Effect of nitrogen reduction by chemical fertilization with green manure (Vicia sativa L.) on soil microbial community, nitrogen metabolism and and yield of Uncaria rhynchophylla by metagenomics.

Uncaria rhynchophylla is an important herbal medicine, and the predominant issues affecting its cultivation include a single method of fertilizer application and inappropriate chemical fertilizer application. To reduce the use of inorganic nitrogen fertilization and increase the yield of Uncaria rhynchophylla, field experiments in 2020-2021 were conducted. The experimental treatments included the following categories: S1, no fertilization; S2, application of chemical NPK fertilizer; and S3-S6, application of chemical fertilizers and green manures, featuring nitrogen fertilizers reductions of 0%, 15%, 30%, and 45%, respectively. The results showed that a moderate application of nitrogen fertilizer when combined with green manure, can help alleviate soil acidification and increase urease activity. Specifically, the treatment with green manure provided in a 14.71-66.67% increase in urease activity compared to S2. Metagenomics sequencing results showed a decrease in diversity in S3, S4, S5, and S6 compared to S2, but the application of chemical fertilizer with green manure promoted an increase in the relative abundance of Acidobacteria and Chloroflexi. In addition, the nitrification pathway displayed a progressive augmentation in tandem with the reduction in nitrogen fertilizer and application of green manure, reaching its zenith at S5. Conversely, other nitrogen metabolism pathways showed a decline in correlation with diminishing nitrogen fertilizer dosages. The rest of the treatments showed an increase in yield in comparison to S1, S5 showing significant differences (p < 0.05). In summary, although S2 demonstrate the ability to enhance soil microbial diversity, it is important to consider the long-term ecological impacts, and S5 may be a better choice.

Chloroplast genome characterization of Uncaria guianensis and Uncaria tomentosa and evolutive dynamics of the Cinchonoideae subfamily.

Uncaria species are used in traditional medicine and are considered of high therapeutic value and economic importance. This work describes the assembly and annotation of the chloroplast genomes of U. guianensis and U. tomentosa, as well as a comparative analysis. The genomes were sequenced on MiSeq Illumina, assembled with NovoPlasty, and annotated using CHLOROBOX GeSeq. Addictionaly, comparative analysis were performed with six species from NCBI databases and primers were designed in Primer3 for hypervariable regions based on the consensus sequence of 16 species of the Rubiaceae family and validated on an in-silico PCR in OpenPrimeR. The genome size of U. guianensis and U. tomentosa was 155,505 bp and 156,390 bp, respectively. Both Species have 131 genes and GC content of 37.50%. The regions rpl32-ccsA, ycf1, and ndhF-ccsA showed the three highest values of nucleotide diversity within the species of the Rubiaceae family and within the Uncaria genus, these regions were trnH-psbA, psbM-trnY, and rps16-psbK. Our results indicates that the primer of the region ndhA had an amplification success for all species tested and can be promising for usage in the Rubiaceae family. The phylogenetic analysis recovered a congruent topology to APG IV. The gene content and the chloroplast genome structure of the analyzed species are conserved and most of the genes are under negative selection. We provide the cpDNA of Neotropical Uncaria species, an important genomic resource for evolutionary studies of the group.

Study on material basis and anti-hypertensive metabolomics of different extraction methods of the Uncaria rhynchophylla Scrophularia Formula.

Hypertension is one of the most challenging public health problems worldwide. Previous studies suggested that the Uncaria rhynchophylla Scrophularia Formula (URSF), a medical institution preparation of the affiliated Hospital of Shandong University of Traditional Chinese Medicine, is effective for essential hypertension. However, the efficacy of URSF for hypertension remains unclear. We aimed to clarify the anti-hypertensive mechanism of the URSF. The material basis of URSF was identified by the LC-MS. We also evaluated the antihypertensive efficacy of URSF on SHR rats by body weight, blood pressure and biochemical indicators. The LC-MS spectrometry-based serum non-targeted metabolomics was used to seek potential biomarkers and relevant pathways for URSF in the treatment of SHR rats. 56 biomarkers were metabolically disturbed in SHR rats in the model group compared with the control group. After URSF intervention, 13 biomarkers showed a recovery in the optimal method compared with the other three groups. We identified 3 metabolic pathways in which URSF is involved: the arachidonic acid metabolism pathway, the niacin and nicotinamide metabolism pathway, and the purine metabolism pathway. These discoveries offer a basis for the study of URSF for the treatment of hypertension.

