. & Vaniot (Asteraceae).

MAIN CONCLUSION: Glandular trichomes of Artemisia argyi H. Lév. & Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév. & Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography-mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4-6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi.

Determination of Mineral Elements in Nanyang Mugwort (Artemisia argyi) Leaves Harvested from Different Crops by Inductively Coupled Plasma Mass Spectrometry and Inductively Coupled Plasma Atomic Emission Spectrometry.

Due to high need for medical purposes, multiple harvests of mugwort (Artemisia argyi) have been extensively applied in China for the increase of mugwort yield recently. However, the investigation on the mineral elements in different crops, which are significantly related to mugwort growth and the clinical efficacy of this medicinal herb, has not been conducted. This study provided an analytical method and quality evaluation for mineral elements in Nanyang mugwort leaves harvested from three different crops. The contents of 35 mineral elements were determined by inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). ANOVA, principal component analysis and factor analysis were applied to evaluate the results. Four principal components were identified and their comprehensive evaluation function was as follows: F = 0.7008Fl + 0.1236F2 + 0.0936F3 + 0.0321F4. The comprehensive scores of the mugwort leaves from different crops were ranked as follows: 3rd crop > 2nd crop ≈ 1st crop. These findings can provide a reference for the quality control and clinical use of mugwort leaves, and a guidance of differential nourishing strategies for different crops.

Comparative Genomics of Spatholobus suberectus and Insight Into Flavonoid Biosynthesis.

Spatholobus suberectus Dunn (S. suberectus), has been widely used in traditional medicines plant source of the Leguminosae family. Its vine stem of which plays an important role in the prevention and treatment of various diseases because it contains various flavonoids. Comparative genome analysis suggested well-conserved genomic components and genetic collinearity between the genome of S. suberectus and other genera of Leguminosae such as Glycine max. We discovered two whole genome duplications (WGD) events in S. suberectus and G. max lineage underwent a WGD after speciation from S. suberectus. The determination of expansion and contractions of orthologous gene families revealed 1,001 expanded gene families and 3,649 contracted gene families in the S. suberectus lineage. Comparing to the model plants, many novel flavonoid biosynthesis-related genes were predicted in the genome of S. suberectus, and the expression patterns of these genes in the roots are similar to those in the stems [such as the isoflavone synthase (IFS) genes]. The expansion of IFS from a single copy in the Leguminosae ancestor to four copies in S. suberectus, will accelerate the biosynthesis of flavonoids. MYB genes are widely involved in plant flavonoid biosynthesis and the most abundant member of the TF family in S. suberectus. Activated retrotransponson positive regulates the accumulation of flavonoid in S. suberectus by introducing the cis-elements of tissue-specific expressed MYBs. Our study not only provides significant insight into the evolution of specific flavonoid biosynthetic pathways in S. suberectus, but also would facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding for alleviating resource shortage of S. suberectus.

Traditional uses, phytochemistry, pharmacology and toxicology of Lamiophlomis rotata (Benth.) Kudo: a review.

Lamiophlomis rotata (Benth.) Kudo is a herbaceous plant of the family Lamiaceae, subfamily Lamioideae. Approximately, 127 chemical constituents have been isolated and identified from L. rotata, including iridoids, flavonoids, phenylethanoid glycosides, polysaccharides, and organic acids. These chemical constituents have extensive pharmacological properties, which include anti-nociceptive, haemostatic, anti-inflammatory, anti-tumour, immunomodulatory, antioxidant, and cardio-protective activities. Documentation of its historical use in traditional medicine and contemporary phytochemical and pharmacological research indicate that L. rotata has significant potential in therapeutic and health care applications. Both whole extracts and individual chemical components isolated from this plant exhibit a wide range of biological activities that warrant further investigation. These investigations can be assisted by careful review of existing traditional knowledge from diverse cultural backgrounds. A new search for chemical and biological markers and reinforced protection of the germplasm resources of L. rotata are also important to ensure targeted and sustainable use of this medicinal resource. The aim of this review was to provide comprehensive information on the botanical characteristics, traditional uses, ethnopharmacology, chemical and pharmacological properties, toxicity profile, and conservation status of L. rotata, to improve understanding of its mechanisms of action so that novel therapeutic agents may be developed from this plant.

Molecular identification of antelope horn by melting curve analysis.

