Chemical Relationship among Genetically Authenticated Medicinal Species of Genus Angelica.

The genus Angelica comprises various species utilized for diverse medicinal purposes, with differences attributed to the varying levels or types of inherent chemical components in each species. This study employed DNA barcode analysis and HPLC analysis to genetically authenticate and chemically classify eight medicinal Angelica species (n = 106) as well as two non-medicinal species (n = 14) that have been misused. Nucleotide sequence analysis of the nuclear internal transcribed spacer (ITS) region revealed differences ranging from 11 to 117 bp, while psbA-trnH showed variances of 3 to 95 bp, respectively. Phylogenetic analysis grouped all samples except Angelica sinensis into the same cluster, with some counterfeits forming separate clusters. Verification using the NCBI database confirmed the feasibility of species identification. For chemical identification, a robust quantitative HPLC analysis method was developed for 46 marker compounds. Subsequently, two A. reflexa-specific and seven A. biserrata-specific marker compounds were identified, alongside non-specific markers. Moreover, chemometric clustering analysis reflecting differences in chemical content between species revealed that most samples formed distinct clusters according to the plant species. However, some samples formed mixed clusters containing different species. These findings offer crucial insights for the standardization and quality control of medicinal Angelica species.

Chemotaxonomic Classification of Peucedanum japonicum and Its Chemical Correlation with Peucedanum praeruptorum, Angelica decursiva, and Saposhnikovia divaricata by Liquid Chromatography Combined with Chemometrics.

The roots of Peucedanum japonicum (Apiaceae) have been used as an alternative to the roots of Saposhnikovia divaricata (Apiaceae) to treat common cold-related symptoms in Korea. However, a variety of Peucedanum species, including the roots of P. praeruptorum or Angelica decursiva (=P. decursivum), have been used to treat phlegm-heat-induced symptoms in China. Hence, as there are differences in the medicinal application of P. japonicum roots between Korea and China, chemotaxonomic classification of P. japonicum was evaluated. Sixty samples derived from P. japonicum, P. praeruptorum, A. decursiva, and S. divaricata were phylogenetically identified using DNA barcoding tools, and chemotaxonomic correlations among the samples were evaluated using chromatographic profiling with chemometric analyses. P. japonicum samples were phylogenetically grouped into the same cluster as P. praeruptorum samples, followed by S. divaricata samples at the next cluster level, whereas A. decursiva samples were widely separated from the other species. Moreover, P. japonicum samples showed higher chemical correlations with P. praeruptorum samples or A. decursiva samples, but lower or negative chemical correlations with S. divaricata samples. These results demonstrate that P. japonicum is more genetically and chemically relevant to P. praeruptorum or A. decursiva and, accordingly, the medicinal application of P. japonicum might be closer to the therapeutic category of these two species than that of S. divaricata.

Quantitative Comparison of the Marker Compounds in Different Medicinal Parts of Morus alba L. Using High-Performance Liquid Chromatography-Diode Array Detector with Chemometric Analysis.

It is thought that the therapeutic efficacy of Morus alba L. is determined by its biological compounds. We investigated the chemical differences in the medicinal parts of M. alba by analyzing a total of 57 samples (15 root barks, 11 twigs, 12 fruits, and 19 leaves). Twelve marker compounds, including seven flavonoids, two stilbenoids, two phenolic acids, and a coumarin, were quantitatively analyzed using a high-performance liquid chromatography-diode array detector and chemometric analyses (principal component and heatmap analysis). The results demonstrated that the levels and compositions of the marker compounds varied in each medicinal part. The leaves contained higher levels of six compounds, the root barks contained higher levels of four compounds, and the twigs contained higher levels of two compounds. The results of chemometric analysis showed clustering of the samples according to the medicinal part, with the marker compounds strongly associated with each part: mulberroside A, taxifolin, kuwanon G, and morusin for the root barks; 4-hydroxycinnamic acid and oxyresveratrol for the twigs and skimmin; chlorogenic acid, rutin, isoquercitrin, astragalin, and quercitrin for the leaves. Our approach plays a fundamental role in the quality evaluation and further understanding of biological actions of herbal medicines derived from various medicinal plant parts.

Chemotaxonomic Monitoring of Genetically Authenticated Amomi Fructus Using High-Performance Liquid Chromatography-Diode Array Detector with Chemometric Analysis.

Amomi Fructus is widely used to treat digestive disorders, and Amomum villosum, A. villosum var. xanthioides, and A. longiligulare are permitted medicinally in national pharmacopeias. However, there are a variety of adulterants present in herbal markets owing to their morphological similarities to the genuine Amomum species. Forty-two Amomi Fructus samples from various origins were identified using internal transcribed spacer and chloroplast barcoding analyses, and then their chromatographic profiles were compared using chemometric analysis for chemotaxonomic monitoring. Among the Amomi Fructus samples, A. villosum, A. longiligulare, A. ghaticum, and A. microcarpum were confirmed as single Amomum species, whereas a mixture of either these Amomum species or with another Amomum species was observed in 15 samples. Chemotaxonomic monitoring results demonstrated that two medicinal Amomum samples, A. villosum and A. longiligulare, were not clearly distinguished from each other, but were apparently separated from other non-medicinal Amomum adulterants. A. ghaticum and A. microcarpum samples were also chemically different from other samples and formed their own species groups. Amomum species mixtures showed diverse variations of chemical correlations according to constituent Amomum species. Genetic authentication-based chemotaxonomic monitoring methods are helpful in classifying Amomi Fructus samples by their original species and to distinguish genuine Amomum species from the adulterants.

DNA Markers to Discriminate Cannabis sativa L. 'Cheungsam' with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes.

Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. Therefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. Thus, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar "Cheungsam" (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars.

Identification and Monitoring of Amomi Fructus and its Adulterants Based on DNA Barcoding Analysis and Designed DNA Markers.

Amomi Fructus is one of the traditional medicines derived from the ripe fruits of the Zingiberaceae family of plants, which include Amomum villosum, A. villosum var. xanthioides, and A. longiligulare. Owing to their highly similar morphological traits, several kinds of adulterants of Amomi Fructus have been reported. Therefore, accurate and reliable methods of identification are necessary in order to ensure drug safety and quality. We performed DNA barcoding using five regions (ITS, matK, rbcL, rpoB, and trnL-F intergenic spacer) of 23 Amomi Fructus samples and 22 adulterants. We designed specific DNA markers for Amomi Fructus based on the single nucleotide polymorphisms (SNPs) in the ITS. Amomi Fructus was well separated from the adulterants and was classified with the species of origin based on the detected SNPs from the DNA barcoding results. The AVF1/ISR DNA marker for A. villosum produced a 270 bases amplified product, while the ALF1/ISF DNA marker produced a 350 bases product specific for A. longiligulare. Using these DNA markers, the monitoring of commercially distributed Amomi Fructus was performed, and the monitoring results were confirmed by ITS analysis. This method identified samples that were from incorrect origins, and a new species of adulterant was also identified. These results confirmed the accuracy and efficiency of the designed DNA markers; this method may be used as an efficient tool for the identification and verification of Amomi Fructus.

Quantitative Interrelation between Atractylenolide I, II, and III in Atractylodes japonica Koidzumi Rhizomes, and Evaluation of Their Oxidative Transformation Using a Biomimetic Kinetic Model.

Analytical methods based on ultraperformance liquid chromatography/ion-trap mass spectrometry (UPLC/ion-trap MS) were developed for quantification of atractylenolide I, II, and III in the methanol extract of Atractylodes japonica rhizomes with a C18 column in an acidified water/acetonitrile gradient eluent in an LC system, and ion-trap MS coupled with electrospray ionization was employed under positive-ion mode. The three atractylenolides were quantified in all A. japonica samples, and the content of atractylenolide I, II, and III showed a significant correlation to each other. Such high correlation was explained by the mechanistic insights into the biosynthetic pathway of atractylenoide III and I from atractylenoide II by using the biomimetic cytochrome P450 model, [Fe(tmp)](CF3SO3) (tmp = meso-tetramesitylporphyrin). Atractylenolides could be transformed by oxidation via the oxidative enzyme in the A. japonica plant. The present study first reports the first oxidative transformation of atractylenolides using the heme iron model complex.

Chemical Differentiation of Genetically Identified Atractylodes japonica, A. macrocephala, and A. chinensis Rhizomes Using High-Performance Liquid Chromatography with Chemometric Analysis.