Uncária Tomentosa (Wild.) D.C. em processos inflamatórios na doença de Alzheimer / Uncária Tomentosa (Wild.) D.C. en procesos inflamatorios en la enfermedad de Alzheimer / Uncária Tomentosa (Wild.) D.C. in inflammatory processes in Alzheimer's disease

Objetivo: explorar os mecanismos envolvidos no desencadeamento e progressão da Doença de Alzheimer (DA) de forma a embasar a sugestão da planta Uncaria Tomentosa (Wild.) como mais uma possiblidade terapêutica coadjuvante para prevenção e tratamento da DA. Método: Trata-se de uma revisão narrativa da literatura realizada com busca de artigos publicados em bases indexadas e diretamente nas revistas de interesse, utilizando-se como descritores "Uncária Tomentosa", "Doença de Alzheimer", e os respectivos termos em inglês. Resultados: com os avanços para a compreensão dos mecanismos moleculares que desencadeiam os efeitos apresentados no desenvolvimento da DA, os diversos mecanismos dos fitocompostos presentes na planta sugerem sua utilização como neuroprotetor, por mecanismos anti-inflamatórios, imunomoduladores e antioxidantes, cujas evidências em literatura são apresentadas para defesa de sua utilização nesta patologia. Conclusão: foram encontradas evidências para sugerir a inclusão da Uncaria tomentosa (Wild.) como possível terapêutica complementar no tratamento da DA. Sua utilização deve ser melhor explorada para aplicação como tratamento complementar as terapêuticas convencionais para a DA

Objective: to explore the mechanisms involved in the triggering and progression of Alzheimer's disease (AD) in order to support the suggestion of the Uncaria Tomentosa (Wild.) plant as another adjuvant therapeutic possibility for the prevention and treatment of AD. Method: This is a narrative review of the literature conducted with a search for articles published on indexed bases and directly in the journals of interest, using as descriptors "Uncária Tomentosa", "Alzheimer's disease", and the respective terms in English. Results: with advances to understand the molecular mechanisms that trigger the effects presented in the development of AD, the various mechanisms of phytocompounds present in the plant suggest its use as neuroprotector, by anti-inflammatory, immunomodulatory and antioxidant mechanisms, whose evidence in the literature is presented to defend its use in this pathology. Conclusion: evidence was found to suggest the inclusion of Uncaria tomentosa (Wild.) as a possible complementary therapy in the treatment of AD. Its use should be better explored for application as a complementary treatment to conventional therapies for AD.

Objetivo: explorar los mecanismos implicados en el desencadenamiento y progresión de la enfermedad de Alzheimer (EA) con el fin de apoyar la sugerencia de la planta Uncaria Tomentosa (silvestre) como otra posibilidad terapéutica adyuvante para la prevención y tratamiento de la EA. Método: Se trata de una revisión narrativa de la literatura realizada con una búsqueda de artículos publicados en bases indexadas y directamente en las revistas de interés, utilizando como descriptores "Uncária Tomentosa", "Alzheimer's disease", y los términos respectivos en inglés. Resultados: con los avances para comprender los mecanismos moleculares que desencadenan los efectos presentados en el desarrollo de la EA, los diversos mecanismos de fitocompuestos presentes en la planta sugieren su uso como neuroprotector, por mecanismos antiinflamatorios, inmunomoduladores y antioxidantes, cuya evidencia en la literatura se presenta para defender su uso en esta patología. Conclusión: se encontró evidencia que sugiere la inclusión de Uncaria tomentosa (Silvestre) como una posible terapia complementaria en el tratamiento de la EA. Su uso debe explorarse mejor para su aplicación como tratamiento complementario a las terapias convencionales para la EA.

