Integrated transcriptome and metabolome analysis provide insights into the mechanism of saponin biosynthesis and its role in alleviating cadmium-induced oxidative damage in Ophiopogon japonicum.

Zhe-Maidong, a cultivar of Ophiopogon japonicus is a prominent traditional herbal medicine rich in saponins. This study explored the mechanism of saponin biosynthesis and its role in alleviating Cd-induced oxidative damage in the Zhe-Maidong cultivar using three experimental groups undergoing Cd stress. In the Cd-contaminated soil treatment, total saponins were 1.68 times higher than those in the control. The saponin content in the Cd-2 and Cd-3 treatments was approximately twice as high as that in the Cd-CK treatment. These findings revealed that Cd stress leads to total saponin accumulation. Metabolomic analysis identified the accumulated saponins, primarily several monoterpenoids, diterpenoids, and triterpenoids. The increased saponins exhibited an antioxidant ability to prevent the accumulation of Cd-induced reactive oxygen species (ROS). Subsequent saponin application experiments provided strong evidence that saponin played a crucial role in promoting superoxide dismutase (SOD) activity and reducing ROS accumulation. Transcriptome analysis revealed vital genes for saponin synthesis under Cd stress, including SE, two SSs, and six CYP450s, positively correlated with differentially expressed metabolite (DEM) levels in the saponin metabolic pathway. Additionally, the TF-gene regulatory network demonstrated that bHLH1, bHLH3, mTERF, and AUX/IAA transcript factors are crucial regulators of hub genes involved in saponin synthesis. These findings significantly contribute to our understanding of the regulatory network of saponin synthesis and its role in reducing oxidative damage in O. japonicum when exposed to Cd stress.

Using Pharmacokinetic-Pharmacodynamic Modeling to Study the Main Active Substances of the Anticancer Effect in Mice from Panax ginseng-Ophiopogon japonicus.

Ginseng Radix et Rhizoma Rubra (Panax ginseng C.A. Mey, Hongshen, in Chinese) and Ophiopogonis Radix (Ophiopogon japonicus (L.f) Ker-Gawl., Maidong, in Chinese) are traditional Chinese herbal pairs, which were clinically employed to enhance the immune system of cancer patients. This study employed the pharmacokinetic and pharmacodynamic (PK-PD) spectrum-effect association model to investigate the antitumor active substances of P. ginseng and O. japonicus (PG-OJ). The metabolic processes of 20 major bioactive components were analyzed using Ultra-Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (UPLC-MS/MS) in the lung tissue of tumor-bearing mice treated with PG-OJ. The ELISA method was employed to detect the levels of TGF-ß1, TNF-α, and IFN-γ in the lung tissue of mice at various time points, and to analyze their changes after drug administration. The results showed that all components presented a multiple peaks absorption pattern within 0.083 to 24 h post-drug administration. The tumor inhibition rate of tumor and repair rate of IFN-γ, TNF-α, and TGF-ß1 all increased, indicating a positive therapeutic effect of PG-OJ on A549 tumor-bearing mice. Finally, a PK-PD model based on the GBDT algorithm was developed for the first time to speculate that Methylophiopogonanone A, Methylophiopogonanone B, Ginsenoside Rb1, and Notoginsenoside R1 are the main active components in PG-OJ for lung cancer treatment.

Quality variation of maidong (Ophiopogon japonicus and Liriope spicata) - A HPTLC-based approach.

The tuberous roots of Ophiopogon japonicus and Liriope spicata are used for the same therapeutic purpose in traditional Chinese medicine and are collectively referred to as maidong medicine. Interestingly, it was observed that the price of tuberous roots varies depending on their location on the plant, and fibrous roots are usually discarded post-harvest. Mislabeling might be of concern due to similarities in morphological features between the two species. Moreover, paclobutrazol has been observed to be heavily applied during the production, and therefore might be of health concern. Overall, maidong might suffer from quality inconsistencies while its metabolomic complexity is influenced by growing region and cultivation practices, botanical species, and plant parts. To address these challenges, this study employed High-Performance Thin Layer Chromatography (HPTLC) approach, in which sample preparation and derivatization procedure were optimized to enable to capture more detailed and comprehensive metabolomic fingerprints. By integrating with rTLC algorithm and Multivariate Data Analysis (MVDA), an improved quality assessment was achieved. Samples were collected from four production regions and supplemented with commercial products from markets. The optimized HPTLC analysis recognized species- and region-specific metabolomic patterns of maidong, uncovering a 4% of mislabelled cases. Moreover, findings highlight the underexplored therapeutic potential of fibrous roots, and comparable therapeutic efficacy between different root types. Additionally, complemented by Liquid Chromatography-Mass Spectrometry (LC-MS) for paclobutrazol residue evaluation, 24.66% of the commercial maidong samples surpassed maximum residue limits of paclobutrazol, raising safety concerns. This research represents a significant analytical advancement, offering a robust, cost-effective, and comprehensive method for maidong quality control, and paving the way for more strict residue regulation and updates to herbal pharmacopoeias and monographs.

Ophiopogon japonicus and its active compounds: A review of potential anticancer effects and underlying mechanisms.

BACKGROUND: Ophiopogon japonicus (Thunb.) Ker Gawl., a well-known Chinese herb, has been used in traditional Chinese medicine for thousands of years. Extensive in vitro and in vivo studies have shown that O. japonicus and its active compounds exhibit potential anticancer effects in a variety of cancer cells in vitro and suppress tumor growth and metastasis without causing serious toxicity in vivo. PURPOSE: This review aims to systemically summarize and discuss the anticancer effects and the underlying mechanisms of O. japonicus extracts and its active compounds. METHODS: The review is prepared following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Various scientific databases including Web of Science, PubMed, Scopus, and Chinese National Knowledge Infrastructure were searched using the keywords: Ophiopogon japonicus, tumor, cancer, carcinoma, content, pharmacokinetics, and toxicity. RESULTS: O. japonicus extracts and the active compounds, such as ruscogenin-1-O-[ß-d-glucopyranosyl(1→2)][ß-d-xylopyranosyl(1→3)]-ß-d-fucopyranoside (DT-13), ophiopogonin B, and ophiopogonin D, exert potential anticancer effects, including the induction of cell cycle arrest, activation of apoptosis and autophagy, and inhibition of metastasis and angiogenesis. In addition, the mechanisms underlying these effects, as well as the pharmacokinetics, toxicity and clinical utility of O. japonicus extracts and active compounds are discussed. Furthermore, this review highlights the research and application prospects of these compounds in immunotherapy and combination chemotherapy. CONCLUSIONS: The traditional herb O. japonicus and its phytochemicals could be safe and reliable anticancer drug candidates, alone or in combination with chemotherapeutic drugs. We hope that this review, which highlights the anticancer properties of O. japonicus, will contribute to drug optimization, therapeutic development, and future studies on cancer therapies based on this medicinal plant.

Comparison of Ophiopogon japonicus and Liriope spicata var. prolifera from Different Origins Based on Multi-Component Quantification and Anticancer Activity.

The tuberous root of Ophiopogon japonicus (Thunb.) Ker-Gawl. is a well-known Chinese medicine also called Maidong (MD) in Chinese. It could be divided into "Chuanmaidong" (CMD) and "Zhemaidong" (ZMD), according to the geographic origins. Meanwhile, the root of Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma (SMD) is occasionally used as a substitute for MD in the market. In this study, a reliable pressurized liquid extraction and HPLC-DAD-ELSD method was developed for the simultaneous determination of nine chemical components, including four steroidal saponins (ophiopojaponin C, ophiopogonin D, liriopesides B and ophiopogonin D'), four homoisoflavonoids (methylophiopogonone A, methylophiopogonone B, methylophiopogonanone A and methylophiopogonanone B) and one sapogenin (ruscogenin) in CMD, ZMD and SMD. The method was validated in terms of linearity, sensitivity, precision, repeatability and accuracy, and then applied to the real samples from different origins. The results indicated that there were significant differences in the contents of the investigated compounds in CMD, ZMD and SMD. Ruscogenin was not detected in all the samples, and liriopesides B was only found in SMD samples. CMD contained higher ophiopogonin D and ophiopogonin D', while the other compounds were more abundant in ZMD. Moreover, the anticancer effects of the herbal extracts and selected components against A2780 human ovarian cancer cells were also compared. CMD and ZMD showed similar cytotoxic effects, which were stronger than those of SMD. The effects of MD may be due to the significant anticancer potential of ophiopognin D' and homoisoflavonoids. These results suggested that there were great differences in the chemical composition and pharmacological activity among CMD, ZMD and SMD; thus, their origins should be carefully considered in clinical application.

