Currently, Japan faces the issue of a super-aging society and consequently aging diseases, syndromes, and conditions such as dementia, sarcopenia, and frailty, and also multifactorial diseases such as life-style-related diseases are increasing. Accordingly, the Japanese Government advocated the National Healthcare Promotion Movement in the twenty-first century [1] with the title of “Health Japan 21,” with aims including extending healthy life expectancy, reducing health disparities, and preventing onset and progression of life-style-related diseases. Japanese traditional medicine (Kampo) has been explored as a potential solution for aging-related diseases, early intervention in pre-symptomatic conditions, and addressing the problem of polypharmacy. Thus, with increased demand for Kampo formulas, their crude drug ingredients must also be supplied. Moreover, sustainable use of crude drugs is fundamentally important in the world. Recently, due to the effects of global climate change and anthropogenic disasters, natural resources have been depleting in many countries, including China. Of the amounts of crude drugs used in Japan, 82.7% are imported from China, and only 10.3% are domestic production [2]. Therefore, medicinal resources of wild and cultivated plants in China must receive close attention. In China, 3000 among 35,000 higher plant species are listed as endangered and 60–70% of endangered plants are used as medicines. Since 2000, the Chinese Government has controlled the collection and export of wild Ephedra and Glycyrrhiza plants, which are the botanical origins of indispensable crude drugs, ephedra herb and glycyrrhiza, to prevent desertification in northern China [3]. Expansion of cultivation is thought to be the best way to resolve this situation; however, problems such as non-conforming quality of cultivated plants as well as mismatching environmental conditions makes this difficult. Even in China, a half of crude drugs prescribed in the Chinese Pharmacopoeia are derived from wild plants [4].

Here, a strategy for sustainable use of crude drugs is considered as follows: (1) construct a management plan for collection and preservation of medicinal plants at a national level; (2) develop alternative crude drug resources to construct a circulating system of crude drugs in Asia; (3) efficient usage of a crude drug with different botanical origins of the same crude drug name; (4) cultivation of medicinal plants, through the process including proposal for suitable species/strains for cultivation such as value-added medicinal plants, establishing effective and efficient cultivation methods, establishing post-harvest processing and preparation methods, and constructing a comprehensive cycle system from production to consumption.

Concerning the above items, except for the first which is a regulatory concern, our group has conducted comprehensive studies, including field investigation on medicinal plants and traditional medicines, and molecular systematic, chemical, and pharmacological analyses on various crude drugs and their related plants. Clearly, it is necessary to maintain safety and efficacy of crude drugs as well as sustainable uses, and to standardize crude drugs through limiting the acceptable range of diversity for quality assurance purposes. This review introduces some of our studies concerning the above strategies.

Expanding cultivation in Japan: genetic and chemical diversity of Paeonia lactiflora and development of brand peony root

Peony root (Paeoniae Radix) has been widely used as an antispasmodic, analgesic, or astringent in Kampo [5]. It is prescribed as the root of Paeonia lactiflora Pallas with no less than 2.0% of paeoniflorin in the Japanese Pharmacopoeia (JP) [6]. Peony root available in the Japanese market (PR, hereafter referring only to crude drugs in the Japanese market) is mainly imported from China and only 2.3% is produced domestically [2]. In China, there are two kinds of peony roots, white peony root (WPR) and red peony root (RPR), which are used for different remedies such as relieving cramps or pains and improving blood stasis or gynecological disease, respectively [5]. The WPR is prescribed as the dried root of P. lactiflora which has been boiled and peeled before drying, while RPR is prescribed as the dried root of P. lactiflora or P. veitchii Lynch in the Chinese Pharmacopoeia (CP) [7]. Most of RPR in the markets is derived from P. lactiflora. Therefore, the difference between WPR and RPR with the same botanical origin has been the object of discussion for many years. First, to clarify genetic and chemical differences between the two, the nucleotide sequences of nrDNA internal transcribed spacer (ITS) and contents of eight main constituents were analyzed for specimens of P. lactiflora, P. veitchii, P. anomala Linn., and P. japonica Miyabe et Takeda (Table S1), and commercial samples of WPR and RPR available in the Chinese market and those of PR in the Japanese market (Table S2). Second, using the same methods, 81 cultivars cultivated in the Toyama Prefectural Medicinal Plants Center (Table S3) were analyzed and selection performed to produce value-added cultivars to build brand peony root.