Antelope horn is a valuable Chinese traditional medicine and widely used in clinic. However, with the deterioration of antelope's living environment and a lot of killing, the saiga population begins falling and in some places plummet. Since the increasing demand of this expensive and good bioactive medicine, the horn of artiodactyla animals is often used as the antelope horn. The adulterated or impostor not only cause damage to clinical medicine but also affect the antelope resources protection and sustainable development. Here, in order to establish a melting curve analysis (MCA) method to distinguish the antelope horn from other animal horns and identify the decoction pieces and Chinese patent medicine in a fast and easy way, animal horns and its decoction pieces, Chinese patent medicines were collected from the market and the DNA of all the collected samples were extracted. The melting curve of two universal fragments (COI and Cyt b) was scanned and Cyt b was selected as feasibility fragment for identifying authentic antelope horn from eight adulterant animal horns. After optimizing the condition for MCA, inspecting the precision and the replication of the method, a reference melting curve modern was established and we performed MCA on the antelope horns, fakes, and adulterants on a 1:1 mix, decoction pieces, and Chinese patent medicine. Thus, this study provides fast and easy methods so that MCA can detect the truth, fakes, and adulterations of antelope horns.

[Authentication of Cuscutae Semen, Raphani Semen and their adulterants by rapid PCR].

To establish an accurate, rapid and efficient method for authenticating Cuscutae Semen and Raphani Semen by using rapid PCR amplification. The samples of Cuscutae Semen, Raphani Semen and their adulterants were collected. The total DNA of the samples has been extracted, and ITS sequence from Cuscutae Semen, Raphani Semen and their adulterants was amplified by PCR and sequenced directionally. These sequences were aligned by using Clustal W. Specific primers were designed and amplified by two-steps PCR amplification method. The rapid PCR methods for authenticating Cuscutae Semen and Raphani Semen were established by optimizing the denatured and annealing temperature, cycle numbers, and etc. When 100 × SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV lamp whereas adulterants showed no florescence. The results indicated that the rapid PCR method can identify Cuscutae Semen and Raphani Semen rapidly. This study provides the technical support for authentication of Chinese medicinal materials.

[Analysis phylogenetic relationship of Gynostemma (Cucurbitaceae)].

The sequences of ITS, matK, rbcL and psbA-trnH of 9 Gynostemma species or variety including 38 samples were compared and analyzed by molecular phylogeny method. Hemsleya macrosperma was designated as outgroup. The MP and NJ phylogenetic tree of Gynostemma was built based on ITS sequence, the results of PAUP phylogenetic analysis showed the following results: (1) The eight individuals of G. pentaphyllum var. pentaphyllum were not supported as monophyletic in the strict consensus trees and NJ trees. (2) It is suspected whether G. longipes and G. laxum should be classified as the independent species. (3)The classification of subgenus units of Gynostemma plants is supported.

[Study on identification of "Digeda" raw materials in Mongolian patent medicine by PCR amplification of specific alleles].

To explore a new method for identification of Mongolian patent medicine (MPM) by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of "Digeda" raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method, psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L rotatum can be identified by specific primers from Digeda-4 Tang, Digeda-8 San, Digeda-4 San, and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San, Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM.

[Application of Rapid PCR Technology on Authentication of Species in Panax Genus].

OBJECTIVE: To establish an accurate, rapid and efficient method for authentication of Panax species by using PCR amplification of specific alleles. METHODS: The samples of Panax species were collected for extracting the total DNA. matK sequence from the Panax species was amplified by PCR and sequenced directionally, and then aligned by using Clustal W. Specific primers were designed and amplified by two-steps PCR amplification method. RESULTS: By optimizing the denatured and annealing temperature and time, cycle numbers, the rapid PCR methods for authentication of Panax species were established respectively. When SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV lamp whereas adulterants were not. CONCLUSION: The rapid PCR method can identify the Panax species rapidly. This study provides the technical support for authentication of Chinese medicinal materials.

Convenient, sensitive and high-throughput method for screening botanic origin.

