Identification of Angelica acutiloba, A. sinensis, and other Chinese medicinal Apiaceae plants by DNA barcoding.

Crude drug Angelicae acutilobae radix is one of the most important crude drugs in Japanese traditional medicine and is used mainly for the treatment of gynecological disorders. In the listing in the Japanese Pharmacopoeia XVIII, Angelicae acutilobae radix is defined as the root of Angelica acutiloba (Apiaceae), which has long been produced on an industrial scale in Japan. With the aging of farmers and depopulation of production areas, the domestic supply has recently declined and the majority of the supply is now imported from China. Due to having only slightly different morphological and chemical characteristics for the Apiaceae roots used to produce dried roots for Chinese medicines, the plant species originating the crude drug Apiaceae roots may be incorrectly identified. In particular, Angelicae sinensis radix, which is widely used in China, and Angelicae acutilobae radix are difficult to accurately identify by morphology and chemical profiles. Thus, in order to differentiate among Angelicae acutilobae radix and other radixes originated from Chinese medicinal Apiaceae plants, we established DNA markers. Using DNA sequences for the chloroplast psbA-trnH intergenic spacer and nuclear internal transcribed spacer regions, Angelicae acutilobae radix and other Chinese Apiaceae roots, including Angelicae sinensis radix, can be definitively identified.

Population genetic structure of wild Angelica acutiloba, A. acutiloba var. iwatensis, and their hybrids by atpF-atpA intergenic spacer in chloroplast DNA and genome-wide SNP analysis using MIG-seq.

Sampling surveys of Angelica acutiloba and A. acutiloba var. iwatensis, which are medicinal plants endemic to Japan, were conducted in the Chubu region in the central area of the main island of Japan. A. acutiloba grows in riverbeds in mountainous areas, while A. acutiloba. var. iwatensis grows on slopes near mountain ridges at 1000 m above sea level or on constantly collapsing rocky slopes and bare fields on developed land along asphalt roads in valleys of mountainous areas. Specimens of two wild Angelica species collected in this region were examined for maternal lineage by DNA polymorphism analysis of the atpF-atpA region for chloroplast DNA using direct sequencing and genomic component analysis by genome-wide SNP using MIG-seq. In this study area, while all A. acutiloba populations were monophyletic in both maternal and ancestral lineages, A. acutiloba var. iwatensis were genetically heterogeneous due to being composed of three maternal and three ancestral lineages to various degrees. In addition, a natural hybrid population with maternal lineage presumed to be A. acutiloba and paternal lineage A. acutiloba var. iwatensis was also found. In the present study, we report that the combined method of atpF-atpA and MIG-seq analyses is a useful tool for determining the population genetic structure of two wild Angelica species and for identifying hybrids.

Ecological and pharmacognostical characteristics in Ephedra saxatilis in the Yarlung Zangbo River Valley of southeastern Tibetan Plateau, China.

In the Yarlung Zangbo River Valley of the southeastern Tibetan Plateau, China (29°07'49.5"N, 92°41'11.0"E, 3256 m above sea level), we found an Ephedra saxatilis community in the xeric steppe with shrubland vegetation habitat of the broad alluvial plain of the river with soil having relatively higher water-soluble cation (Ca2+, 8.62; K+, 1.94; Mg2+, 2.38 mmol/100 g dry soil weight) and nitrogen (NO3-, 21.78; NH4+, 1.82 mmol/100 g dry soil weight) content. The ranges of ephedrine and pseudoephedrine in 13 E. saxatilis samples were as follows: ephedrine, not detected-3.03 of dry weight (%DW) and pseudoephedrine, not detected-1.36%DW. The 13 E. saxatilis plants collected in the study area showed intraspecific variability of ephedrine and pseudoephedrine with 6 samples containing ephedrine and pseudoephedrine, 6 samples containing only ephedrine, and 1 sample containing only pseudoephedrine.

Genetic and Chemical Diversity of Commercial Japanese Valerian.

In order to investigate the relationship between the chemical composition of essential oils and haplotypes of the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) in Valerianae Fauriei Radix (Japanese Valerian; JV), we analyzed the DNA sequence and GC-MS metabolome of JV from Japanese markets and of herbal specimens from related species. DNA analysis revealed that JV products from Japan consisted of three haplotypes, namely AH-1, -2 and -5 reported in our previous study. The GC-MS metabolome revealed five chemotypes (J1, J2, C, K and O), of which J1, J2 and C were detected in the JV products from Japan. Chemotypes J1 and J2, with kessyl glycol diacetate (KGD) as the main volatile component, were found in the products of Japanese origin whereas chemotype C, with 1-O-acetyl-2,10-bisaboladiene-1,6-diol (ABD), was found in the products of Chinese and Korean origin. The haplotypes were correlated with the chemotypes: haplotype AH-1 for chemotype J1, AH-2 for chemotype J2 and AH-5 for chemotype C, suggesting that the chemical diversity of JV is not attributed to the environmental factors rather to the genetic factors. Since KGD and ABD were reported to have sedative effects and nerve growth factor (NGF)-potentiating effects, respectively, understanding the chemotypes and selecting an appropriate one would be important for the application of JV. The psbA-trnH haplotypes could be useful DNA markers for the quality control and standardization of JV.

Relationship between ephedrine alkaloid profile in Ephedra gerardiana and soil characteristics of glacial landforms in southeastern Tibetan Plateau, China.

In the Kaluxung River catchment of the southeastern Tibetan Plateau in China, we identified three Ephedra gerardiana communities on different soils and glacial landforms from 4842 to 4899 m above sea level: a moraine community located on constantly collapsing sandy gravel alpine steppe slopes with exposed bedrock on the outer slope of the terminal moraine of the Qiangyong Glacier on Mt. Kaluxung; an outwash plain community located on a gentle alpine steppe slope with exposed bedrock at the terminal end of the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang; and a river terrace community located in an alpine meadow on a rock-scattered flat river terrace along a glacier-fed river in the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang. Based on the finding of identical DNA sequences of the intergenic spacers of chloroplast trnT-trnF and trnS-trnfM regions for all Ephedra specimens examined in this study, the E. gerardiana in this study were considered to comprise a genetically homogeneous population. Analysis of the relationship between ephedrine alkaloid profiles of these three communities and soil characteristics showed that the river terrace community in wet alpine meadow had significantly lower ephedrine content than did the moraine and outwash plain communities in dry alpine steppe (moraine community, 1.52 ± 0.44; outwash plain community, 1.42 ± 0.68; river terrace community, 0.33 ± 0.65%DW), but pseudoephedrine content showed the reverse pattern (moraine community, 0.86 ± 0.30; outwash plain community, 0.73 ± 0.60; river terrace community, 1.50 ± 0.71%DW). In addition, total alkaloid (ephedrine and pseudoephedrine) content in the river terrace community (1.83 ± 0.24%DW) was significantly lower than that in the moraine community (2.38 ± 0.64%DW) and outwash plain community (2.15 ± 0.55%DW).

Identification of Lagopus muta japonica food plant resources in the Northern Japan Alps using DNA metabarcoding.