Simultaneous Extraction and Determination of Characteristic Steroidal Saponins and Homoisoflavonoids in Zhejiang Ophiopogon japonicus.

Zhejiang Ophiopogonjaponicus (ZOJ) is a specific variety of Ophiopogon japonicus with characteristic steroidal saponins and homoisoflavonoids, which are also main pharmacodynamic constituents with clinical effects, including curing inflammation and cardiovascular diseases. However, few analysis methods were applied to simultaneously and quantitatively determine two kinds of its constituents, and hazardous organic solvents are mostly used for extraction. In this study, a new validated simultaneous extraction and determination method for four characteristic steroidal saponins and homoisoflavonoids in ZOJ was established by ionic liquid-ultrasonic extraction (IL-UAE) combined with HPLC-DAD-ELSD analysis, which can be used for the quality control of ZOJ. Chromatographic separation was performed with a DAD wavelength at 296 nm, and the ELSD parameters of the drift tube temperature (DTT), atomizer temperature (AT), and nitrogen gas pressure (NGP) were set at 20% heating power, 70 °C, and 25 psi, respectively. The optimal IL-UAE conditions were 1 mol/L [Bmim]CF3SO3 aqueous solution, a liquid-material ratio of 40 mL/g, and an ultrasonic time of 60 min. The proposed method is reliable, reproducible, and accurate, which were verified with real sample assays. Consequently, this work will be helpful for the quality control of ZOJ. It can also present a promising reference for the simultaneous extraction and determination of different kinds of constituents in other medicinal plants.

Quality Evaluation of Ophiopogon japonicus from Two Authentic Geographical Origins in China Based on Physicochemical and Pharmacological Properties of Their Polysaccharides.

Ophiopogon japonicus is widely used as a tonic herb in China. According to the origins, MaiDong of Chinese materia medica can be classified as Zhe MaiDong (Ophiopogon japonicus in Zhejiang), Chuan MaiDong (Ophiopogon japonicus in Sichuan), Duanting Shan MaiDong (Liriope muscari), and Hubei MaiDong (Liriope spicata). In terms of quality control, polysaccharides-based evaluations have not yet been conducted. In this study, microwave-assisted extraction (MAE) was used for the preparation of polysaccharides from 29 batches of MaiDong. HPSEC-MALLS-RID and HPAEC-PAD were employed to investigate their molecular parameters and compositional monosaccharides, respectively. The ability to scavenge ABTS radicals and immune promotion abilities, in terms of nitric oxide releasing and phagocytosis on RAW 264.7 macrophages, were also compared. The results showed that polysaccharides in different MaiDong varied in molecular parameters. All polysaccharides mainly contained fructose and glucose with small amounts of arabinose, mannose, galactose, and xylose. For polysaccharides of Zhe MaiDong and Chuan MaiDong, the molar ratio of Fru to Glc was roughly 15:1 and 14:1, respectively. Zhe MaiDong exhibited better antioxidant and immune promotion activity, and so did that of fibrous roots. The pharmacological activity, however, did not account for the variation in growth years. Finally, indicators for quality control based on multivariate statistical analysis included: yield, antioxidant activity, the content of fructose, and RI signal. It was concluded that MaiDong's fibrous roots had similar components to the root, and their quality was not significantly affected by growth age. This may provide some guidance for the cultivation and use of MaiDong.

The Effects of Lycium chinense, Cuscuta chinensis, Senna tora, Ophiopogon japonicus, and Dendrobium nobile Decoction on a Dry Eye Mouse Model.

Background and objective: Dry eye disease (DED) is a relatively common disorder associated with abnormal tear film and the ocular surface that causes ocular irritation, dryness, visual impairment, and damage to the cornea. DED is not a life-threatening disease but causes discomfort and multifactorial disorders in vision that affect daily life. It has been reported that all traditional medicinal plants exhibit anti-inflammatory effects on several diseases. We hypothesized that the decoction ameliorated ocular irritation and decreased cytokine expression in the cornea. This study aimed to investigate the molecular mechanisms of DED and discover a therapeutic strategy to reduce corneal inflammation. Material and Methods: We used a DED mouse model with extraorbital lacrimal gland (ELG) excision and treated the mice with a decoction of five traditional medicines: Lycium chinense, Cuscuta chinensis, Senna tora, Ophiopogon japonicus, and Dendrobium nobile for 3 months. The tear osmolarity and the ocular surface staining were evaluated as indicators of DED. Immunohistochemistry was used to detect the level of inflammation on the cornea. Results: After treatment with the decoction for three months, epithelial erosions and desquamation were reduced, the intact of corneal endothelium was maintained, and tear osmolarity was restored in the eyes. The IL-1ß-associated inflammatory response was reduced in the cornea in the DED model. Conclusions: These data suggested that a mixture of traditional medicines might be a novel therapy to treat DED.

Interaction of the main active components in Shengmai formula mediated by organic anion transporter 1 (OAT1).

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a classical traditional Chinese medicine prescription, which is widely used in the treatment of cardiovascular and cerebrovascular diseases. Our previous studies have demonstrated that some components in SMF can interact with each other through breast cancer resistance protein, sodium taurocholate co-transporting polypeptide, organic anion transporting polypeptide 1B1 and 1B3. Organic anion transporter 1 (OAT1) is highly expressed in kidney, mediating the elimination of many endogenous and exogenous substances. However, the interaction between the main active components in SMF and OAT1 is not clear. AIM OF THE STUDY: This study aimed to investigate the interactions of the major bioactive components in SMF mediated by OAT1. MATERIALS AND METHODS: Four main fractions, namely, ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), fructus schisandrae total lignans (STL), and 12 active components, namely, ginsenoside Rg1, Re, Rd and Rb1, ophiopogonin D and D', methylophiopogonanone A and B, schizandrol A and B, schizandrin A and B, were selected to explore the interactions of SMF with OAT1 using cell and rat models. RESULTS: The above four main fractions in SMF all exhibited inhibitory effects on the uptake of 6-carboxyfluorescein (6-CF), a classic substrate of OAT1. Among the 12 main effective components, only ginsenoside Re, Rd, and methylophiopogonanone A showed inhibition of 6-CF uptake. Additionally, we found that schizandrin B was transported by HEK293-OAT1 cells, and schizandrin B uptake was markedly inhibited by GTS, OTS, OTF, ginsenoside Re, Rd, and methylophiopogonanone A. In rats, ginsenoside Re, Rd, and methylophiopogonanone A jointly increased the AUC(0-t), AUC(0-∞), and Cmax of schizandrin B, but they decreased its clearance in plasma and excretion in urine. CONCLUSIONS: Ginsenoside Re, Rd, and methylophiopogonanone A were the potential inhibitors of OAT1, and may interact with some drugs serving as OAT1 substrates clinically. Schizandrin B was a potential OAT1 substrate, and its OAT1-mediated transport was inhibited by ginsenoside Re, Rd, and methylophiopogonanone A. OAT1-mediated interactions of the main active components in SMF can be regarded as one of the important compatibility mechanisms of traditional Chinese medicine preparations.

Oxidation of methylophiopogonanone A on the surface of TLC plate.

Methylophiopogonanone A (MOPNA, 1) is a characteristic homoisoflavonoid, having two methyl groups on the A ring, isolated from Ophiopogon Root (enlarged part of the root of Ophiopogon japonicus Ker-Gawler, Liliaceae). Although MOPNA is chemically stable in various organic solvents, such as acetone, chloroform, methanol and dimethyl sulfoxide, it gave a spot of higher polarity in addition to the spot of MOPNA on the surface of TLC plate. The spot was isolated and the structure of the major compound was revealed to be a compound with an oxygen atom at C-6 of the A ring. This oxidation on the surface of TLC plate was observed for synthetic intermediates of MOPNA having two methyl groups on a phloroacetophenone-type ring, suggesting that the methyl groups enhanced susceptibility to air oxidation. The structure of the major oxidation product was similar to those of humulone and sufflomin A. Humulone has two prenyl units and sufflomin A has two C-glucosyl moieties instead of the two methyl groups, respectively. As dialkylated phloroacetophenone derivatives seem susceptible to oxidation, air oxidation might be involved in the formation of these compounds.

Spirostane saponins with a rearranged A/B ring system isolated from the rhizomes of Ophiopogon japonicus.

In this study, the popular food and medicinal herb Ophiopogon japonicus was investigated alongside a 70% ethanol extract of its rhizomes, revealing twenty-three steroidal glycosides with four undescribed steroidal saponins, named ophiopogonols A-D. Among them, ophiopogonols A-B are two unusual spirostanols with a rearranged A/B ring system (5/7/6/5/5/6 ring system) that have not previously been identified in plants. The chemical structures of all isolated steroidal glycosides were elucidated by comprehensive analysis through chemical methods, HRESIMS, and NMR spectroscopy. Further, putative biosynthetic pathways for ophiopogonols A-B were proposed. In addition, based on traditional applications of O. japonicus, cytotoxic effects of the isolates were evaluated using human large cell lung carcinoma cells (NCI-H460 cells). Sprengerinin C displayed a remarkable cytotoxic effect with IC50 values of 2.1 ± 0.8 µM by inducing apoptosis and G2/M phase cycle arrest in the NCI-H460 cell line.

MDG, an Ophiopogon japonicus polysaccharide, inhibits non-alcoholic fatty liver disease by regulating the abundance of Akkermansia muciniphila.

MDG, a polysaccharide derived from Ophiopogon japonicus, displays a protective effect against obesity and non-alcoholic fatty liver disease (NAFLD). However, there is no definitive evidence proving the specific mechanism of MDG against NAFLD. The results showed MDG supplementation ameliorated lipid accumulation, liver steatosis, and chronic inflammation in high-fat diet-induced NAFLD mice. Besides, MDG increased the abundance and diversity of microbial communities in the gut. These effects were mediated by the colonization of fecal microbiota. Further investigation revealed that Akkermansia muciniphila levels correlated negatively with NAFLD development, and lipid metabolism-related signaling might be the key regulator. Our study suggested that MDG treatment could inhibit obesity and the NAFLD process by modulating lipid-related pathways via altering the structure and diversity of gut microbiota. In addition, Akkermansia miniciphila might be a promising candidate in future research into NAFLD.

Ophiopogon japonicus inhibits white spot syndrome virus proliferation in vivo and enhances immune response in Chinese mitten crab Eriocheir sinensis.

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control. Herbal medicines have been widely used to treat a number of viral infections, which could offer a rich reserve for antiviral drug discovery. Here, we evaluated the inhibition activities of 30 herbal medicines against WSSV in Chinese mitten crab Eriocheir sinensis. A WSSV infection model in E. sinensis was firstly established in order to determine the antiviral effects of the plant extracts and to explore the potential action mechanisms. Results showed that the highest anti-WSSV activity was obtained by the treatment of Ophiopogon japonicus extract (93.03%, 100 mg/kg). O. japonicus treatment decreased viral loads in a dose-dependent manner and significantly improved the survival of WSSV-challenged crabs. O. japonicus reduced the expression of vital genes in viral life cycle in vivo, particularly for the immediate-early stage gene ie1. Further results indicated that O. japonicus could repress the JAK-STAT signaling pathway to block ie1 transcription. Moreover, O. japonicus could modulate certain immune genes such as the myosin, toll-like receptor, crustin, and prophenoloxidase in the interactions between WSSV and crabs. The up-regulated expression of pro-autophagic factors (Gabarap and Atg7) and elevated levels of antioxidant enzymes (SOD, CAT and GSH) suggested that O. japonicus may induce autophagy and attenuate WSSV-induced oxidative stress. Taken together, O. japonicus could inhibit WSSV proliferation and improve the survival of WSSV-challenged crabs. Thus, O. japonicus may have the potential to be developed as a preventive or therapeutic agent against WSSV, and its effective compounds merit further isolation and identification.

Paclobutrazol exposure induces apoptosis and impairs autophagy in hepatocytes via the AMPK/mTOR signaling pathway.

Paclobutrazol (PBZ), one of the most widely used plant growth retardants in vegetables, fruits, and traditional Chinese medicine ingredients, exposes people to adverse events. In this study, HepaRG hepatocytes were cultured and exposed to PBZ (360 µM) in vitro to determine its mechanism. Results showed that PBZ exposure inhibited cell viability in a time- and dose-dependent manner and increased the oxidative stress and apoptosis ratio in HepaRG cells. These data revealed that the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) has an important role in PBZ-induced cell apoptosis, which is mediated by impaired autophagy and blocked by the AMPK activator. In conclusion, PBZ exposure induces apoptosis and impairs autophagy in hepatocytes via the AMPK/mTOR signaling pathway.