Characterization and Comparative Analysis of Chloroplast Genomes in Five Uncaria Species Endemic to China.

Uncaria, a perennial vine from the Rubiaceae family, is a typical Chinese traditional medicine. Currently, uncertainty exists over the Uncaria genus' evolutionary relationships and germplasm identification. The complete chloroplast genomes of four Uncaria species mentioned in the Chinese Pharmacopoeia and Uncaria scandens (an easily confused counterfeit) were sequenced and annotated. The findings demonstrated that the whole chloroplast genome of Uncaria genus is 153,780-155,138 bp in full length, encoding a total of 128-131 genes, containing 83-86 protein-coding genes, eight rRNAs and 37 tRNAs. These regions, which include eleven highly variable loci and 31-49 SSRs, can be used to create significant molecular markers for the Uncaria genus. The phylogenetic tree was constructed according to protein-coding genes and the whole chloroplast genome sequences of five Uncaria species using four methods. The topology of the two phylogenetic trees showed no difference. The sequences of U. rhynchophylla and U. scandens are clustered in one group, while the U. hirsuta and U. macrophylla are clustered in another group. U. sessilifructus is clustered together with the above two small clades. New insights on the relationship were revealed via phylogenetic research in five Uncaria species. This study will provide a theoretical basis for identifying U. rhynchophylla and its counterfeits, as well as the species of the Uncaria genus. This research provides the initial chloroplast genome report of Uncaria, contributes to elucidating the chloroplast genome evolution of Uncaria in China.

Current Research of Phytochemical, Medicinal and Non-Medicinal Uses of Uncaria gambir Roxb.: A Review.

Uncaria gambir Roxb. is a plant from Southeast Asia and is widely used as an alternative medicine with various applications. This plant has been widely used in traditional medicine. This paper aims to provide information on U. gambir, a summary of data on phytochemicals and on medical and nonmedical activities. Phytochemical studies reveal biologically active constituents such as flavonoids, phenolics, and alkaloids. Various studies have shown that extracts and compounds obtained from U. gambir have medical uses for their antioxidant, antibacterial, anti-helminthic, anticancer, antifungal, anti-inflammatory, anti-hyperglycemic, anti-hyperuricemic, anti-lipid peroxidation, antihyperlipidemic and other properties. In addition, this extract has other uses, such as adsorbent for dyes and metal ions, as well as corrosion inhibition. Thus, U. gambir, which is commonly used in traditional medicine, is a potential plant for many therapeutic applications and prospects for drug development as well as other applications such as adsorbent and corrosion inhibition.

Environmentally benign cinchonain IIa from Uncaria laevigata for corrosion inhibition of Q235 steel in HCl corrosive medium: Experimental and theoretical investigation.

Traditional corrosion inhibitors make great contribution to metal protection, but also cause environmental pollution. To solve the problem, plant extracts as green corrosion inhibitors have attracted much attention in recent years. Plants are good raw materials for corrosion inhibitors and also meet the requirements of industry. However, they have not been successfully applied in industry due to the unknown composition of the effective corrosion inhibitors and large dosage thereof. Therefore, cinchonain IIa was separated from Uncaria laevigata with abundant sources and low cost from nature in this work. Here we hypothesized that cinchonain IIa could show good corrosion inhibition performance for Q235 steel in the acidic medium. Through experiments and theoretical calculation, we studied the corrosion inhibition effect of cinchonain IIa on Q235 in 1 M HCl solution at 298 K for 48 h. Electrochemical experiments revealed that the inhibition efficiency of 200 mg/L cinchonain IIa in 1 M HCl for Q235 steel was 94.08% for 48 h. It even showed over 93% corrosion inhibition efficiency and durable protection performance to 28 d. Surface observations indicated that cinchonain IIa were firmly attached to the steel surface by forming a protective film. Moreover, quantum chemical calculation and molecular dynamics simulation revealed the inhibition mechanism at molecular and atomic level. Compared with some plant extracts, here we demonstrate that the outstanding advantages of cinchonain IIa include sustained protective effect, small dosage, and low toxicity. Accordingly, it may be used as a green industrial corrosion inhibitor with great potential in oilfield acidification and acid pickling.