Genetic diversity of Paeonia species and peony root

The ITS sequences of specimens of the four Paeonia species as well as crude drug samples were of the same length, in which ITS1, 5.8S rRNA gene, and ITS2 regions were 267, 164, and 221 bp, respectively. Almost all the samples of WPR, RPR, and PR were identified as P. lactiflora, except for a few RPR samples from Sichuan Province that were identified as P. veitchii [8]. Significant intra-species polymorphism of the ITS sequences was detected within P. lactiflora. Clustering analysis on the basis of sequence similarity showed that P. lactiflora formed a large group distinct from P. veitchii, P. anomala, P. japonica, and P. suffruticosa Andrews (Fig. 1). Within the P. lactiflora group, there were two main subgroups: I and II. The P. lactiflora plants cultivated in southern China and Chinese WPR produced in Anhui, Zhejiang, and Sichuan provinces, as well as Japanese PR including Japanese medicinal cultivars “Bonten (S34)” and “Kitasaisho (S31),” belonged to subgroup I (WPR-type); whereas, P. lactiflora plants growing wild in northern China (P1, P2) and Mongolia (P3), and Chinese RPR (D12-D15) fell into subgroup II (RPR-type). Most horticultural varieties from Japan belonged to subgroup II.

Fig. 1

Clustering analysis based on the similarity of ITS sequence. D: Crude drug sample; sky blue in color: Chinese WPR, red color: Chinese RPR, purple color: Japanese PR. P: Plant specimen, S: Cultivar (S31: Kitasaisho, S34: Bonten)

The ITS sequences deposited in the International Nucleotide Sequence Database included two main types, differing by three nucleotides at positions 69, 458, and 523: type 1 showed three cytosines (C–C–C; U27682) [9], while type 2 had thymine (T), adenine (A), and T (T–A–T; JN572150) [10] at the three sites. According to the nucleotides at these three sites, crude drug samples and plants were divided into two main groups corresponding to the two subgroups (Fig. 1). One group showed T69-A458-T523 at the three sites, which was the same as for type 2. The other group showed additive nucleotides (double nucleotides detected at the same site) of Y (T & C), M (A & C), and Y at the three sites (Y69-M458-Y523), respectively. The two Japanese medicinal cultivars showed a sequence identical to a type of sequence from WPR (AB920144) of subgroup I. The five PR samples produced in Nara Prefecture (D7, D9, D10, D42, and D44), usually called “Yamato Shakuyaku,” were divided into two subgroups: I and II. Moreover, the PR from Niigata Prefecture (D6) was a mixture of individuals belonging to two subgroups, indicating Japanese PR was not only derived from medicinal cultivars of P. lactiflora.

Chemical diversity of Paeonia species and peony root

The contents of eight components—paeoniflorin (P1), albiflorin (P13), 1,2,3,4,6-penta-O-galloyl-β-d-glucose (P19), (+)-catechin (P20), paeonol (P21), gallic acid (P22), methylgallate (P23), and benzoic acid (P24)—were quantitatively analyzed to clarify chemical properties of P. lactiflora, P. veitchii, P. anomala, and the crude drug samples including WPR, PR, and RPR (Fig. 2). Within the commercial samples derived from P. lactiflora, RPR samples produced in northern China had obviously higher contents of P1, P20, and P21 but lower content of P13 compared to WPR produced in southern China and most PR produced in Japan [8] (Fig. 3). Among 11 WPR samples, eight contained less than the 2.0% of paeoniflorin specified in the JP. Comparing high-performance liquid chromatography (HPLC) chromatograms of these samples with those of the PR collected from Japanese markets, commonly showed a conspicuous peak at retention time around 10.4 min. Further analysis using liquid chromatography (LC)/mass spectrometry (MS) clearly indicated that this notable peak was paeoniflorin sulfonate (P2), which could be produced by traditional processing with sulfur fumigation [11]. This indicated that the WPR available in Chinese markets was usually processed by sulfur fumigating, resulting in an extremely low P1 content; whereas the PR available in the Japanese market was not treated with this process. Apart from the WPR samples in which P2 was detected, quantitative data of the six constituents excluding P23 and P24 in Paeonia specimens and commercial samples was subjected to principal component analysis (PCA) and the PCA score plot showed four separate clusters of all samples. Besides the respective groups of P. veitchii and P. anomala, samples derived from P. lactiflora were clearly classified into two groups: one group included RPR (RPR group) and the other group was composed of WPR, PR produced in China, and most PR produced in Japan (WPR/PR group). The former was characterized by high P21, P20, and P1 contents, and the latter by a relatively high content of P13. Moreover, the P. veitchii group was far from the other groups and had high contents of P1, P19, and P22. The grouping in the PCA score plot was in accordance with clustering based on the similarity of ITS sequences. This result indicated that WPR and RPR were not only geographically isolated, but also genetically and chemically separated.

Fig. 2

Structures of reference standard compounds. P1, paeoniflorin; P2, paeoniflorin sulfonate; P3, 4-O-methylpaeoniflorin; P4, salicylpaeoniflorin; P5, benzoylpaeoniflorin; P6, mudanpioside C; P7, galloylpaeoniflorin; P8, mudanpioside J; P9, oxypaeoniflorin; P10, benzoyloxypaeoniflorin; P11, 6’-O-vanillyloxypaeoniflorin; P12, mudanpioside E; P13, albiflorin; P14, 4-epi-albiflorin; P15, paeonivayin; P16, paeoniflorol; P17, 4’-hydroxypaeoniflorigenone; P18, lactiflorin; P19, 1,2,3,4,6-penta-O-galloyl-β-d-glucose; P20, (+)-catechin; P21, paeonol; P22, gallic acid; P23, methyl gallate; P24, benzoic acid; P25, paeonolide; P26, paeonibenzofuran; P27, quercetin; P28, quercetin-3-O-β-d-glucopyranoside; P29, (2R)-(-)-naringenin-7-O-β-d-glucopyranoside

Fig. 3

Contents of 8 components in the different types of peony root and the roots of four related species including P. lactiflora, P. veitchii, P. anomala and P. japonica. D: crude drug sample; +: samples processed by sulfur-fumigation. P: plant specimens; P9, P1 and P7 were collected in Zhejiang, Inner Mongolia and Sichuan, respectively. Contents of P1 and P13 are shown in upper part and those of P19-P24 are shown in lower part

In addition, monoterpenoid profiling was performed using LC coupled with ion trap and time-of-flight MS (LC–IT-TOF-MS) to precisely characterize and quantify different types of peony root and the roots of related Paeonia species. The MS/MS fragmentation patterns of monoterpenoids with paeoniflorin (PF)-, albiflorin (AF)-, and sulfonated paeoniflorin (PFS)-type of skeletons were elucidated, which provided basic clues enabling subsequent identification of 35 monoterpenoids in LC–MS profiles of Paeonia species (Fig. 2). Mudanpioside C (P6) was the characteristic component of P. lactiflora, and 4-O-methyl-paeoniflorin (P3) was only detected in P. veitchii and P. anomala. Notably, six PFS-type monoterpenoids were detected in sulfur-fumigated WPR samples [12]. Quantification of 15 main monoterpenoids including 10 PF-type (P1, P3–P10, and P12), three AF-type (P13–P15), and two other types, a new compound paeoniflorol (P16) and lactiflorin (P18), in 56 samples revealed that five monoterpenoids [P1, benzoylpaeoniflorin (P5), galloylpaeoniflorin (P7), oxypaoniflorin (P9), and P13] predominated in all samples, but with quite different relative contents. Of the samples derived from P. lactiflora, RPR samples showed higher contents of five PF-type monoterpenoids [salicylpaeoniflorin (P4), P6, mudanpioside J (P8), P9, and benzoyloxypaeoniflorin (P10)] as well as P1 compared to WPR/PR, but comparatively lower contents of AF-type monoterpenoids [a new compound, 4-epi-albiflorin (P14), paeonivayin (P15), and P13] (Fig. 4). For the two other types of monoterpenoids, P16 had a high content in RPR, whereas P18 had a high content in WPR and PR. The above differences in monoterpenoid profiles between WPR and RPR might be some of the reasons for their different traditional uses and different bioactivities including anti-allergic effects. The samples derived from P. veitchii had the highest P4 and P7 contents, very low P9 and P10 contents, and no P6, and clearly differed from those derived from other sources [12].