Nontargeted metabolomic analysis and multiple criteria decision-making method induced robust quality markers screening for the authentication of herbal medicines from different origins by taking Ophiopogon japonicus (L. f.) Ker-Gawl. as a case study.

A key segment in medicinal plant authentication is the establishment of quality markers that embody the intrinsic metabolites difference independent of instruments and experiment conditions. A strategy integrating nontargeted metabolomics and multicriteria decision-making model for robust quality markers discovery is presented and applied to authenticate Ophiopogon japonicus (L. f.) Ker-Gawl. First, an ultra-performance liquid chromatography/quadrupole time-of-flight MSE approach was established for global metabolites profiling and identification. Second, multivariate statistical analysis was performed to explore potential quality markers of different origins of ophiopogonis radix. Third, potential quality markers were ordered and filtered by multicriteria decision-making model to infer robust quality markers and further validated in different instruments and experiment conditions by validation model. Fourth, the validation model using the robust quality markers managed to discriminate the origins of ophiopogonis radix samples procured from the herbal markets. Consequently, two robust quality markers, cixi-ophiopogon B and ophiopogonin D, were discovered and further validated on different instruments and experiment conditions. This integrated strategy provided a practical solution for reliable and convenient authentication of geo-authentic herb.

Genomic Sequence Resource of Alternaria alternata Strain B3 Causing Leaf Blight on Ophiopogon japonicus.

Black spot of Ophiopogon japonicus is an economically important disease, which can result in significant losses of both yield and quality of this traditional Chinese medicinal plant. The disease is caused by the small-spored fungal pathogen Alternaria alternata, a necrotrophic fungus that is ubiquitously distributed in the environment. Here, we present the draft whole-genome sequence of A. alternata strain B3 that caused black spot on O. japonicus. The assembly consists of 76 contigs with an estimated genome size of 33.8 Mb. Furthermore, we identified genes that may be associated with the pathogenicity, such as carbohydrate-active enzymes, secreted proteins, and secondary metabolite gene clusters. This genome resource will provide a useful source for future research on the evolution of pathogenicity of A. alternata and phylogenomic analysis in delineating phylogenetic lineages within the genus Alternaria.

First Draft Genome Sequence Resource of Colletotrichum liriopes Causing Leaf Anthracnose on Ophiopogon japonicus.

Leaf anthracnose of Ophiopogon japonicus is an important disease that can significantly reduce the quality and economic value of this traditional Chinese medicinal plant. The disease is caused by Colletotrichum liriopes, a necrotrophic fungus that belongs to the Glomerellaceae family of the Sordariomycetes class. Here, we present the draft whole-genome sequence of the C. liriopes strain A2 that caused leaf anthracnose on O. japonicus. The assembly consists of 407 contigs with an estimated genome size of 53.1 Mb. Furthermore, we identified 670 carbohydrate-active enzymes, 1,377 secreted proteins, and 60 secondary metabolite gene clusters, which may be associated with the pathogenicity of this pathogen. This genome resource will provide a valuable resource for future research on the pathogenesis of C. liriopes and comparative genome analyses within genus Colletotrichum.

Differences in the Hemolytic Behavior of Two Isomers in Ophiopogon japonicus In Vitro and In Vivo and Their Risk Warnings.

Ophiopogonin D (OPD) and Ophiopogonin D' (OPD') are two bioactive ingredients in Ophiopogon japonicus. Previously published studies have often focused on the therapeutic effects related to OPD's antioxidant capacity but underestimated the cytotoxicity-related side effects of OPD', which may result in unpredictable risks. In this study, we reported another side effect of OPD', hemolysis, and what was unexpected was that this side effect also appeared with OPD. Although hemolysis effects for saponins are familiar to researchers, the hemolytic behavior of OPD or OPD' and the interactions between these two isomers are unique. Therefore, we investigated the effects of OPD and OPD' alone or in combination on the hemolytic behavior in vitro and in vivo and adopted chemical compatibility and proteomics methods to explain the potential mechanism. Meanwhile, to explain the drug-drug interactions (DDIs), molecular modeling was applied to explore the possible common targets. In this study, we reported that OPD' caused hemolysis both in vitro and in vivo, while OPD only caused hemolysis in vivo. We clarified the differences and DDIs in the hemolytic behavior of the two isomers. An analysis of the underlying mechanism governing this phenomenon showed that hemolysis caused by OPD or OPD' was related to the destruction of the redox balance of erythrocytes. In vivo, in addition to the redox imbalance, the proteomics data demonstrated that lipid metabolic disorders and mitochondrial energy metabolism are extensively involved by hemolysis. We provided a comprehensive description of the hemolysis of two isomers in Ophiopogon japonicus, and risk warnings related to hemolysis were presented. Our research also provided a positive reference for the development and further research of such bioactive components.

Chemical Comparison of Ophiopogonis radix and Liriopes radix Based on Quantitative Analysis of Multiple Components by HPLC Coupled with Electrospray Ionization Tandem Triple Quadrupole Mass Spectrometry.

BACKGROUND: Ophiopogonis radix and Liriopes radix are well known for the treatment of dry coughs and phthisis. Liriopes radix is occasionally used as a substitute for Ophiopogonis radix in various prescriptions due to the extremely similar pharmacological activities and clinical efficacies, but they are regarded as two different remedies in the Chinese Pharmacopoeia. Accordingly, the establishment of a reliable analytical approach for the discrimination and quality evaluation of Ophiopogonis and Liriopes is required. OBJECTIVE: To establish a simple, accurate, and reliable method that can simultaneously determine multiple components in Ophiopogonis radix and Liriopes radix. To comprehensively compare the chemical compositions of the two herbs and find markers for discrimination and quality assessments. METHOD: An HPLC-ESI-triple quadrupole (QQQ)-MS/MS method was developed for simultaneous characterization and quantification of chemical components in the two herbs. The results were further analyzed by PLS discriminant analysis to provide more information about the chemical differences, as well as to evaluate the quality of each sample. RESULTS: A total of 23 compounds have been characterized and quantified in 31 batches of herbs from different geographical regions, among which liriopesides B, sprengerinin A, ophiopogonin B, and ophiopogonanone E contribute mostly. The contents of homoisoflavonoids were much higher in Ophiopogonis radix than in Liriopes radix, but the levels of steroidal saponins followed a contrary trend. CONCLUSIONS: Simultaneous determination of multiple components by HPLC-QQQ-MS/MS coupled with chemometrics analysis is an acceptable strategy to evaluate and control the quality of Ophiopogonis radix and Liriope radix. HIGHLIGHTS: Simultaneous determination of 12 steroidal saponins and 11 homoisoflavonoids in both Ophiopogonis radix and Liriope radix by using HPLC-QQQ-MS/MS in positive ion mode, as well as the quality control study.

Investigating the authenticity of Ophiopogonis Radix and its Chinese patent medicines by using a nucleotide signature.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonis Radix (Maidong), derived from the dried root tuber of Ophiopogon japonicus (Thunb.) Ker Gawl., has been widely used in the treatment of chronic inflammatory and cardiovascular diseases. However, Ophiopogonis Radix is often adulterated with some species because of morphological similarities. Adulterants circulating in herbal markets are a latent threat to the clinical safety and consumers' interest. AIM OF THE STUDY: We aimed to develop a nucleotide signature for identification of Ophiopogonis Radix and its Chinese patent medicines. MATERIALS AND METHODS: A total of 255 ITS2 sequences representing 39 species and 4 varieties were used to develop a nucleotide signature of Ophiopogonis Radix. The nucleotide signature was used to investigate 17 commercial crude drugs and eight batches of Chinese patent medicines. RESULTS: A 69 bp nucleotide signature unique to Ophiopogonis Radix was found. The survey revealed that 2 of 17 crude drug samples were adulterants detected as Liriopes Radix (Shanmaidong). Fortunately, no adulterants were detected in the eight batches of Chinese patent medicines. CONCLUSIONS: The newly developed nucleotide signature could be efficiently applied to identify Ophiopogonis Radix and its Chinese patent medicines, aiding in the authentication, quality control, and supervision of processed products in herbal markets.