GJD Modulates Cardiac/Vascular Inflammation and Decreases Blood Pressure in Hypertensive Rats.

Gedan Jiangya decoction (GJD) (aqueous ethanol extract), a traditional Chinese medicine formula which contain six botanical drugs (Uncaria rhynchophylla (Miq.) Miq., Salvia miltiorrhiza Bunge, Pueraria lobata (Willd.) Ohwi, Eucommia ulmoides Oliv., Prunella vulgaris L., and Achyranthes bidentata Blume) was designed to treat hypertension; however, the underlying mechanism of action is unclear. This study aimed to determine the mechanisms of action of GJD in the treatment of hypertension in spontaneously hypertensive rats (SHR). Male SHRs were randomly divided into five groups: GJD doses were low (1.36 g/kg/d), medium (2.72 g/kg/d), and high (5.44 g/kg/d), captopril (13.5 mg/kg/d), and SHR groups, with Wistar-Kyoto rats (WKY) serving as the control. Every rat was gavaged once a day. The ALC-NIBP, a noninvasive blood pressure device, measured systolic (SBP) and diastolic (DBP) blood pressures. Six weeks following treatment, all rats were anesthetized. The blood samples were obtained from the abdominal aorta and then serum isolated to assess endothelin-1 and angiotensin II, interleukin-1beta, interleukin-6, and TNF-alpha. The left ventricular and thoracic aortas were taken for HE staining, immunohistochemistry, RT-qPCR, and western blot examination. Following GJD therapy, SBP and DBP were significantly lowered, as were serum levels of endothelin-1 and angiotensin II. The thickness of the left ventricular and thoracic aorta walls reduced, as did type I collagen, type III collagen, and alpha-SMA expression in the left ventricular and aortic tissues. The GJD treatment significantly reduced serum levels of the inflammatory markers interleukin-1beta, interleukin-6, and TNF-alpha. Furthermore, interleukin-1 beta, interleukin-6, TNF-alpha, TAK1, and NF-κB/p65 levels were significantly reduced in left ventricular and aortic tissues, whereas IkB-alpha levels were significantly elevated. GJD has a dose-dependent effect on all parameters. In conclusion, GJD has been shown to lower blood pressure, improve cardiovascular remodeling, and reduce inflammation via regulating NF-κB in SHRs.

[Two new monoterpenoid indole alkaloids from hook-bearing branches of Uncaria sessilifructus].

The present study investigated the chemical constituents from Uncaria sessilifructus and their neuroprotective activities. The compounds were separated and purified from the 90% ethanol extract of U. sessilifructus by various chromatographic methods, including silica gel, Sephadex LH-20, and semi-preparative HPLC. Seven compounds were obtained, and their structures were identified as uncanidine J(1), uncanidine K(2), 17-O-ethylhirsutine(3), tetrahydroalstonine(4), akuammigine(5), hirsutine(6), and hirsuteine(7) by physicochemical properties and various spectral techniques, including UV, IR, MS, and NMR. Compounds 1 and 2 are two new compounds. Compound 3 is a new natural product, and compound 4 was isolated from U. sessilifructus for the first time. In addition, the isolated compounds were evaluated for their neuroprotective effects on oxygen and glucose deprivation/reoxygenation(OGD/R) injury in primary cortical neurons in rats. The results showed that compounds 1-7 had different degrees of protective effects on OGD/R injury. The EC_(50) values of compounds 2-4 were(0.17±0.03),(1.70±0.38), and(1.79±0.23) µmol·L~(-1), respectively.

Mechanisms of natural products as potential antiepileptic drugs.