The rhizome of Atractylodes japonica, which is a herbal medicine used for gastrointestinal therapeutics, has been categorized with A. macrocephala rhizome or A. chinensis rhizome based on different therapeutic criteria in Korea, China, and Japan. In the present study, 61 A. japonica, A. macrocephala, and A. chinensis rhizomes were collected from Korea and China and were genetically identified by internal transcribed spacer sequencing analysis. Chromatographic profiles were obtained from high-performance liquid chromatography analysis of the methanol and hot-water extracts of Atractylodes rhizomes and chemical differentiation of the rhizomes was carried out using chemometric statistical analyses such as principal component analysis, hierarchical clustering analysis, and Pearson's correlation coefficient analysis. The results from chromatographic profiles and chemometric analyses demonstrate that A. japonica rhizomes showed apparent chemical differences from A. macrocephala and A. chinensis rhizomes in the methanol extracts. In contrast, no clear distinction was apparent for the hot-water extracts of Atractylodes rhizomes, especially A. chinensis rhizomes. These results indicate that there is a clear chemical difference between A. japonica and A. macrocephala rhizomes; however, the chemical diversity of A. chinensis rhizome shows different chemical relationships with A. japonica or A. macrocephala rhizome, dependent on the chemical features.

Identification and monitoring of Korean medicines derived from Cinnamomum spp. by using ITS and DNA marker.

In this study, we identified and evaluated the genetic relationships among Cinnamomum plants, which are used in traditional medicine. We also attempted to monitor the distribution of traditional medicines derived from Cinnamomum cassia by using DNA barcoding and a species-specific DNA marker. Plants of the genus Cinnamomum, and in particular C. cassia, are commonly used as medicinal herbs in the form of Cinnamomi Ramulus, Cinnamomi Cortex, and Cassiae Cortex Interior. However, it is difficult to distinguish among different Cinnamomum species based on morphological features, and so to overcome this limitation, nucleotide sequences of the internal transcribed spacer (ITS) region of Cinnamomum DNA were determined and compared. On the basis of the discrepancy in determined ITS sequences, a 408-bp product, amplified by the primer pair CC F1/CC R3, was developed as a C. cassia-specific DNA marker. Using the developed DNA marker in combination with the ITS 2 nucleotide sequence, we monitored imported and commercially supplied medicinal products derived from Cinnamomum plants in markets in Korean, China, and Japan. The results revealed that most of the specimens monitored were derived from C. cassia.

Application of Partial Internal Transcribed Spacer Sequences for the Discrimination of Artemisia capillaris from Other Artemisia Species.

Several Artemisia species are used as herbal medicines including the dried aerial parts of Artemisia capillaris, which are used as Artemisiae Capillaris Herba (known as "Injinho" in Korean medicinal terminology and "Yin Chen Hao" in Chinese). In this study, we developed tools for distinguishing between A. capillaris and 11 other Artemisia species that grow and/or are cultured in China, Japan, and Korea. Based on partial nucleotide sequences in the internal transcribed spacer (ITS) that differ between the species, we designed primers to amplify a DNA marker for A. capillaris. In addition, to detect other Artemisia species that are contaminants of A. capillaris, we designed primers to amplify DNA markers of A. japonica, A. annua, A. apiacea, and A. anomala. Moreover, based on random amplified polymorphic DNA analysis, we confirmed that primers developed in a previous study could be used to identify Artemisia species that are sources of Artemisiae Argyi Folium and Artemisiae Iwayomogii Herba. By using these primers, we found that multiplex polymerase chain reaction (PCR) was a reliable tool to distinguish between A. capillaris and other Artemisia species and to identify other Artemisia species as contaminants of A. capillaris in a single PCR.

Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools.

Atractylodes rhizomes have been used as the herbal medicine "Changchul" or "Baekchul," according to their clinical purpose, in Korea, China, and Japan. Among the Atractylodes species, the medicinal use of Atractylodes japonica has been controversial, as it is categorized as both Changchul and Baekchul in those countries, and, moreover, parts of the rhizome have been differently used, depending on age of the plant, in Korea. Chromatographic fingerprinting by using HPLC combined with chemometric analyses and internal transcribed spacer (ITS) sequencing analysis were conducted to classify and identify 34 crude drugs derived from Atractylodes rhizomes. The identification of the samples, authenticated by their morphological features as A. japonica Koidz. (Changchul and Baekchul), A. chinensis Koidz., and A. macrocephala Koidz., was confirmed as A. japonica, A. chinensis, and A. macrocephala by ITS sequencing. The results from chemometric analyses showed that the chemical components of the crude drugs from A. japonica were significantly different from those from A. macrocephala but were similar to those from A. chinensis. The analyses also suggested that the categorization by age of A. japonica as Changchul or Baekchul is not recommended. The results indicate that A. japonica should be categorized as "Changchul" and should not be further categorized by age.

Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants.

Some plants in the genus Artemisia have been used for medicinal purposes. Among them, Artemisia iwayomogi, commonly referred to as "Haninjin," is one of the major medicinal materials used in traditional Korean medicine. By contrast, Artemisia capillaris and both Artemisia argyi and Artemisia princeps, referred to as "Injinho" and "Aeyup," respectively, are used to treat diseases different from those for which "Haninjin" is prescribed. Therefore, the development of a reliable method to differentiate each Artemisia herb is necessary. We found that a random amplified polymorphic DNA (RAPD) method can be used to efficiently discriminate a few Artemisia plants from one another. To improve the reliability of RAPD amplification, we designed primer sets based on the nucleotide sequences of RAPD products to amplify a sequence-characterized amplified region (SCAR) marker of A. iwayomogi. In addition, we designed two other primer sets to amplify SCAR markers of "Aeyup" (A. argyi and A. princeps) along with "Injinho" (A. capillaris) and Artemisia japonica, which are also traded in Korean herbal markets. Using these three primer sets, we developed a multiplex PCR method concurrently not only to discriminate A. iwayomogi from other Artemisia plants, but also to identify Artemisia plants using a single PCR process.

Application of the multiplex PCR method for discrimination of Artemisia iwayomogi from other Artemisia herbs.

Some plants classified in the genus Artemisia are used for medicinal purposes. In particular, A. iwayomogi, which is referred to as 'Haninjin,' is used as an important medicinal material in traditional Korean medicine. However, A. capillaris, and both A. argyi and A. princeps, referred to as 'Injinho' and 'Aeyup,' respectively, are used for purposes other than those for which 'Haninjin' is utilized. However, it is occasionally difficult to differentiate 'Haninjin' from 'Injinho' and/or 'Aeyup' on the basis of their morphological features, particularly when in the dried and/or sliced form. Therefore, the development of a reliable method by which to discriminate 'Haninjin' from other Artemisia herbs, especially 'Injinho' and 'Aeyup,' is clearly necessary. We recently determined that the RAPD (random amplified polymorphic DNA) technique can be used to discriminate efficiently between some Artemisia herbs. In particular, when applied to RAPD, the non-specific UBC primer 391 (5'-GCG AAC CTC G-3') was demonstrated to amplify PCR products specific to A. iwayomogi. Based on the nucleotide sequences of the PCR product, we designed a 2F1 (5'-ACC TCG GAC CTA AAT ACA-3')/ 2F3 (5'-TTA TGA TTC ATG TTC AAT TC-3') primer set to amplify a SCAR (sequence-characterized amplified region) marker of A. iwayomogi. Employing this primer set, along with two other primer sets amplifying SCAR markers of 'Aeyup' (A. argyi and A. princeps) and both 'Injinho' (A. capillaris) and A. japonica, which are classified into the same subgroup in a phenogram constructed from RAPD analysis, we developed a multiplex PCR method by which A. iwayomogi could be discriminated with certainty from other Artemisia herbs. Via this method, we determined not only whether the tested Artemisia herb was A. iwayomogi, but also which Artemisia herbs were tested concurrently with A. iwayomogi.

Development of SCAR marker for discrimination of Artemisia princeps and A. argyi from other Artemisia herbs.

Some Artemisia herbs are used for medicinal purposes. In particular, A. princeps and A. argyi are classified as 'Aeyup' and are used as important medicinal material in traditional Korean medicine. On the other hand, A. capillaris and A. iwayomogi, which are classified as 'Injinho' and 'Haninjin', respectively, are used for other purposes distinct from those of 'Aeyup'. However, sometimes 'Aeyup' is not clearly discriminated from 'Injinho' and/or 'Haninjin'. Furthermore, Artemisia capillaris and/or A. iwayomogi have been used in place of A. princeps and A. argyi. In this study, we developed an efficient method to discriminate A. argyi and A. princeps from other Artemisia plants. The RAPD (random amplified polymorphic DNA) method efficiently discriminated various Artemisia herbs. In particular, non-specific primer 329 (5'-GCG AAC CTC C-3'), which shows polymorphism among Artemisia herbs, amplified 838 bp products, which are specific to A. princeps and A. argyi only. Based on nucleotide sequence of the primer 329 product, we designed a Fb (5'-CAT CAA CCA TGG CTT ATC CT-3') and R7 (5'-GCG AAC CTC CCC ATT CCA-3') primer-set to amplify a 254 bp sized SCAR (sequence characterized amplified regions) marker, through which A. princeps and A. argyi can be efficiently discriminated from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi.