Diterpenoids from the Aerial Parts of Isodon serra with Selective Cytotoxic Activity.

Four new diterpenoids, isodosins A-D (1-4), together with nine known compounds (5-13) were isolated and identified from the aerial parts of Isodon serra (Maxim.) Hara. The structures of the new diterpenoids were elucidated based on the analysis of HR-ESI-MS data, 1D/2D-NMR-spectroscopic data, and electronic circular dichroism (ECD) calculations. Cytotoxicities of compounds 2, 3, 5, 6, and 9 against the HepG2 and H1975 cell lines were evaluated with the MTT assay. As a result, compounds 2, 3, and 6 revealed higher levels of cytotoxicity against HepG2 cells than against H1975 cells. Moreover, compund 6 demonstrated the most efficacy in inhibiting the proliferation of HepG2 cells, with an IC50 value of 41.13 ± 3.49 µM. This effect was achieved by inducing apoptosis in a dose-dependent manner. Furthermore, the relationships between the structures and activities of these compounds are briefly discussed.

[Analysis of chemical compositions in different parts of Isodon japonicus using UPLC-Q-TOF-MS technique combined with chemometrics].

In order to analyze the similarities and differences of chemical compositions between the roots and stems and leaves of Isodon japonicus(IJ), this study utilized UPLC-Q-TOF-MS technology to systematically characterize its chemical compositions, analyzed and identified the structure of its main compounds, and established a method for simultaneous determination of its content by refe-rence substance. A total of 34 major compounds in IJ, including 14 reference compounds, were identified or predicted online. Moreover, an UPLC-UV content determination method was developed for 11 compounds [danshensu, caffeic acid, vicenin-2,(1S,2S)-globoidnan B, rutin,(+)-rabdosiin,(-)-rabdosiin,(1S,2S)-rabdosiin, shimobashiric acid C, rosmarinic acid, and pedalitin]. The method exhibited excellent separation, stability, and repeatability, with a wide linear range(0.10-520.00 µg·mL~(-1)) and high linearity(R~2>0.999). The average recovery rates ranged from 94.72% to 104.2%. The principal component analysis(PCA) demonstrated a clear difference between the roots and stems and leaves of IJ, indicating good separation by cluster. Furthermore, the orthogonal partial least squares discriminant analysis(OPLS-DA) model was employed, and six main differentially identified compounds were identified: rosmarinic acid, shimobashiric acid C, epinodosin, pedalitin, rutin, and(1S,2S)-rabdosiin. In summary, this study established a strategy and method for distinguishing different parts of IJ, providing a valuable tool for quality control of IJ and a basis for the ratio-nal utilization and sustainable development of IJ.

Identification and functional characterization of three cytochrome P450 genes for the abietane diterpenoid biosynthesis in Isodon lophanthoides.

MAIN CONCLUSION: We identify two ferruginol synthases and a 11-hydroxyferruginol synthase from a traditional Chinese medicinal herb Isodon lophanthoides and propose their involvement in two independent abietane diterpenoids biosynthetic pathways. Isodon lophanthoides is a traditional Chinese medicinal herb rich in highly oxidized abietane-type diterpenoids. These compounds exhibit a wide range of pharmaceutical activities, yet the biosynthesis is barely known. Here, we describe the screening and functional characterization of P450s that oxidize the abietane skeleton abietatriene. We mainly focused on CYP76 family and identified 12 CYP76AHs by mining the RNA-seq data of I. lophanthoides. Among the 12 CYP76AHs, 6 exhibited similar transcriptional expression features as upstream diterpene synthases, including root or leaf-preferential expression pattern and highly MeJA inducibility. These six P450s were considered as first-tier candidates and functionally characterized in yeast and plant cells. In yeast assays showed that both CYP76AH42 and CYP76AH43 were ferruginol synthases hydroxylating the C12 position of abietatriene, whereas CYP76AH46 was characterized as a 11-hydroxyferruginol synthase which catalyzes two successive oxidations at C12 and C11 of abietatriene. Heterologous expression of three CYP76AHs in Nicotiana benthamiana resulted in the formation of ferruginol. qPCR analysis showed CYP76AH42 and CYP76AH43 were mainly expressed in the root, which was consistent with the distribution of ferruginol in the root periderms. CYP76AH46 was primarily expressed in the leaves where barely ferruginol or 11-hydroxyferruginol was detected. In addition to distinct organ-specific expression pattern, three CYP76AHs exhibited different genomic structures (w or w/o introns), low protein sequence identities (51-63%) and were placed in separate subclades in the phylogenetic tree. These results suggest that the identified CYP76AHs may be involved in at least two independent abietane biosynthetic pathways in the aerial and underground parts of I. lophanthoides.

Synthesis of 1-O-acyl- and 1-oxo-kamebanin analogues and their cytotoxic activity.

A series of 1-O-acyl- and 1-oxo-kamebanin analogues were prepared from kamebanin, isolated from Rabdosia excisa and their cytotoxicity was assayed on HL60 promyelocytic leukemia cells and HCT116 human colon cancer cells. The structure-activity relationship study showed that the presence of 1-O-acyl groups of a C3-C5 carbon chain increased the cytotoxic activity.

Di- and Triterpenoids from the Rhizomes of Isodon amethystoides and Their Anti-inflammatory Activities.

Amethystoidesic acid (1), a triterpenoid with an unprecedented 5/6/6/6 tetracyclic skeleton, and six undescribed diterpenoids, amethystoidins A-F (2-7), were isolated from the rhizomes of Isodon amethystoides along with 31 known di- and triterpenoids (8-38). Their structures were fully elucidated via extensive spectroscopic analysis including 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) calculations. Compound 1 is the first example of a triterpenoid possessing a rare ring system (5/6/6/6) derived from a contracted A-ring and the 18,19-seco-E-ring of ursolic acid. Compounds 6, 16, 21, 22, 24, and 27 significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, which could be partly mediated by the downregulation of LPS-induced inducible nitric oxide synthase (iNOS) protein expression.

Cytotoxic C-20 non-oxygenated ent-kaurane diterpenoids from Isodon wardii.

Twenty new ent-kaurane diterpenoids, wardiisins A-T (1-20), along with two previously undescribed artefactual compounds (21 and 22) and twelve known analogues (23-34), were isolated from the aerial part of Isodon wardii. Their structures were elucidated by comprehensive analysis of spectroscopic data and single-crystal X-ray diffraction, and most of them were found to bear unusual C-12 oxygenation. Compounds 4, 7, 8, 19, 20, 21 exhibited remarkable cytotoxicity against the cancer cell lines HL-60, SMMC-7721, A-549, MDA-MB-231, and SW480, with IC50 values ranging from 0.3 to 5.2 µM. Moreover, 7 was found to induce G2/M cell cycle arrest and promote apoptosis in SW480 cell lines.

Antiproliferative Activities of Diterpenes from Leaves of Isodon trichocarpus against Cancer Stem Cells.

Two new diterpenes named trichoterpene I (1) and trichoterpene II (2) were isolated from the extract from the leaves of Isodon trichocarpus together with 19 known diterpenes. Their chemical structures were elucidated on the basis of chemical and physicochemical properties. Among them, oridonin (3), effusanin A (4), and lasiokaurin (9) with the α,ß-unsaturated carbonyl moiety showed antiproliferative activities against breast cancer MDA-MB-231 and human astrocytoma U-251 MG cells [i.e., non-cancer stem cells (non-CSCs)] and their cancer stem cells (CSCs) isolated by sphere formation. In particular, compound 4 (IC50 = 0.51 µM) showed a higher antiproliferative activity against MDA-MB-231 CSCs than against MDA-MB-231 non-CSCs. The antiproliferative activity toward CSCs of compound 4 was equal to adriamycin (positive control, IC50 = 0.60 µM).

Guidongnins I-J: Two New 6,7-seco-7,20-Olide-ent-kaurene Diterpenes with Unusual Structures from Isodon rubescens.

Two undescribed ent-kaurene diterpenes, named guidongnins I (1) and J (2), were isolated from the medicinal plant Isodon rubescens. Compound 1 was determined to contain an unprecedented 23 carbons in the skeleton by bearing an extra isopropyl group at C-17 out of the diterpenoid parent structure, and compound 2 was the first example of 6,7-seco-7,20-olide-ent-kaurenes with two fused-tetrahydrofuran rings formed between C-6 and C-19/C-20 through oxygen bridges. Their structures, including their absolute configurations, were determined using the analyses of the spectroscopic and X-ray diffraction data. Guidongnins I (1) and J (2) were assessed for their anti-cancer activities against the growth of various cancer cell lines, and 2 displayed cytotoxic potency against HepG2 at IC50 27.14 ± 3.43 µM.

[Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway].

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-ß1(TGF-ß1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-ß1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Scopariusicides D-M, ent-clerodane-based isomeric meroditerpenoids with a cyclobutane-fused γ/δ-lactone core from Isodon scoparius.

Scopariusicides D-M (1-10), ten new ent-clerodane-based meroditerpenoids with a cyclobutane-fused γ/δ-lactone core, were isolated from Isodon scoparius. Their structures were determined by comprehensive analysis of spectroscopic data, single-crystal X-ray diffraction, chemical transformation, and TDDFT ECD calculation. A plausible biosynthetic pathway of 1-10 was proposed in which the asymmetrical cyclobutane ring was formed via a crossed "head-to-tail" intermolecular [2 + 2] cycloaddition in anti/syn facial approaches between an ent-clerodane lactone and a cis-4-hydroxycinnamic acid. Bioactivity evaluation manifested that 5 exhibited significant neuroprotective effect against corticosterone-induced injury in PC12 cells, while 6 and 7 exhibited moderate immunosuppressive activity against human T cell proliferation stimulated by anti-CD3/anti-CD28 mAb.

Structurally diverse diterpenoids from Isodon oresbius and their bioactivity.

Twelve new diterpenoids, isoresbins A-L (1-12), together with twenty-eight known ones, were isolated from the aerial parts of Isodon oresbius. Their diverse structures included 6,7-seco-ent-kaurane, 7,20-epoxy-ent-kaurane, 6,7:8,15-diseco-ent-kaurane, and abietanes skeletons, which were elucidated by spectroscopic data interpretation, single-crystal X-ray diffraction, and quantum chemical calculation. Isoresbins A (1) and B (2) possessed a new rearranged 15(8 â†’ 11)-abeo-6,7-seco-ent-kaurane skeleton. 1 and 5 promoted lysosomal function, which was evaluated by LysoTracker Red staining and DQ-ovalbumin dequenching assay. 1 showed cytotoxicity against six human tumor cell lines with IC50 values in 2.07-4.04 µM range. Moreover, 1 induced damage of mitochondrial membrane potential, G2/M cell cycle arrest and apoptosis in SW480 cells.

Screening and Capability Assessment of Tyrosinase Inhibitors in Isodon excisoides by Ultrafiltration Coupled with UHPLC-Q-TOF-MS and Molecular Docking Technology.

Tyrosinase inhibitors can alleviate the harm to the liver caused by tyrosinase. How to effectively screen out natural tyrosinase inhibitors becomes a focus. In this study, Isodon excisoides was first extracted with the ultrasound optimized by response surface methodology. Then, a method combined ultrafiltration with ultra-liquid chromatography mass spectrometry (UHPLC/MS) was built to screen and identify tyrosinase inhibitors. The binding energies of active ingredients to tyrosinase were calculated by molecular docking. The reliability of the results was validated by the IC50 of enzyme inhibition assay. As a result, the binding energies of 7 components including excisanin B, lasiokaurin, rabdophyllin G, rabdoserrin B, rabdosin D, rabdosinate and weisiensin were lower than that of resveratrol. It was indicated that these components had high tyrosinase inhibitory activity. The IC50 values of lasiokaurin and excisanin B were 177 and 142 µmol/mL, which were less than that of resveratrol (183 µmol/mL). It showed that this way was simple, rapid, reliable and effective, which provided a new strategy to screen natural bioactive compounds from plants.

Cytotoxic ent-Kaurane Diterpenoids from Rabdosia Rubescens.

One new (1) and 11 reported ent-kaurane diterpenoids (2-12) were received from the ethanol extract of the air-dried aerial parts of Rabdosia rubescens collected in Jiyuan. Their structures were determined in accordance with high resolution electrospray ionization mass spectroscopy, one dimensional (1D) and two-dimensional (2D) NMR spectroscopy and the data published in the literature. The cytotoxic activity of these isolated compounds was assessed against SMMC-7721, A-549, H-1299 and SW-480 cancer cell lines. Compounds 2-6 revealed significant cytotoxic activity on lung cancer cell lines A549 with IC50 values from 6.2 to 28.1 µM. Analysis of structure-activity relationship of these tested compounds indicated the carbonyl at C-15 and hydroxy at C-1 together could be crucial groups for inhibiting lung cancer cell lines A549 proliferation.

A practical technique for rapid characterisation of ent-kaurane diterpenoids in Isodon serra (Maxim.) Hara by UHPLC-Q-TOF-MS/MS.

INTRODUCTION: The diterpenoids are the most important active constituents that contribute to the pharmacological efficacy of Isodon serra (Maxim.) Hara. Clinical studies have revealed that diterpenoids possess multiple features, e.g. antitumour, antitubercular and anti-ischemic activities. Therefore, the identification and detection of diterpenoids may be equally important for understanding the pharmacological basis of diterpenoids and enhancing the product quality control of I. serra. OBJECTIVES: The purpose of this study was to develop a practical analysis approach of rapid characterisation using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) for the structure characterisation of the ent-kaurane diterpenoids from I. serra. METHODOLOGY: The analytical strategy was as follows: first, ent-kaurane diterpenoids were detected by a novel on-line data acquisition approach, i.e. sequential window acquisition of all theoretical fragment-ion spectra (SWATH). Second, the MS of eight ent-kaurane diterpenoids was explored, and their mass spectrum cleavage pathways were summarised and determined. Finally, the methanol extract of I. serra was studied using SWATH and identified by extracted ion chromatography (XIC). RESULTS: Compared to the traditional information-dependent acquisition (IDA) method, SWATH significantly improved the hit rate of ent-kaurane diterpenoids. With support from UHPLC separation and specific detection by tandem mass spectrometry (MS/MS), 48 ent-kaurane diterpenoids were successfully characterised and classified as ent-kaurane diterpenoids from a complex matrix. CONCLUSIONS: These combined qualitative methods were used to provide a potential approach for the characterisation of traditional Chinese medicine (TCM) and its preparations. Meanwhile, the SWATH provided a novel and reliable method for the structural characterisation of ent-kaurane diterpenoids from other complicated TCMs.

3,4-Seco-Isopimarane Diterpenes from the Twigs and Leaves of Isodon Flavidus.

Three isopimarane diterpenes [fladins B (1), C (2), and D (3)] were isolated from the twigs and leaves of Chinese folk medicine, Isodon flavidus. The chemical structures were determined by the analysis of the comprehensive spectroscopic data, and the absolute configuration was confirmed by X-ray crystallographic analysis. The structures of 1-3 were formed from isopimaranes through the rearrangement of ring A by the bond break at C-3 and C-4 to form a new δ-lactone ring system between C-3 and C-9. This structure type represents the first discovery of a natural isopimarane diterpene with an unusual lactone moiety at C-9 and C-10. In the crystal of 1, molecules are linked to each other by intermolecular O-H···O bonds, forming chains along the b axis. Compounds 1-3 were evaluated for their bioactivities against different diseases. None of these compounds displayed cytotoxic activities against HCT116 and A549 cancer cell lines, antifungal activities against Trichophyton rubrum and T. mentagrophytes, or antiviral activities against HIV entry at 20 µg/mL (62.9-66.7) µM. Compounds 1 and 3 did not show antiviral activities against Ebola entry at 20 µg/mL either; only 2 was found to show an 81% inhibitory effect against Ebola entry activity at 20 µg/mL (66.7 µM). The bioactivity evidence suggested that this type of compound could be a valuable antiviral lead for further structure modification to improve the antiviral potential.

Preparation, Characterization, and Evaluation of Liposomes Containing Oridonin from Rabdosia rubescens.

Due to the remarkable anti-tumor activities of oridonin (Ori), research on Rabdosia rubescens has attracted more and more attention in the pharmaceutical field. The purpose of this study was to extract Ori from R. rubescens by ultrasound-assisted extraction (UAE) and prepare Ori liposomes as a novel delivery system to improve the bioavailability and biocompatibility. Response surface methodology (RSM), namely Box-Behnken design (BBD), was applied to optimize extraction conditions, formulation, and preparation process. The results demonstrated that the optimal extraction conditions were an ethanol concentration of 75.9%, an extraction time of 35.7 min, and a solid/liquid ratio of 1:32.6. Under these optimal conditions, the extraction yield of Ori was 4.23 mg/g, which was well matched with the predicted value (4.28 mg/g). The optimal preparation conditions of Ori liposomes by RSM, with an ultrasonic time of 41.1 min, a soybean phospholipids/drug ratio of 9.6 g/g, and a water bath temperature of 53.4 °C, had higher encapsulation efficiency (84.1%). The characterization studies indicated that Ori liposomes had well-dispersible spherical shapes and uniform sizes with a particle size of 137.7 nm, a polydispersity index (PDI) of 0.216, and zeta potential of -24.0 mV. In addition, Ori liposomes presented better activity than free Ori. Therefore, the results indicated that Ori liposomes could enhance the bioactivity of Ori, being proposed as a promising vehicle for drug delivery.

[Diterpenoids from Rabdosia flexicaulis].

The genus Rabdosia is famous for the abundance of diverse and novel ent-kaurane diterpenoids. However, only a few ent-kauranoids have been discovered from R. flexicaulis since the investigation on its chemical constituents is not systematic. To find novel bioactive diterpenoids, the ethyl acetate extract of the above ground part of R. flexicaulis in Daofu County, Sichuan Province was obtained by column chromatography. One new compound and five known ones were identified as flexicaulin E(1), forrestin B(2), inf-lexarabdonin D(3), 7α-hydroxydehydroabietic acid(4), 15-hydroxydehydroabietic acid(5), and pomiferin F(6) by spectral techniques. Compounds 1-3 were the ent-kaurane diterpenoids isolated from this species for the first time. Compounds 4-6, aromatic abie-tanoids, were isolated from the genus Rabdosia for the first time.

[Main components in different varieties of Xihuangcao by UPLC-Q-TOF-MS and UPLC-DAD].

This study established an ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) method to analyze the main components in different varieties of Xihuangcao and established a UPLC-DAD method to simultaneously determine the five active components(caffeic acid, rosmarinic acid, schaftoside, isoschaftoside, and oridonin).The chromatographic separation was performed on a Waters ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm) with a gradient elution of methanol(B)-water containing 0.1% formic acid(A) at a flow rate of 0.3 mL·min~(-1).The column temperature was 30 ℃.The Q-TOF-MS discriminant analysis was performed under positive electrospray ion mode and the split ratio was 1∶1. Quantitative analysis was carried out by UPLC-DAD.The determination wavelength was set at 245 nm.Thirty-two main components of Xihuangcao were separated and identified by UPLC-Q-TOF-MS, where 19 were identified in Rabdosia serra, nine in R.nervosa, 10 in R.lophanthoides, 15 in R.lophanthoides var.graciliflora, 10 in R.lophanthoides var.gerardianus, and seven in R.stracheyi.The UPLC-DVD method was developed for simultaneously determining five active components in different varieties of Xihuangcao.The standard curves for five compounds showed good linearity with correlation coefficients higher than 0.999 0.The precision, repeatability, and stability were good.The average recoveries(n=6) were between 97.01% and 102.7% with RSD<3.0%.The results of UPLC-Q-TOF-MS analysis provided a scientific basis for the use of R.stracheyi as a medicinal material of Xihuangcao and the equivalent use of R.lophanthoides var.gerardianus with R.lophanthoides var.graciliflora to some extent.The UPLC-DAD method for simultaneously determining five active components is simple, rapid, and accurate.This study can provide the basis for the quality control of different varieties of Xihuangcao.

Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana.

Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara-7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.

An integrative method based on UHPLC-Q-TOF-MS/MS combined with metabolomics to authenticate Isodon rubescens.

Genuine regional drugs have played a vital role in clinical use for a long time. There are differences in traditional Chinese medicines (TCM) from different regions based on their chemical composition. Differences in chemical composition may lead to deviations in therapeutic effects. To our knowledge, to date, there are no effective methods for distinguishing genuine regional drugs based on the differences in their chemical composition. Therefore, establishing an analytical platform for distinguishing the compounds used in TCM from various geographical locations is essential. In this work, an integrated platform based on UPLC-Q-TOF-MS/MS combined with plant metabolomics approach was established for comprehensively distinguishing genuine regional drugs. Isodon rubescens (Hemsl.) Hara, a widely used herbal medicine of China, was chosen for this study and 24 samples from four geographical locations in China were collected. A total of 60 ent-kaurane diterpenoids were tentatively identified, and then the samples from four geographical origins were distinguished using PCA (principal component analysis) and PLS-DA (partial least squares discrimination analysis). Different compounds were identified among the samples collected from the four geographical locations, and 12 of them were regarded as marker compounds responsible for the distinction. Our study highlights the essence and predictive ability of metabolomics in detecting minute differences in the same varieties of TCM samples based on the levels and compositions of their metabolites. These results showed that the analysis using UHPLC-Q-TOF-MS/MS combined with metabolomics could be applied to distinguish the geographical origins and varieties of TCM.