In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

The genus Anemarrhena Bunge: A review on ethnopharmacology, phytochemistry and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Anemarrhena asphodeloides Bunge. (Asparagaceae) yields Anemarrhenae Rhizoma, which has a long history to be used as a traditional medicine to treat various ailments, like cold-induced febrile disease with arthralgia, hematochezia, tidal fever and night sweats by Yin deficiency, bone-steaming, cough, and hemoptysis. It is also used as an ingredient of healthy food, wine, tea, biological toothpaste. Its importance is demonstrated by large scale to treat kinds of diseases in eastern Asian countries. The aim of this review is to provide up-to-date information about phytochemistry, pharmacology, and toxicology of Anemarrhena asphodeloides based on scientific literatures. It will build up a new foundation for further study on mechanism and development of better therapeutic agent and healthy product from Anemarrhena asphodeloides. MATERIAL AND METHODS: All the available information on Anemarrhena asphodeloides was collected via electronic search (using PubMed, SciFinder Scholar, CNKI, TPL (www.theplantlist.org), Google Scholar, Baidu Scholar, and Web of Science). RESULTS: Comprehensive analysis of the literatures searched through sources available above confirmed that the ethnomedical uses of Anemarrhena asphodeloides had been recorded in China, Japan, and Korea for thousands of years. The phytochemical investigation revealed the presence of steroidal saponins, flavonoids, phenylpropanoids, alkaloids, steroids, organic acids, anthraquinones, and others. Crude extracts and pure compounds from Anemarrhena asphodeloides exhibited significant pharmacological effects on the nervous system and the blood system. They also showed valuable bioactivities, such as antitumor, anti-oxidation, anti-microbial, anti-virus, anti-inflammation, anti-osteoporosis, anti-skin aging and damage as well as other activities. CONCLUSIONS: In light of long traditional use and modern phytochemical and pharmacological studies summarized, Anemarrhena asphodeloides has demonstrated a strong potential for therapeutic and health-maintaining purposes. Both the extracts and chemical components isolated from the plant showed a wide range of biological activities. Thus more pharmacological mechanisms on main active compounds (TBII, TAIII, mangiferin and other ingredients) are necessary to be explored. In addition, as a good source of the traditional medicine, clinical studies of main therapeutic aspects (e.g. diabetes, Alzheimer׳s disease, Parkinson׳s disease, etc.), toxicity and adverse effect of Anemarrhena asphodeloides will also undoubtedly be the focus of future investigation.

Commercialized non-Camellia tea: traditional function and molecular identification.

Non-Camellia tea is a part of the colorful Chinese tea culture, and is also widely used as beverage and medicine in folk for disease prevention and treatment. In this study, 37 samples were collected, including 33 kinds of non-Camellia teas and 4 kinds of teas (Camellia). Traditional functions of non-Camellia teas were investigated. Furthermore, non-Camellia teas of original plants were characterized and identified by molecular methods. Four candidate regions (rbcL, matK, ITS2, psbA-trnH) were amplified by polymerase chain reaction. In addition, DNA barcodes were used for the first time to discriminate the commercial non-Camellia tea and their adulterants, and to evaluate their safety. This study showed that BLASTN and the relevant phylogenetic tree are efficient tools for identification of the commercial non-Camellia tea and their adulterants. However, some sequences from original plants have not been found and there is a limitation of sequence number of original plants in GenBank. Submitting more original plant sequences to the GenBank will be helpful for evaluating the safety of non-Camellia teas.

[Study on identification of cistanche hebra and its adulterants by PCR amplification of specific alleles based on ITS sequences].

To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.

[Study on fluorescence sequencing typing technology identification of raw materials in liuwei dihuang pill].

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.

[Study on molecular identification of water extracts from Gelsemium elegans and Lonicera japonica and its close species by specific PCR amplification].

OBJECTIVE: To explore the new method of discriminating Gelsemium elegans from Lonicera japonica and its close species by using specific PCR amplification. METHOD: Thirteen samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. PsbA-rnnH sequence from G. elegans was amplified by PCR and sequenced unidirectionally, ClustulW was used to align psbA-trnH sequences of the G. elegans and L. japonica and its close species from GenBank database. RESULT: All samples were amplified by PCR with specific primer, DNA from G. elegans would be amplified 97 bp whereas PCR products from all of Lonicera samples had not bands. CONCLUSION: Specific PCR amplification can be used to identify G. elegans from L. japonica and its close species successfully and is an efficient molecular marker for authentication of G. elegans and L. japonica and its close species.

[Effect of PCR enhancer in molecular authenticate of Chinese herbal medicine].

To evaluate the effect of PCR enhancer on molecular identification of Chinese herbal medicine, and select the optimal enhancers suitable for traditional Chinese medicine (TCM), genomic DNA from 180 kinds of Chinese herbal medicine was extract by CTAB method and alkaline lysis method, respectively. PCR success rate of five universal fragments (ITS2, psbA-trnH, rbcL, matK, trnL-trnF) was compared in a PCR system with and without enhancer. PCR efficiency of Real-time PCR was also compared in a PCR system with and without enhancer. Results showed that PCR success rate of ITS2,psbA-trnH, rbcL fragment was increased by using PVP and BSA. The PCR efficiency was decreased by PCR enhancer in Real-time PCR system. The results indicate that BSA and PVP as PCR enhancer can dramatically increase PCR success rate and genotyping accuracy in TCM molecular authentication.

[Study on identification of Astragali Radix and Hedysari Radix by PCR amplification of specific alleles].

To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles, 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted, trnL-trnF sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature, dNTP, etc were optimized. All samples were amplified by PCR with specific primer, DNA from Astragali Radix would be amplified 136 bp, whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix.