Application of two-dimensional reversed phase countercurrent chromatography × high-performance liquid chromatography to bioactivity-guided screening and isolation of α-glucosidase inhibitors from Rheum palmatum L.

In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α-glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation (1D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis (2D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1H NMR and 13C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4'-O-(6″-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α-glucosidase inhibitors in Rheum palmatum L. for the first time.

Anti-Aging of the Nervous System and Related Neurodegenerative Diseases With Chinese Herbal Medicine.

Human beings have always pursued a prolonged lifespan, while the aging of the nervous system is associated with a large variety of diseases. Pathological aging of the nervous system results in a series of neurodegenerative diseases and can cause disability and death in the elderly. Therefore, there is an urgent need for the prevention and treatment of nervous system aging. Chinese herbal medicines have a long history, featuring rich and safe ingredients, and have great potential for the development of anti-aging treatment. We searched the publications on PubMed with key words "anti-aging of the nervous system" and "Chinese herbal medicine" in recent 10 years, and found sixteen Chinese herbal medicines. Then by comparing their popularity of use as well as active components based on the research articles, five common Chinese herbal medicines namely Ginseng Radix, Lycii Fructus, Astragali Radix, Coptidis Rhizoma and Ginkgo Folium, were confirmed to be the most related to anti-nervous system aging and neural degenerative diseases. At the same time, the active ingredients, research models, action mechanisms and curative effects of these five common Chinese herbal medicines were reviewed. From the five common Chinese herbal medicines reviewed in this paper, many encouraging effects of Chinese herbal medicines on treating nervous system aging and related diseases were revealed and more potent herbs would be explored with the help of the proposed possible mechanisms.

Matrix solid-phase dispersion combined with micro-fractionation bioactivity evaluation screening polymethoxylated flavones from Citrus peels.

Polymethoxyflavones were a unique class of natural and safe flavonoids containing two or more methoxy groups, which were also the most abundant edible part in Citrus peel. The optimum condition in the process of selective extraction of polymethoxylated flavones from Citrus peel by matrix solid-phase dispersion (MSPD) was as follows: SBA-15 as adsorbent, ethyl acetate as eluent, the mass ratio of adsorbent to sample 1:1, and the mixture of sample and adsorbent was ground for 3 min. Twelve antioxidants were successfully screened by micro-fractionation bioactivity evaluation assay, in which four of them were flavonoid glycosides, seven of them were polymethoxylated flavones, and one was phenylpropanoid. 1-sinapoly-ß-D-glucopyranoside (1) was reported for the first time in Citrus peel. And antioxidant capacity of 1-sinapoly-ß-D-glucopyranoside, 5, 7, 8, 3', 4', 5'-hexamethoxyflavone (6), hexamethoxyflavone (11), and 5, 6, 7, 4'-tetramethoxyflavone (7) were reported for the first time. Nobiletin (compound 8), 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone (9) and tangeretin (10) were isolated and purified by countercurrent chromatography combined with preparative liquid chromatography. Antioxidant activity evaluation indicated that the three isolated polymethoxylated flavones owned similar antioxidant activity. This study indicated that MSPD combined with micro-fractionation bioactive evaluation was efficient in screening bioactive compounds for rapid extraction and effective pinpointing bioactive substances in natural products.

A comprehensive strategy for prediction and quality evaluation of standardized planting herbs based on plant metabolomics coupled with extreme learning machine: Astragali Radix as an example.

INTRODUCTION: Standardizing the planting process is an effective way to control the quality stability of herbal resources, which are susceptible to external environmental factors (e.g., moisture, soil, etc.). However, how to scientifically and comprehensively assess the effects of standardized planting on plant quality and quickly test unknown samples has not been addressed. OBJECTIVE: The aim of this study was to determine and compare the metabolite levels of herbs before and after standardized planting, to quickly distinguish their sources, and to evaluate their quality, using the typical herb Astragali Radix (AR) as an example. METHODS: In this study, an efficient strategy using liquid chromatography-mass spectrometry (LC-MS) based on plant metabolomics combined with extreme learning machine (ELM) has been developed to efficiently distinguish and predict AR after standardized planting. Moreover, a comprehensive multi-index scoring method has been developed for the comprehensive evaluation of the quality of AR. RESULTS: The results confirmed that AR after standardized planting was significantly differentiated, with a relatively stable content of 43 differential metabolites, mainly including flavonoids. An ELM model was established based on LC-MS data, and the accuracy in predicting unknown samples could reach more than 90%. As expected, higher total scores were obtained for AR after standardized planting, indicating much better quality. CONCLUSION: A dual system for evaluating the impact of standardized planting on the quality of plant resources has been established, which will significantly contribute to innovation in the quality evaluation of medicinal herbs and support the selection of optimal planting conditions.

Recent discovery of tyrosinase inhibitors in traditional Chinese medicines and screening methods.

ETHNOPHARMACOLOGICAL RELEVANCE: Tyrosinase, the key rate-limiting enzyme for melanogenesis, is one of the main targets for skin senescence and some pigmented skin diseases, such as albinism and melanoma. Tyrosinase inhibitors are capable of reducing melanin generation and deposition in the skin through blocking the reaction chain of formation. Thus, it has been used for anti-melanoma and showed the potential to be developed into novel skin whitening and spot removing products. With the trend of back-to-nature, natural tyrosinase inhibitors are receiving more and more attention. Traditional Chinese medicines (TCMs) as the promising source of novel chemotypes and pharmacophores, are huge treasures for the discovery of natural tyrosinase inhibitors characterized with green, safe, and highly efficient. AIM OF THIS REVIEW: This review aims to provide a systematic overview of natural tyrosinase inhibitors and a detailed summary of the types of TCMs from which they originate. In addition, this paper also highlights the screening methods developed for exploring tyrosinase inhibitors in recent years, compares the advantages and disadvantages of various methods under the guidance of different screening principles, and predicts their applications in the future. MATERIALS AND METHODS: Relevant literature have been obtained using the keywords "tyrosinase inhibitors", "traditional Chinese medicines", "whitening", and "screening" in scientific databases, such as "PubMed", "SciFinder", "Web of Science", "Elsevier", "China Knowledge Resource Integrated databases". Information was also collected from Chinese pharmacopoeia, Chinese herbal classics books, "Google Scholar", "Baidu Scholar", and other literature sources, etc. RESULTS: An overview about the tyrosinase inhibitors derived from TCMs since 2002 has been compiled via the above-mentioned sources. Up to now, 186 components, mainly belonging to flavonoids, lignans, terpenoids, Diels-Alder adducts, simple phenylpropanoids and stilbenes, from 61 kinds of TCMs have been reported to possess tyrosinase inhibitory activity, among which flavonoids are mainly focused on. Furthermore, on the basis of bioactive detection strategies, the screening methods for tyrosinase inhibitors have been classified into bioaffinity-based, intrinsic enzymatic-based, and computer-aided drug design (CADD). Precisely because screening approaches are essential for rapid identification of tyrosinase inhibitors from TCMs, the principles, advantages and disadvantages, and specific applications of each method are presented along with a comparison of applicability. CONCLUSIONS: The summary of TCMs-derived inhibitors gives a clue on the discovery of candidates with the property to whiten the skin. Meanwhile, the outlook of developed screening methods provides technical references for the efficient exploration of safer and more effective tyrosinase inhibitors from TCMs.

High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities.

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.

Comprehensive two-dimensional countercurrent chromatography × gas chromatography characterization of Artemisia argyi essential oil.

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

[Comparison on volatile components between Artemisiae Verlotori Folium and Artemisiae Argyi Folium based on GC-MS and chemometrics].

Artemisiae Argyi Folium is commonly used in clinical practice. Artemisiae Verlotori Folium, the dried leaves of Artemisia verlotorum, is often used as a folk substitute for Artemisiae Argyi Folium in Lingnan area. In this study, gas chromatography-triple quadrupole mass spectrometry(GC-MS) was used to detect the volatile oil components of 27 samples of Artemisiae Verlotori Folium and 13 samples of Artemisiae Argyi Folium, and the volatile components were compared between the two species. The internal standard method was combined with multi-reaction monitoring mode(MRM) to determine the content of six major volatile components. Hierarchical clustering analysis(HCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were carried out for the content data. The results showed that the Artemisiae Argyi Folium samples had higher content and more abundant volatile oils than the Artemisiae Verlotori Folium samples. Artemisiae Argyi Folium mainly had the components with lower boiling points, while Artemisiae Verlotori Folium mainly had the components with higher boiling points. Terpenoids were the main volatile components in Artemisiae Verlotori Folium(mainly sesquiterpenoids) and Artemisiae Argyi Folium(monoterpenoids). In addition, Artemisiae Argyi Folium had higher content of oxygen-containing derivatives than Artemisiae Verlotori Folium. Furthermore, the stoichiometric analysis showed that the two species could be distinguished by both HCA and OPLS-DA, indicating that the volatile components of the two were significantly different. This study can provide a scientific basis for the quality evaluation and data support for the local rational application of Artemisiae Verlotori Folium in Lingnan.

[Historical evolution of Xuanfu Daizhe Decoction].

Xuanfu Daizhe Decoction, first seen in Zhang Zhongjing's Treatise on Cold Damage Diseases, was composed of seven medicinal materials: Inulae Flos, Glycyrrhizae Radix, Ginseng Radix, Zingiberis Rhizoma Recens, Haematitum, Pinelliae Rhizoma and Jujubae Fructus. It was used to treat gastric fullness and hardness and belching due to the wrong treatment of typhoid fever and sweating. With detailed records and description in ancient medical books, Xuanfu Daizhe Decoction was widely adopted in clinical practice by physicians of later generations, which expanded its main therapeutic functions. By comprehensive collation of ancient and modern literature on Xuanfu Daizhe Decoction, this paper systematically explored the historical evolution of the prescription from the source, composition, dosage, processing, clinical application, function interpretation and decocting method. It was found that the composition and processing method of the prescription in the past dynasties were relatively consistent, and there was a gradual decrease in dosage in clinical application. In ancient times, Xuanfu Daizhe Decoction was mainly used to treat nausea, vomiting, hiccups, constipation, etc., while modern clinicians mainly used it for digestive diseases such as reflux esophagitis and gastritis. Through the analysis and textual research, the composition, dosage, processing, function evolution and decocting method of this prescription were determined, which provided reference for the research and development of compound preparations of Xuanfu Daizhe Decoction.

Discovery of potential hypoglycemic metabolites in Cassiae Semen by coupling UHPLC-QTOF-MS/MS combined plant metabolomics and spectrum-effect relationship analyses.

Cassiae Semen (CS) is consumed as fried tea or medicinal food in Asian areas. Its two commercial forms, namely raw and fried CS, exert different clinical applications, in which fried CS is commonly applied as a functional tea for losing weight. To prevent confusion in the use of the two forms of CS, a comprehensive strategy by combining plant metabolomics and spectrum-effect relationship analyses was developed for the fast and efficient discrimination of raw and fried CS, and further for the discovery of the potential hypoglycemic metabolites of CS to control its quality. First, the plant metabolic profiling of raw and processed samples was performed by UHPLC-QTOF-MS/MS. A total of 1111 differential metabolites were found to well distinguish the raw and fried CS after analyzing by MPP software. Subsequently, α-glucosidase inhibition of raw and fried CS was investigated. As a result, fried CS demonstrated much stronger α-glucosidase inhibition activity than the raw sample. By analyzing the spectrum-effect relationship with GRA, BCA, and PLSR, 14 potential hypoglycemic-related compounds were discovered. As anticipated, they were also found as the differential metabolites of the raw and fried samples with a potential hypoglycemic effect, which might be beneficial for the quality control of CS tea. Additionally, molecular docking analysis was conducted to reveal the underlying inhibition mechanisms of the four most critical constituents, including physcion, chrysoobtusin, aurantio-obtusin, and obtusifolin. This study provides a powerful tool for the discrimination of processed samples and fast screening of the active constituents in complex natural products on a high-throughput basis.

A Strategy for Pinpointing Natural Bioactive Components Using Two-Dimensional Bioassay Profilings Combined with Comprehensive Two-Dimensional Countercurrent Chromatography × High-Performance Liquid Chromatography.

Inspired by the interpretation of two-dimensional (2D) nuclear magnetic resonance spectra, an efficient strategy was proposed for pinpointing bioactive components from complex natural products. An off-line comprehensive countercurrent chromatography (CCC) × high-performance liquid chromatography (HPLC) was employed to achieve a 2D chemical chromatogram, and 2D bioassay profilings were obtained from bioassays of the eluent of the first dimension (1D) CCC and the eluent of the second dimension (2D) HPLC. Then 2D chemical chromatograms and 2D bioassay profilings were matched for pinpointing bioactive natural components from complex matrices. Thus, bioactive components in a complex matrix could be efficiently analyzed, separated, and bioactivity-determined. This experimental scheme was successfully demonstrated with a traditional medicinal herb Polygonum cuspidatum Sieb. et Zucc. The feasibility of this 2D strategy was verified with tyrosinase inhibition assay, α-glucosidase inhibition assay, DPPH radical scavenging assay, and ABTS•+ decolorization assay. Eight natural inhibitors were successfully pinpointed and identified from P. cuspidatum. Both pieceid-2″-O-gallate (10) and vanicoside B (20) were screened and identified as natural tyrosinase inhibitors for the first time. Meanwhile, vanicoside B (20) was also found as the strongest α-glucosidase inhibitor among all the isolated components. Most of the compounds exhibited much higher radical scavenging activities. Compared with traditional methodology based on one-dimensional chromatographic separation, the present 2D strategy would be more precise, efficient, and convenient to screen and separate bioactive compounds from complex matrices.

In silico screening of off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography for four saponins isolation.

Being restrained by the limited peak capacity, one-dimensional chromatography usually leads to an unsatisfactory separation with low purity of compounds in a complex mixture. To obtain more highly pure targets for standard reference and to discover new substances for structural elucidation, two-dimensional chromatography is more and more prevalent in many fields. As few metrics on assessment of the preparative capability of two-dimensional chromatographic separations are reported, a methodology of in silico screening of various two-dimensional chromatographic separations with a minimal number of experiments was demonstrated in this work, which was based on three descriptors including the occupation rate of peaks and system homogeneity of a two-dimensional separation space, and the minimal distance of all nearest-neighbor distances of peaks. Combining the advantages of counter-current chromatography and liquid chromatography, we elaborated the methodology by employing off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography to be in silico screened for separation of four saponins from Panax notoginseng at an analytical scale to simulate the case of preparative scale transfer. The predictive results were presented by two-dimensional contour plots and verified by experiments. The result showed that the experimental results were in general accord with the predictive results.

Beneficial effects of monounsaturated fatty acid-rich blended oils with an appropriate polyunsaturated/saturated fatty acid ratio and a low n-6/n-3 fatty acid ratio on the health of rats.

BACKGROUND: The effects of dietary fat on health are influenced by its fatty acid profile. We aimed to determine the effects of monounsaturated fatty acid (MUFA)-rich blended oils (BO) containing a balance of polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) and with a low n-6/n-3 PUFA ratio on the health of rats fed normal or high-fat diets. The BO was obtained by mixing red palm oil, rice bran oil (RO), tea seed oil and flaxseed oil in appropriate proportions. RESULTS: BO consumption reduced the serum low-density lipoprotein cholesterol (LDL-C), non-esterified fatty acid (NEFA), insulin (INS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), high-sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), lipid peroxide (LPO) and oxidized LDL (ox-LDL) concentrations and the homeostasis model assessment of insulin resistance (HOMA-IR); it increased the high-density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) concentrations, and the bone mineral density (BMD) versus control oil-containing normal and high-fat diets. BO also reduced the triglyceride (TG), hs-CRP, MDA, ox-LDL and reactive oxygen species (ROS) concentrations; and increased the serum HDL-C and SOD, and BMD versus RO-containing high-fat diets. Finally, BO reduced the glucose (GLU) and INS, and HOMA-IR; it increased HDL-C, SOD, femoral weight and BMD versus RO-containing normal diets. CONCLUSION: BOs with an appropriate fatty acid profile have beneficial effects on the glucolipid metabolism, inflammation, oxidative stress and bone quality of rats when included in both normal and high-fat diets. © 2022 Society of Chemical Industry.

A review on the chemical constituents and pharmacological efficacies of Lindera aggregata (Sims) Kosterm.

Lindera aggregata (Sims) Kosterm. (L. aggregata), which belongs to the genus Lindera in the family Lauraceae, is widely distributed in Asia and the temperate, tropical regions of North America. Its roots and leaves have been used for thousands of years as traditional Chinese medicine and/or functional food. To further explore its underlying nutritional value, this review provided a comprehensive insight into chemical constituents and pharmacological effects on L. aggregata. The phytochemical investigation of different parts of L. aggregata led to the identification of up to 349 components belonging to sesquiterpenoids, alkaloids, flavonoids, essential oils, and other compounds. Among them, sesquiterpenoids, flavonoids, and alkaloids are assessed as representative active ingredients of L. aggregata. A wide variety of pharmacological effects of L. aggregata, such as anti-hyperlipidemic, anti-tumor, anti-inflammatory, analgesic, and anti-oxidant, have been proved in vitro and in vivo. In summary, this review aims to provide a scientific basis and reference for further research and utilization of L. aggregata and lay the foundation for developing functional foods with potential active ingredients for the prevention and management of related diseases.

A novel high-resolution monophenolase/diphenolase/radical scavenging profiling for the rapid screening of natural whitening candidates from Peaonia lactiflora root and their mechanism study with molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Paeonia lactiflora is a traditionally-used whitening medicine in China for thousands of years. Although some tyrosinase inhibitors and/or antioxidants such as 1,2,3,4,6-pentagalloylglucose, gallic acid, have been isolated and identified, their tyrosinase inhibition pathway (monophenolase or diphenolase inhibition, or both two) have not been systematically studied and the underlying tyrosinase inhibition mechanism has not been revealed yet. Moreover, the exploring of new natural tyrosinase inhibitors and antioxidants is urgently needed. AIM OF THE STUDY: This review aimed to develop a new microplate-based high-resolution tyrosinase inhibition profiling assay and establish a furthermore triple high-resolution monophenolase/diphenolase/radical scavenging profiling for accelerating identification bioactive compounds from complicated plant extract. MATERIALS AND METHODS: The targeted isolation and structure elucidation were performed with high-performance liquid chromatography-high-resolution mass spectrometry and preparative high-performance liquid chromatography. It allows to be a proof of concept with the root of Paeonia lactiflora crude extract as a natural whitening herbal drug. RESULTS: The result showed that galloylpaeoniflorin specifically inhibited monophenolase activity. While 1,2,3,4,6-pentagalloylglucose, gallic acid and catechin demonstrated the inhibition towards both monophenolase and diphenolase. Among them, 1,2,3,4,6-pentagalloylglucose can inhibit monophenolase activity was reported for the first time. In addition, antioxidant properties were attributed to catechin, 1,2,3,4,6-pentagalloylglucose and gallic acid. Due to its low content and complicated configuration in the root of Paeonia lactiflora, a new potential tyrosinase inhibitor and radical scavenger which tentatively identified as hexagalloylglucose by high-resolution MS was still need further verification. What's more, the molecular docking unveiled that bioactive enzymatic inhibitors mainly interacted with amino acid catalytic residues of tyrosinase via H-bonds and van der wals, which may be helpful to understand their inhibition mechanisms with tyrosinase in the skin whitening. CONCLUSIONS: The platform provided a promising and efficient strategy for the rapid screening of whitening active components from natural sources.

New insights into metabolomics profile generation in fermented tea: the relevance of bacteria and metabolites in Fuzhuan brick tea.

BACKGROUND: The contribution of bacteria to fermented tea is not clear and the associated research is relatively limited. To reveal the role of microorganisms in fermented tea processing, the microbial community and metabolites of Fuzhuan brick tea (FBT), a Chinese traditional fermented tea, were revealed via high-throughput sequencing and liquid chromatography-mass spectrometry (LC-MS). RESULTS: In FBT, bacterial communities had a higher abundance and diversity, Lactococcus and Bacillus were the main bacteria, and Eurotium was the predominant fungus. The predictive metabolic function indicated the pathways of cellular growth, environmental information, genetics and material metabolism of bacterial communities were abundant, whereas the fungal community predictive metabolic function was almost saprotroph. Using LC-MS, 1143 and 536 metabolites were defined in positive and negative ion mode, respectively. There were essential correlations between bacterial populations and metabolites, such that Bacillus was correlated significantly with 44 metabolites (P < 0.05) and Enterococcus was significantly associated with 15 metabolites (P < 0.05). Some of the main active components were significantly correlated with the bacteria, such as Enterococcus, Lactococcus and Carnobacterium. CONCLUSION: Not only Eurotium, but also the bacteria were involved in the changes of metabolomics profile in fermented FBT. The present study assists in providing new insights into metabolomics profile generation in fermented tea. The present research lays a foundation for controlling the FBT fermentation by artificial inoculation to improve quality. © 2021 Society of Chemical Industry.

Herbal Textual Research on Coptidis Rhizoma in Famous Classical Formulas / 中国实验方剂学杂志

Through the combing of ancient books of Chinese herbal medicine in the past dynasties， a textual research of Coptidis Rhizoma involved the name， origin， medicinal parts， producing area， quality evaluation， harvesting and processing methods in famous classical formulas was conducted in this paper. After textual research， the mainstream varieties of Coptidis Rhizoma in the Ranunculaceae family before Tang and Song dynasties were Coptis chinensis and C. chinensis var. brevisepala， after the Ming and Qing dynasties， C. deltoidea， C. teeta and C. omeiensis were gradually praised. In ancient times， the authentic producing area of Coptidis Rhizoma has the characteristics of gradually moving to the west. The eastern Coptidis Rhizoma was highly praised in the early stage， while in the later stage， western Coptidis Rhizoma like chicken feet was highly praised. In the early stage， western Coptidis Rhizoma probably originated from C. chinensis and its genus， while Coptidis Rhizoma like chicken feet was cultivated， and no wild species has been found so far. As Coptidis Rhizoma has mixed use of multiple origins in ancient books of past dynasties， based on the current shortage of market resources in C. teeta and C. deltoidea， there are also endangered and protected plants of C. chinensis var. brevisepala and C. omeiensis， combined with the mainstream medicines and resources of past generations， it is recommended to choose C. chinensis as the base of the formulas. In ancient times， there were many processing methods for Coptidis Rhizoma， such as frying and wine-， ginger-， honey-processed. In the process of developing famous classical formulas， the appropriate processing specifications of Coptidis Rhizoma should be selected based on the original source records and the requirements of the medicinal material.

[Herbalogical study on historical evolution of collection, processing and efficacy of Citri Exocarpium Rubrum and Citri Grandis Exocarpium].

In ancient times, the original plants of Citri Exocarpium Rubrum and Citri Grandis Exocarpium had experienced succession and change, including tangerine(Citrus reticulata), pomelo(C. grandis), and Huazhou pomelo(C. grandis 'Tomentosa'), a specific cultivar of C. grandis produced in Huazhou, Guangdong. Before the Qing Dynasty, tangerine was the main original plant, while Huazhou pomelo came to the fore in the Qing Dynasty. In the 1950 s and 1960 s, the producing area of Huazhou pomelo was destroyed, and thus it had to be supplemented with pomelo. From then on, C. grandis 'Tomentosa' and C. grandis were both listed as the original plants of Citri Grandis Exocarpium in the Chinese Pharmacopoeia. This paper reviewed the historical evolution of the collection, processing, and efficacy of Citri Exocarpium Rubrum and Citri Grandis Exocarpium. The research showed that:(1)The harvest time of the original plants of Citri Grandis Exocarpium and Citri Grandis Exocarpium had changed from maturity to immaturity. The collection and processing of Citri Exocarpium Rubrum was first recorded in the Illustrated Classics of Materia Medica in the Song Dynasty. During the Ming and Qing Dynasties, the mesocarp of Citri Exocarpium Rubrum needed to be removed completely, and Citri Grandis Exocarpium from C. grandis 'Tomentosa' was processed into different specifications such as seven-piece, five-piece, and single piece. Furthermore, processed young fruits of Huazhou pomelo appeared.(2)Citri Exocarpium Rubrum and Citri Grandis Exocarpium were processed with carp skin for the first time in the Master Lei's Discourse on Medicinal Processing. It was suggested that carp skin might be helpful for eliminating bones stuck in throat. During the Song, Jin, and Yuan Dynasties, some other processing methods such as ba-king, stir-frying, and salt-processing appeared. Honey, soil, ginger juice, and alum were firstly used as adjuvants for the processing in the Ming and Qing Dynasties. Citri Exocarpium Rubrum was mainly prepared with salt in order to improve the effect of lowering Qi, while it was unnecessary for Citri Grandis Exocarpium from C. grandis 'Tomentosa' because of its obvious effect of lowering Qi and eliminating phlegm. The stir-frying and honey-frying methods helped reduce the strong effect of Citri Grandis Exocarpium from C. grandis 'Tomentosa'.(3)According to the application of Citri Exocarpium Rubrum and Citri Grandis Exocarpium in history, their medicinal use began in Han and Tang Dynasties, developed in Song, Jin, and Yuan Dynasties, and matured in Ming and Qing Dynasties. Citri Grandis Exocarpium from C. grandis 'Tomentosa' was originally applied in Ming and Qing Dynasties, and it still plays an important in role treating COVID-19 nowadays. Moreover, Citri Grandis Exocarpium from C. grandis had cold medicinal property, while Citri Grandis Exocarpium from C. grandis 'Tomentosa' had warm medicinal property, and thus they should not be treated the same. At present, Huazhou pomelo has a certain production scale. Therefore, it is recommended that in the next edition of Chinese Pharmacopoeia, only C. grandis 'Tomentosa' should be included as the original plant of Citri Grandis Exocarpium, and C. grandis should be deleted. The results are conducive to the further development and utilization of Citri Exocarpium Rubrum and Citri Grandis Exocarpium, and support the rational use of Citri Grandis Exocarpium and its processed products.

TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization.

BACKGROUND: To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization. METHODS: TLR2-/- and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iµ-Cµ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iµ-Cγ, Iµ-Cα or Iµ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2-/- mice (with or without IL21 treatment). RESULTS: The lungs of TLR2-/- mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2-/- compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2-/- mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency. CONCLUSION: These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

[Textual research principles and suggestions on function and indication of ancient classical formulas].

Indication and functions is an important key information in the research and development of the ancient classical formulas, which directly affects the clinical positioning of the compound formulas and their reasonable, effective and safe use after marke-ting. It is also the embodiment of the ultimate vitality of ancient classical formulas. Due to the particularity of ancient classical formulas, it is of great significance to accurately define and describe the functions and indications of classical formulas to exert their unqiue advantages. Based on the analysis of the provenances, classification of clinical indications, and functions of 100 prescriptions in the Catalogue of Ancient Classical Formulas(First Batch), this paper summarized the incompleteness, irregularity and inconsistency in the original text, the differences in terminology between ancient and modern times, and the evolution of the meanings of prescriptions in different dynasties. In addition, under the guidance of the general principle of textual researh on ancient classical formulas, which is to inherit the essence, make the past serve the present, link up the past and the present, and bulid consensus, this paper proposed the following ideas: respecting the original meanings and provenances of ancient classical formulas, taking full consideration of the development and evolution, serving the current clinical application and health needs, accurately linking up the ancient and modern terminologies, standardizing the expression of traditional Chinese medicine(TCM) terminology, highlighting the characteristics of TCM, attaching importance to the textual research principles and suggestions of post-marketing evidence-based and clinical positioning research, so as to determine the the functions and indications of the ancient classical formulas in a scientific, standardized and reasonable way and better guide the clinical application of the classical formulas.