Fig. 4

Contents of 11 monoterpenoids in the different types of peony root and the roots of four related species including P. lactiflora, P. veitchii, P. anomala and P. japonica. A and B: Contents of peoniflorin-type monoterpenoids, P5, P7, P9 and P12 in A, and P3, P4, P6, P8 and P10 in B; C: Contents of albiflorin-type monoterpenoids, P13–P15. *: plant specimens; +: samples processed by sulfur-fumigation

The same analytical methods as peony root were applied to two medicinal and 61 horticultural cultivars of P. lactiflora, which were genetically of RPR- and WPR-types. There were high similarities between RPR- and WPR-types of cultivars in regard to the eight main constituents (Fig. S1) [13]. Moreover, monoterpenoid composition of the respective P. lactiflora cultivars showed high similarity regardless of RPR- or WPR-type (Fig, 4) [12], differing from the situation between commercial WPR and RPR.

Anti-allergic activity of peony root and P. lactiflora horticultural cultivars and their active compounds

Because several studies have reported anti-allergic activity of peony root [14], a bioactivity screening experiment concerning the inhibitory effect against immunoglobulin E (IgE)-mediated mast cell degranulation was performed to compare the activity of RPR from the Inner Mongolia Autonomous Region of China and PR from Japan and to determine a characteristic cultivar with anti-allergic activity among 17 horticultural cultivars. Among them, a RPR sample and two cultivars, “Edulis Superba (ES)” and “Harunoyosooi (HY),” significantly inhibited β-hexosaminidase release stimulated by 2,4-dinitrophenylated bovine serum albumin on IgE-sensitized rat basophil leukemia-2H3 cells at a concentration of 1.0 mg/mL. In order to elucidate the active compounds, bioassay-guided fractionations were subsequently conducted on the RPR sample and cultivar ES, and anti-allergic activities of isolated compounds were, respectively, examined. From the 60% and 80% aqueous methanol subfractions of methanol extract of RPR sample that showed activity, 29 compounds were isolated and identified as three new monoterpenoids [P14, P16, and 4′-hydroxypaeoniflorigenone (P17)], 14 known monoterpenoids, five flavonoids, and seven other types of compounds [15]. In the same way, 26 compounds were isolated from the ethyl acetate- and n-butanol-soluble fractions of methanol extract of cultivar ES, including one new norneolignan, paeonibenzofuran (26), five monoterpenoids, 10 flavonoids, and 10 other types of compounds [16]. Among compounds isolated from the RPR sample, nine monoterpenoids (P1, P4, P5, P7, P8, P10, P11, P16, and P18), as well as P19 and P23, showed moderate anti-allergic activities (Table S4). Among monoterpenoids, a new compound P16 exhibited the greatest effectiveness (IC50 41.17 μM), followed by P4 and P7. However, AF-type monoterpenoids had no activity [15]. Moreover, mudanpioside E (P12), quercetin (P27), and quercetin-3-O-β-d-glucopyranoside (P28) isolated from cultivar ES, showed potent inhibitory activity (IC50 40.34, 25.05, and 42.55 μM, respectively), followed by paeonolide (P25), (2R)-(-)-naringenin-7-O-β-d-glucopyranoside (P29), and P26 [16]. Based on these experimental results, two horticultural cultivars with moderate anti-allergic activity were selected as promising candidates with potential as medicinal resources of RPR, as well as the active components being clarified.

Development of brand peony root in Toyama―selection from horticultural cultivars and establishment of appropriate post-harvest processing methods

A medicinal cultivar “Bonten (BT)” belonging to WPR-type has been widely cultivated in Toyama Prefecture. To find new resources of peony root of RPR-type from P. lactiflora cultivars, with both horticultural and medicinal utilities, PCA was performed on quantitative data of six constituents (P1, P13, and P19–P22) from 63 P. lactiflora cultivars added to those from commercial samples except P2-containing samples, and Paeonia specimens. In the PCA score plot, samples and specimens derived from P. lactiflora were clearly classified into two groups: RPR and WPR/PR [13]. Three horticultural cultivars were included in the RPR group; the two cultivars ES and HY among them showed anti-allergic activity as described in the previous section, and were nominated as candidates for brand peony roots.

Next, with the aim to determine the influence of different post-harvest processing methods of boiling, peeling, drying, and storing on chemical composition and morphological features of the produced peony root and to establish an appropriate and practicable method for production of brand peony root with superior quality, 15 kinds of processing methods were applied to the 15 groups of fresh roots of cultivar BT after 4 years of cultivation. The roots produced were analyzed using HPLC and the contents of eight components (P1, P13, and P19–P24) and internal root color were compared (Fig. 5). The results showed that low temperature (4 °C) storage of fresh roots for approximately 1 month after harvest resulted in stable and high content of P1, possibly due to suppression of enzymatic degradation including enzymes involved in paeoniflorin hydrolysis [17]. This storage also prevented discoloration of roots and facilitated production of desirable bright color in roots, traditionally believed to be of high quality. In addition, quantitative 1H nuclear magnetic resonance (qHNMR) analysis demonstrated that sucrose content increased significantly in root after low-temperature storage for 1 month, resulting in increased ethanol extract yield [18]. Boiling treatment triggered decomposition of polygalloylglucoses and led to significantly increased contents of P19, P22, and P23, shown by monitoring gallotannin changes using LC-IT-TOF–MS. Peeling treatment resulted in decreased P13 content; additionally in the group without boiling, the P20 content also decreased. For roots washed and boiled after low-temperature storage, P19 content was slightly higher for no peeling compared to peeling treatment, and for drying at 30 °C using a drying machine compared with indoor drying. As a result, the optimized processing method to produce high contents of main active components in the root was low-temperature storage for approximately 1 month after harvest, followed by washing, boiling, and drying at 30 °C with a drying machine [17]. Similar experiments to those described above were applied to two P. lactiflora cultivars: ES and HY. Following application of the optimized processing method, the main constituents were quantified and compared among three cultivars, with the order for levels of P1 and P13 of ES > HY > BT, and for P20 of HY > ES > BT. Moreover, P23 was found in cultivars ES and BT. Although P21 was not detected following the optimized processing roots of the three cultivars, ES and HY contained P21 in the dried roots treated without boiling. Using the above results, we selected suitable cultivars and processing methods. Recognizing the potential of these discoveries, a collaborative project between academia and local government is currently in progress in Toyama Prefecture to produce brand peony root.

Fig. 5

Contents of 7 components in the roots produced by 15 kinds of post-harvest processing methods. Average contents and S.D. of P1 and P13 are shown in upper part and average contents of P19, P20, P22–P24 are shown in lower part (n = 5). Photo of cross-sections of the roots treated with different processing methods is shown

Development of alternative crude drug resources: quality assessment of plant resources of glycyrrhiza, ephedra herb, and saposhnikovia root and rhizome from Mongolia

Around 3000 species of vascular plants are distributed in Mongolia: 812 species are efficacious medicinal plants, and 200 species have been used as formulaic ingredients in Mongolian traditional medicine [19]. Mongolian medicinal plants are also attractive as sources of crude drugs used in traditional Chinese medicine and Kampo, such as Glycyrrhiza uralensis Fischer, Ephedra sinica Stapf, E. equisetina Bunge, and Saposhnikovia divaricata (Turcz.) Schischk. Qualities of these plants were assessed in a collaborative study with Mongolian researchers to inform an ongoing conservation program for the efficient usage of medicinal plants by the Mongolian Government.

Resources of glycyrrhiza (Glycyrrhizae Radix)

For glycyrrhiza, one of the crude drugs with export restricted by the Chinese Government, to reveal chemical characteristics of G. uralensis growing in Mongolia, eight major bioactive constituents in the underground parts were quantitatively analyzed and compared with glycyrrhiza produced in China [20]. Most of the 15 specimens from eastern, southern, and western Mongolia contained 26.95–58.55 mg/g of glycyrrhizin, exceeding the criterion assigned in the JP [6], and was highest in the sample from Tamsagiyn Hooloy, Dornod Province, eastern Mongolia. The total content of three flavanones (liquiritin apioside, liquiritin, and liquiritigenin) was in the range of 3.00–26.35 mg/g and of three chalcones (isoliquiritin apioside, isoliquiritin, and isoliquiritigenin) was 1.13–10.50 mg/g. The content of glycyrrhizin and subtotal contents of flavanones and chalcones from Mongolian G. uralensis were obviously lower than those of the crude drugs available in the Japanese market derived from Chinese wild specimens, but higher than those derived from cultivated specimens in the Chinese market. Glycycoumarin, a constituent specific to G. uralensis, was detected in all Mongolian specimens and its content was higher in samples from Sergelen and Tamsagiyn Hooloy, which were comparable to that of Tohoku-kanzo in Japan derived from wild Chinese G. uralensis. Thus, Mongolian G. uralensis could be a glycyrrhiza resource and was generally of JP grade. However, the producing area should be taken into consideration for ensuring high quality.

Resources of ephedra herb (Ephedrae Herba)

After a field survey of Ephedra plants in Mongolia, molecular and chemical assessments on plant specimens were conducted to clarify whether they could be an alternative resource of ephedra herb used in Japan [21]. The distribution of E. sinica, E. equisetina, E. monosperma Gmelin ex C. A. Meyer, E. przewalskii Stapf, E. glauca Regel, E. regeliana Florin, E. lomatolepis Schrenk, and unknown Ephedra sp. (assumed to be E. dahurica Turz.) was confirmed. Among them, E. sinica and E. equisetina are prescribed as the botanical origin of ephedra herb in JP [6]. On the basis of nucleotide sequences of nuclear 18S rRNA gene and chloroplast trnK gene, E. sinica, E. equisetina, and E. monosperma presented completely identical sequences to the corresponding species from China. Since the population in southwestern Mongolia showed a high likelihood of hybrid origin, further sequence analysis of the partial nuclear ITS1 region was conducted to obtain detailed evidence of hybridization status as well as to elucidate the marker sequences of pure lines of each species [22]. As a result, the ITS1 sequences from all eight Ephedra species were roughly divided into five types: I–V. E. equisetina and E. monosperma had similar sequences (Type V), differing from other species. Although the remaining five species possessed similar sequences, they were divided into four types based on the nucleotides at four informative sites. Among them, type II sequences had additive nucleotides at four sites observed in E. sinica, Ephedra sp., E. glauca, and E. regeliana, which provided useful information for tracing hybrid origin. Morphological, genetic and distribution evidences suggested that hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii were involved in these events. Integrated with trnK-, 18S-, and ITS-sequence types, pure lines of each species were proposed. The pure line (type I [AB600683]) of E. sinica was only found in eastern areas, while E. equisetina, which consisted of pure line, was found in the mountainous region of central–western areas. Quantitative analysis of five ephedrine alkaloids [ephedrine (E1), pseudoephedrine (E2), norephedrine, norpseudoephedrine, and methylephedrine] revealed that almost all Mongolian Ephedra plants, except E. przewalskii and E. lomatolepis, contained high amounts of total ephedrine alkaloids (TAs), with range 1.86–4.90% of dry weight [21]. The E. sinica (types I and II) contained 1.95–4.16% TAs and showed a higher percentage of E1 in TAs than other species. Within E. sinica, types I and II specimens collected from eastern grassland areas showed a high proportion of E1 in TAs (mean 51.4% and 54.0%, respectively); whereas, type II specimens from central areas showed a high proportion of E2 in TAs (mean 55.5%). The proportions of E1 and E2 might be affected more by growing environment than genetic factors. Ephedra equisetina had the highest content of TAs (3.98–4.90%), with more than 90% of TAs being E2. Both E. sinica and E. equisetina satisfied the JP criterion [6] of containing no less than 0.7% of summed content of E1 and E2, with 1.43–3.68% and 3.81–4.59%, respectively. Therefore, these two species growing in Mongolia were a suitable new resource of ephedra herb used in Japan. Given that the pure line of E. sinica is limited to eastern areas, and E. equisetina has a limited distribution, promoting the cultivation of selected specimens in suitable regions is crucial for ensuring a stable supply of ephedra herb and supporting environmental preservation.

Resources of saposhnikovia root and rhizome (Saposhnikoviae Radix)

Saposhnikovia root and rhizome (SR) derived from Saposhnikovia divaricata (Turcz.) Schischk. have been used as a diaphoretic, febrifuge, analgesic, and anti-phlogistic in China and Japan, including frequently as an ingredient in Kampo formulas [5]. In recent years, natural SR resources have been depleted because of increasing demand, therefore, a large amount of SR derived from cultivated plants has become available in the Chinese and Japanese markets. However, some of these do not meet the CP and JP requirements due to lower amounts of prim-O-glucosylcimifugin (S1) and 4′-O-β-d-glucosyl-5-O-methylvisamminol (S3) and the higher yield of dilute-ethanol-soluble extract [6, 7]. For sustainable utilization of SR resources through high-performance cultivation by selecting suitable plant resources and cultivation areas, Mongolia was chosen because of the large population of wild growing S. divaricata, especially in the eastern part including Khentii and Dornod Provinces, and field investigation and metabolomic analysis were performed on collected plant specimens.

To evaluate the quality of Mongolian S. divaricata, metabolomic profiling of all root parts of 43 specimens from Mongolia, as well as eight SR samples and two plant specimens from China, were conducted using LC-IT-TOF–MS. The LC–MS profiles of the 70% methanol extracts of the specimens and SR samples showed uniformity, and 13 chromones and 17 coumarins were tentatively identified [23]. Among them, a new compound, 3′-O-(6″-O-malonyl)-glucosylhamaudol (S10) [24] and 17 known compounds were isolated and unambiguously identified. Orthogonal partial least squares-discriminant analysis (OPLS-DA) based on LC–MS data revealed that Mongolian specimens were clearly distinguished from Chinese SR and characterized by an abundance of S1. Moreover, Mongolian specimens could be discriminated by their growing regions based on the content of eight compounds (S1–S3, S6, S7, and S9–S11) (Fig. 6A). Specimens from Khalkhgol in far eastern Mongolia contained higher amounts of dihydrofurochromones S1, cimifugin (S2), and S3, while those from Holonbuir contained relatively higher amounts of hamaudol (S6), 3′-O-acetylhamaudol (S7), 3′-O-angeloylhamaudol (S9), and praeruptorin B (S11) [23].

Fig. 6

Quantification of metabolites in Saposhnikovia divaricata roots from Mongolia by HPLC–DAD. A Structures of reference standard compounds; B Average contents and S.D. of S1 and S3 in Mongolian specimens and saposhnikovia root and rhizome from China; C OPLS-DA of Mongolian S. divaricata spec