DNA metabarcoding was employed to identify plant-derived food resources for the Japanese rock ptarmigan (Lagopus muta japonica), which is registered as a natural living monument in Japan, in the Northern Japanese Alps in Toyama Prefecture, Japan, in July to October, 2015-2018. DNA metabarcoding using high-throughput sequencing (HTS) of rbcL and ITS2 sequences from alpine plants found in ptarmigan fecal samples collected in the study area. The obtained sequences were analyzed using a combination of a constructed local database and the National Center for Biotechnology Information (NCBI) database, revealed that a total of 53 plant taxa were food plant resources for ptarmigans. Of these plant taxa, 49 could be assigned to species (92.5%), three to genus (5.7%), and one to family (1.9%). Of the 23 plant families identified from the 105 fecal samples collected, the dominant families throughout all collection periods were Ericaceae (99.0% of 105 fecal samples), followed by Rosaceae (42.9%), Apiaceae (35.2%), and Poaceae (21.0%). In all of the fecal samples examined, the most frequently encountered plant species were Vaccinium ovalifolium var. ovalifolium (69.5%), followed by Empetrum nigrum var. japonicum (68.6%), Kalmia procumbens (42.9%), Tilingia ajanensis (34.3%) and V. uliginosum var. japonicum (34.3%). A rarefaction analysis for each collection period in the study revealed that the food plant resources found in the study area ranged from a minimum of 87.0% in July to a maximum of 97.5% in September, and that 96.4% of the food plant taxa were found throughout the study period. The findings showed that DNA metabarcoding using HTS to construct a local database of rbcL and ITS2 sequences in conjunction with rbcL and ITS2 sequences deposited at the NCBI, as well as rarefaction analysis, are well suited to identifying the dominant food plants in the diet of Japanese rock ptarmigans. In the windswept alpine dwarf shrub community found in the study area, dominant taxa in the Ericaceae family were the major food plant s for Japanese rock ptarmigans from July to October. This plant community therefore needs to be conserved in order to protect the food resources of Japanese rock ptarmigans in the region.

Characterization of inter-annual changes in soil microbial flora of Panax ginseng cultivation fields in Shimane Prefecture of Western Japan by DNA metabarcoding using next-generation sequencing.

Panax ginseng C.A.Mey. (Araliaceae) cultivation suffers from the inability to cultivate the same fields continuously for long durations due to replant failure. The main cause of replant failure is considered to be the annual change in the soil microbial flora, especially the invasion and settlement of pathogenic microorganisms of soil-borne diseases. We analyzed the soil bacterial and fungal flora and inter-annual changes in their composition over 5 years in ginseng cultivation fields on Daikonshima Island, Shimane Prefecture of Western Japan by DNA metabarcoding using next-generation sequencing. Bacteria such as Sphingomonas sp., Bacillus sp., and Betaproteobacteria and the fungus Mortierella sp. were consistently detected throughout the cultivation period. The inter-annual compositional changes of the bacterial flora, especially two members of the family Burkholderiaceae, one member of the phylum Actinobacteria, one member of the genus Candidatus Koribacter, and one member of the genus Sphingomonas, corresponded to the cultivation period, whereas those of the fungal flora showed random changes, suggesting that the growth of ginseng may be greatly affected by changes in the bacterial flora. Therefore, a greater understanding of the bacterial flora could provide valuable information for the cultivation of ginseng. The absence of pathogenic microorganisms associated with soil-borne diseases, which have been reported as causative agents of the main diseases of ginseng, in all soil sampling sites throughout the entire cultivation period in this study proves, for the first time, that traditional cultivation management employing empirical methods and chemical control is an effective approach to control these pathogens. Therefore, the DNA metabarcoding of the bacterial flora could provide valuable information for cultivation management, specifically in detecting and controlling soil-borne pathogens responsible for ongoing cultivation damage in long-term cultivation of medicinal plants.

Identification of Valeriana fauriei and other Eurasian medicinal valerian by DNA polymorphisms in psbA-trnH intergenic spacer sequences in chloroplast DNA.

In order to differentiate among Valeriana fauriei Briq. and other Eurasian medicinal valerian (V. dioica L., V. hardwickii Wall., V. jatamansi Jones, and V. officinalis L.), we attempted to establish DNA markers. DNA sequences for the psbA-trnH intergenic spacer region of chloroplast DNA (psbA-trnH) and 18S ribosomal RNA, internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA, internal transcribed spacer 2 (ITS2), and 28S ribosomal RNA of nuclear DNA in V. fauriei and other Eurasian medicinal valerian were compared. Using partial sequences of psbA-trnH (nucleotide positions 1-75 from the 5' end of the intergenic spacer region), V. fauriei and other Eurasian medicinal valerian could be correctly identified to the species level. In addition, the partial sequences of psbA-trnH in V. fauriei contained five different haplotypes, and it was possible to distinguish the origins of valerian from Japan and Eurasia (China and Korea). On the other hand, individuals had heterogeneous sequences of ITS1 and ITS2, making it impossible to use direct sequencing and DNA markers of ITS1 and ITS2 to distinguish species and origins of V. fauriei and other Eurasian medicinal valerian.

Environmental and soil characteristics in Ephedra habitats of Uzbekistan.

In Uzbekistan, Ephedra distachya L., E. equisetina Bunge, E. foliata Boiss. ex C. A. Mey., E. lomatolepis Schrenk, and E. strobilacea Bunge show species specificity for habitat environments and physical and chemical characteristics of habitat soils. Furthermore, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. distachya was found growing from 80 to 200 m above sea level (a.s.l) in the Plateau Ustyurt on the desert steppe of cliffs on soil having relatively higher loss on ignition (19.8-33.8%) and water-soluble cations (Ca2+, 5.14-133.13; Mg2+, 0.85-3.18; and Na+, 2.27-8.33 mmol/100 g dry soil weight) than for other Ephedra habitats. E. strobilacea was found growing on the flat sandy Kyzylkum desert at 94 m a.s.l. and had habitat soil that was the driest with the lowest loss on ignition (2.9%) and highest Na+ (9.05 mmol/100 g dry soil weight) of all the Ephedra habitat soils. On dry steppe from 1054 to 1819 m a.s.l., E. foliata, E. lomatolepis, and E. equisetina formed not only a single community but also a complex community on constantly collapsing sandy gravel slope with relatively higher Ca2+ (3.40-17.44 mmol/100 g dry soil weight) soil content. Notably, E. equisetina grew on the dry steppe of constantly collapsing sandy gravel slopes, in rocky areas, on sandy gravel floodplains of rivers, and on stable humus soil at the base of coniferous trees in a wide range of habitats from dry steppe to coniferous forest zones at altitudes ranging from 1392 to 1819 m a.s.l., as reflected in the greater variability than for other Ephedra habitats in the parameters of loss on ignition (1.4-34.8%), pH (7.1-9.6), NO3- (0.08-35.17 mmol/100 g dry soil weight), Ca2+ (0.24-17.44 mmol/100 g dry soil weight), Mg2+ (not detected-1.25 mmol/100 g dry soil weight), and Na+ (0.13-5.19 mmol/100 g dry soil weight). Ephedrine alkaloids were not detectable in E. strobilacea, E. foliata, and E. lomatolepis. Almost all E. distachya contained only pseudoephedrine (1.25-1.59% of dry weight, %DW), while E. equisetina contained from 1.31 to 2.05%DW ephedrine and from 1.29 to 2.80%DW pseudoephedrine. Ephedrine and pseudoephedrine in E. equisetina showed a statistically significant negative correlation with soil Cl- and Mg2+, respectively.

Physical and chemical characteristics of soils in Ephedra gerardiana and E. pachyclada habitats of Kali Gandaki Valley in Central Nepal.

In the Kali Gandaki Valley in Central Nepal, Ephedra gerardiana and E. pachyclada show species specificity for physical and chemical characteristics of soils. Here, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. gerardiana grew in moist alpine scrub and upper alpine meadow from 3735 to 4156 m a.s.l., while E. pachyclada grew in the lower Caragana steppe and dry alpine scrub from 2629 to 3671 m a.s.l. The soil texture of E. gerardiana and E. pachyclada collection sites were classified as loam or sandy loam mainly composed of sand and silt. Loss on ignition (%) of soil in E. gerardiana habitats (28.4-35.0%) was markedly higher than for that in E. pachyclada habitats (14.2-17.2%). E. pachyclada soil (pH 8.4-9.2) was more alkaline than that for E. gerardiana (pH 8.5). The five ions (Cl-, SO42-, Ca2+, Mg2+, and Na+) in soil of E. pachyclada (Cl-, 0.01-18.97 mmol/100 g dry soil weight; SO42-, 1.95-83.33; Ca2+, 3.79-77.91; Mg2+, 1.28-27.9; Na+, 0.94-34.49) were markedly higher than those of E. gerardiana (Cl-, 0.18-0.29; SO42-, 0.07-0.08; Ca2+, 4.19-4.59; Mg2+, 0.22-0.58; Na+, 0.93-1.40). The main factor contributing to strongly alkali soils for each species was different between E. gerardiana and E. pachyclada: CaCO3 for E. gerardiana and CaSO4, MgSO4, NaCl, or a combination of these for E. pachyclada. The total ephedrine and pseudoephedrine content in E. gerardiana and E. pachyclada ranged from 1.67-1.88%DW and 1.95-4.80%DW, respectively. Both E. gerardiana and E. pachyclada were amenable for use a raw material source for extraction of ephedrine and pseudoephedrine, and the ephedrine content of both species showed a statistically significantly positive correlation with Mg2+ and Na+ contents of the soil.

Ecological, phylogenetical, and pharmacognostical characteristics of Aconitum kiyomiense endemic to Hida highlands, Takayama city, Gifu Prefecture, Japan.

Aconitum kiyomiense Kadota (Ranunculaceae) is endemic to Takayama city, Gifu Prefecture, central Japan. We collected specimens from marshes and flood plains at altitudes ranging from 852 to 1085 m and from a new habitat consisting of a mesic meadow in the subalpine belt (1681 m). Glabrous pedicels and flowering sequence of inflorescence were used for identification, but intra-species variations in the pilus of pedicels (glabrous, pilose, and chimeric types) were observed. Although the flowering sequence has been reported as both indeterminate and determinate, all specimens in the present study were determinate. No intra-species variation was detected via partial nuclear internal transcribed space, and sequences did not match another 17 East Eurasian continent subgenus Aconitum species. The chloroplast trnL-trnF intergenic spacer region (trnL-trnF) showed three different haplotypes. The trnL-trnF dominant haplotype sequence was identical to that of A. kusnezoffii growing on the Eurasian continent, suggesting that A. kiyomiense is more primitive than other Japanese aconitum and a relic species of the Eurasian continent. We report the first detection of aconitine alkaloids in the tuberous roots, which exhibited aconitine alkaloid contents varying from 0.32 to 4.05 mg/g dry weight (mg/g) for aconitine, 0.02 to 4.12 mg/g for hypaconitine, undetectable to 0.05 mg/g for jesaconitine, and 0.42 to 3.76 mg/g for mesaconitine. The variation of aconitine alkaloid components and contents appeared to be random and did not vary with inflorescence phenotype, trnL-trnF haplotype, environmental habitat conditions, or the geographic region of the collection sites. Since most populations showed no genetic intra-variation, it will be necessary to maintain the continuity of habitats and designate areas for conservation of genetic diversity at the population level.

Evaluation of raw nepodin extraction from Rumex japonicus and R. obtusifolius and their DNA polymorphisms.

Nepodin, found in the roots of Rumex japonicus Houtt. (Polygonaceae), inhibits osteoclast differentiation and has an antidiabetic effect. We propose nepodin as an ingredient of new functional foods or as a drug candidate for reducing the risk of reduced locomotion resulting from diseases such as osteoporosis. Although there are no previous reports of R. obtusifolius L., which is found throughout Japan, having roots containing nepodin, we found nepodin in the roots of this species. Therefore, R. obtusifolius as well as R. japonicus was considered a candidate raw material for nepodin extraction. We also discuss the suitability of R. japonicus and R. obtusifolius as sources of raw nepodin for cultivation on the Ryukyu Islands. In this study, all specimens on the Ryukyu Islands were identified as R. japonicus. Conversely, all specimens on mainland Japan were R. obtusifolius. The DNA sequence of the chloroplast trnL-trnF intergenic spacer region and partial nuclear internal transcribed spacer was consistent with the identification of R. japonicus and R. obtusifolius by morphological characteristics of the perianth segments. Therefore, to avoid erroneous identification and misuse of the plant species used for extraction of raw materials, it is preferable to develop DNA markers for these two regions. The content of nepodin varied from undetectable to 0.34% of the fresh weight (%FW) in R. japonicus and from undetectable to 0.21%FW in R. obtusifolius. From a pharmacological perspective, as plants that might be suitable as raw materials for nepodin extraction, it became clear that both R. japonicus and R. obtusifolius can be used with the same expected extraction efficiency. Based on our findings, R. obtusifolius could not be confirmed as inhabiting the Ryukyu Islands. For this reason, to conserve the endemic genetic characteristics of the Ryukyu Islands and to prevent genetic pollution by R. obtusifolius, only R. japonicus should be cultivated on the Ryukyu Islands.

Preliminary survey of taxonomical problems, pharmacognostical characteristics, and chloroplast DNA polymorphisms of the folk medicinal herb Artemisia campestris from the Ryukyu Islands, Japan.

Artemisia campestris L. (Compositae) occurs naturally along the coastline of the Ryukyu Islands and has been traditionally used as a folk medicine for the treatment of liver and kidney disorders. The authors obtained specimens from the Ishigaki and Kume Islands of the Ryukyu Islands, Japan, and from the USA. A survey of the literature revealed that the Japanese name for A. campestris is Niitaka-yomogi or Riukiu-yomogi. Two distinct overall plant-form phenotypes were identified: an erect phenotype with long, upright, and straight main axis and assurgent branches; and a prostrate phenotype, having branches that are longer than the main axis and which grow along the ground. Except for the number of ray flowers, most of the flower head characters in the erect phenotypes were significantly larger than those in the prostrate phenotypes. In this experiment, the flower heads contained only small amounts of either capillarisin (<0.01-0.11 of the dry weight, % DW) and 6,7-dimethylesculetin (<0.01-0.30% DW), or none at all. DNA polymorphisms at two sites of the rpl16-rpl14 spacer region (nucleotide position 181-189 and 291-300 from the 5' end) revealed the existence of four different haplotypes. The number of adenines at nucleotide positions 291-300 appeared to be polymorphic within A. campestris from the Ryukyu Islands. Conversely, geographic differences between specimens from the Ryukyu Islands and USA manifested as a nine-base deletion at nucleotide positions 181-189. From a pharmacognostical context, the use of A. campestris flower heads as a substitute for Artemisiae capillaris Flos is not effective.

Identification of Curcuma plants and curcumin content level by DNA polymorphisms in the trnS-trnfM intergenic spacer in chloroplast DNA.

With the goal of developing an accurate plant identification method, molecular analysis based on polymorphisms of the nucleotide sequence of chloroplast DNA (cpDNA) was performed in order to distinguish four Curcuma species: C. longa, C. aromatica, C. zedoaria, and C. xanthorrhiza. Nineteen regions of cpDNA were amplified successfully via polymerase chain reaction (PCR) using total DNA of all Curcuma plants. Using the intergenic spacer between trnS and trnfM (trnSfM), all four Curcuma plant species were correctly identified. In addition, the number of AT repeats in the trnSfM region was predictive of the curcumin content in the rhizome of C. longa.

The sequences of the plastid gene rpl16 and the rpl16-rpl14 spacer region allow discrimination among six species of Scutellaria.

Dried aerial parts of Scutellaria galericulata L. (Labiatae; marsh skullcap) and Scutellaria lateriflora L. (Labiatae; mad dog skullcap) are mainly used as skullcap, a medicinal herb, in Europe and the United States. The respective dried aerial parts of the two species are difficult to distinguish morphologically from each other. We attempted to discriminate among six species (Scutellaria altissima L. (Labiatae), Scutellaria baicalensis Georgi (Labiatae), Scutellaria galericulata, Scutellaria incana Spreng. (Labiatae), Scutellaria indica L. (Labiatae) and Scutellaria lateriflora) of Scutellaria, which include three medicinal species (Scutellaria galericulata, Scutellaria lateriflora and Scutellaria baicalensis), by comparing the respective nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparisons of these sequences allowed us to identify each of the six species unequivocally.