The universal epileptogenic cascade remains unknown and most modern treatments focus on the reduction of symptoms and the prevention of seizure recurrence. Experimental studies have demonstrated that herbal medicines may act as antiepileptogenic agents. In this study, the possibilities of plants with antiepileptic properties were reviewed and discussed on their structures and related mechanism of actions. This work constituted a literature review of medicinal plants showing antiepileptic properties by literature searching in Science Direct, PubMed and Wiley Online Library. The keywords of search included epilepsy, antiepileptogenesis, antiepilepsy, natural compounds, extract, herbal medicines and medicinal plants in epilepsy treatment. Only articles published in English were reviewed. Mechanism of action of the natural plants were described according to experimental studies. From the databases, we found 135 natural plants with antiepileptic properties. In this review, the highly studied natural plants were selected. These included Acorus calamus, Bacopa monnieri, Boerhaavia diffusa, Curcuma longa, Gastrodia elata, Ginseng, Uncaria rhynchophylla, Pinellia ternatae, Withania somnifera, Magnolia bark and Resveratrol-related products. From the evidences, natural products may potentially be developed as antiepileptic or antiepileptogenic agents. However, several issues in drug development should be considered such as safety, formulations, pharmacokinetic characteristics and possible interactions.

Rapid screening of hepatotoxic components in Uncariae Ramulus Cum Uncis based on "component-target-pathway" network.

As a multi-base source traditional Chinese medicine, the hepatotoxicity of Uncariae Ramulus Cum Uncis (URCU) has been reported repeatedly in recent years. The lack of clarity of toxic components and toxicity mechanisms is a key issue that needs to be addressed. In this article, a "component-target-pathway" network strategy was established to firstly predicting the hepatotoxic components and the toxicity mechanism of URCU. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and data post-processing technology were used to classify and identify the main components in Uncaria rhynchophylla (Miq.) Miq. ex Havil. (UR) and Uncaria sinensis (Oliv.) Havil. (US). Then, the potential hepatotoxic components were screened by network pharmacology and molecular docking. As a result, 40 components and 39 ingredients were identified in UR and US, respectively. Cadambine, rhynchophylline, corynoxeine, isocorynoxeine, strictosamide and mitraphylline were screened as the potential hepatotoxic ingredients contained both in UR and US. The network pharmacology showed that the potential hepatotoxic components could affect the IL-17 signaling pathway by regulating related targets such as MAPK1 and MAPK14, which might lead to the occurrence of liver injury. This study not only provided a reasonable strategy for the rapid screening of hepatotoxic components in URCU, but also supplied reference and guidance for the rational clinical application and scientific supervision of URCU.

[Secondary metabolites of endophyte Hypoxylon sp. HD-2014 from Uncaria rhynchophylla].

This study aims to investigate the compounds in the product of rice fermented with endophyte Hypoxylon sp.HD-2014 from Uncaria rhynchophylla.To be specific, normal-phase, MCI, Sephadex LH-20, and semi-preparative high performance liquid chromatography(HPLC) was used to yield 12 compounds.Through spectral data analysis and comparison with previous reports, they were identified as methyl(E)-5-hydroxymethyl-6,7-dihydroxy-4,5-epoxy-octanoate-2-ene(1),(2E,6E)-nona-2,6,8-triene-4,5-diol(2), 8-O-(R)-methoxynodulisporin A(3), 3-nitropropionic acid(4), 3-nitropropionic acid methyl ester(5), 3,4-dihydroxy-phenylethanol(6), 2,4-dichlorobenzoic acid(7), cis-4-hydroxyscytalone(8), 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one(9), isosclerone(10), 4H-1-benzopyran-4-one-2,3-dihydro-5-hydroxy-8-(hydroxyl-methyl)-2-methy(11), and 5-methylmellein(12), respectively.Compounds 1 and 2 were identified for the first time.In vitro cytotoxicity test indicated that compounds 1-12 had no significant inhibitory effect on A549 and HepG2 cells.Antimicrobial susceptibility testing revealed that compound 3 showed synergistic effect with the positive control chloramphenicol.

Biotransformation and pharmacokinetic studies of four alkaloids from Uncaria rhynchophylla in rat plasma by ultra-performance liquid chromatography with tandem mass spectrometry.

Uncaria rhynchophylla (Miq.) Miq. ex Havil (U. rhynchophylla), a traditional Chinese medicine that has been officially included in the Chinese Pharmacopeia, is used to treat cardiovascular and central nervous system diseases. The major alkaloids isolated from U. rhynchophylla are two pairs of epimer including rhynchophylline (RIN) and isorhynchophylline (IRN), along with corynoxeine (CN) and isocorynoxeine (ICN). An ultra-performance liquid chromatography with tandem mass spectrometry method (UHPLC-MS/MS), which was highly accurate, stable and sensitive, was established and validated for the simultaneous determination of four alkaloid compounds (RIN, IRN, CN, ICN) in rat plasma samples after oral administration of RIN, IRN, CN, ICN and U. rhynchophylla extract. In this study, the biotransformation and pharmacokinetics of RIN, IRN, CN and ICN were determined for the first time. An ACQUITY UPLC®HSS T3 column (1.8 µm, 2.1 mm × 100 mm) was used to complete the chromatographic separation within 3 min. The isocratic mobile phase was composed of 0.1 % formic acid water (v/v) and acetonitrile, and the flow rate was 0.2 mL/min. The multireaction monitoring mode was adopted, and the tandem mass spectrometry in the positive ion mode was detected by the electrospray ionization source. The method was fully verified and linear at a wide concentration (r > 0.9913), and the linear concentration range was 0.1552-124.5 ng/mL. The intraday and interday precisions of the four analytes were lower than 8.20 % and 13.42 %, respectively. The accuracy range was - 2.64 % and 13.63 %. The extraction recoveries of the analytes exceeded 83.74 %, and the matrix effect range was 98.43-103.7 % in the plasma samples. The four alkaloids can be quickly absorbed into the blood (Tmax, 0.22-3.83 h) and cleared comparatively slowly (T1/2, 7.67-12.13 h). This method has been successfully applied to the biotransformation and pharmacokinetic studies of SD rat plasma after oral administration of U. rhynchophylla extracts. This proved that RIN with IRN and CN with ICN can transform into each other in vivo. The results are of great significance for determining the mechanism of action and guiding the clinical application of U. rhynchophylla extracts.

[Steroids from Uncaria rhynchophylla].

Thirteen steroids(1-13) were isolated from the non-alkaloid constituents of Uncaria rhynchophylla by column chromatography on silica gel, ODS, Sephadex LH-20, and preparative HPLC chromatography, and their structures were elucidated by analyses of the MS and NMR spectral data. All the compounds were isolated from the genus Uncaria for the first time, and 1 was a new compound. The ~1H-NMR and ~(13)C-NMR data of two compounds(12 and 13) in deuteron-chloroform were completely assigned. This study enriched the steroid constituents of U. rhynchophylla and provided scientific references for the elucidation of active constituents and further development and utilization of U. rhynchophylla.

Uncaria Rhynchophylla attenuates angiotensin â ¡-induced myocardial fibrosis via suppression of the RhoA/ROCK1 pathway.

Uncaria rhynchophylla (UR), a traditional Chinese medicine, has been proven effective in treating hypertensive patients in China. However, the mechanisms of action of UR in reducing hypertension and myocardial fibrosis are still unclear. The purpose of this study was to explore the role of UR in an angiotensin Ⅱ (Ang Ⅱ) induced mouse model. The mice were randomly divided into 5 groups and infused with Ang Ⅱ (500 ng/kg/min) or saline, then administered UR (0.78, 1.56 or 3.12 g/kg/d) or saline for 4 weeks. UR treatment significantly attenuated the elevation of blood pressure caused by Ang Ⅱ. It enhanced myocardial function and attenuated the increase in the heart weight index and the pathological changes in the Ang Ⅱ-induced hypertensive mice. Furthermore, UR treatment inhibited cardiac fibrosis and significantly down-regulated collagen I, collagen Ⅲ, and α-SMA protein expression in cardiac tissues. UR also attenuated the expression of RhoA, ROCK1, CTGF, and TGF-ß1. In cultured cardiac fibroblasts stimulated with Ang Ⅱ, UR significantly down-regulated the expression of Collagen I, Collagen III, RhoA, ROCK1, and α-SMA. In summary, UR can significantly attenuate Ang Ⅱ-induced hypertension and cardiac fibrosis, partly via suppression of the RhoA/ROCK1 signaling pathway.

A novel pentacyclic triterpene from the canes of Uncaria sessilifructus (Rubiaceae).

One novel pentacyclic triterpene, 24-dimethoxymethyl-3ß,6ß,19α- trihydroxy -12-en-28-oic acid (1), along with six known compounds 2-7, were isolated from the canes of Uncaria sessilifructus Roxb. Their structures were determined according to spectroscopic and spectrometric analysis. The anti-inflammatory activities of the isolated compounds (1-7) were scanned against NO production in LPS-activated RAW 264.7 macrophages by MTS assay, however no activities were observed.

Safety and biological activity evaluation of Uncaria rhynchophylla ethanolic extract.

Uncaria rhynchophylla (UR) belongs to the Rubiaceae family, and its dried hooks are usually used in traditional medicine. It is effective in treating diseases related to the central nervous system. This study aimed to evaluate the safety of UR extract, investigate its antimutagenic and antioxidative activities, and elucidate its active components. Extraction and fractionation of the UR extract resulted in yields of 6.71%, 0.037%, 0.042%, 0.152%, 0.332%, and 5.132%, for hexane, ether, DCM, EtOAC, and aqueous fractions, respectively. The four indole alkaloids, total phenolic content (TPC), and total flavonoid content (TFC) of UR extract and its subfractions were measured. Alkaloid content was highest in the UR extract. TPC was the highest in the EtOAC fraction (373.7 ± 20.9 mg gallic acid equivalent (GAE)/g), whereas TFC was the highest in the UR extract (33.5 ± 2.4 mg quercetin equivalent (QE)/g). To assess the safety of UR extract mutagenicity, cytotoxicity, and oxidative stress inducibility assays were performed. The UR extract (2000 µg/plate) showed excellent antimutagenic activity (above 90%) against BaP in both TA98 and TA100 strains. The UR extract exhibited efficient DPPH (RC50 239.2 ± 16.5 µg/mL) and ABTS scavenging activity (RC50 458.7 ± 25.0 µg/mL). The UR extract (150 µg/mL) showed cytoprotective activity (65.6% ± 9.2%) against t-BHP. Among the subfractions, the EtOAC fraction possessed the strongest activities, overall. UR generally showed excellent biological activity at nontoxic concentrations (determined in vitro in current work), although the chemical composition of UR requires further investigation prior to its potential future use.

Rapid discovery of potentially vasodilative compounds from Uncaria by UHPLC/Q-Orbitrap-MS based metabolomics and correlation analysis.

The genus Uncaria belongs to the family of Rubiaceae, which contains approximately 34 species. It has been widely used as a traditional Chinese medicine (TCM) in China to treat hypertension, fevers, headaches, gastrointestinal illness, epilepsy, wounds, and ulcers. Uncaria rhynchophylla. (Miq.) Miq. ex Hvail.(URM) and Uncaria hirsuta Havil.(UHH) are mainly used as remedies for hypertension, which both belong to the resource of "Gou-teng" in the Chinese Pharmacopoeia. However, the authentic antihypertensive components of Uncaria still have not been fully elucidated until now. In this work, we firstly explored and compared the vasorelaxation effect of URM and UHH on the isolated rat mesenteric artery ring. Then, the variations of metabolite profiles between URM and UHH samples were investigated by UHPLC/Q-Orbitrap-MS, and 16 different metabolites have been found through multivariate statistical analysis. Further, the potential vasodilative compounds which include corynoxeine, isocorynoxeine, isorhynchophylline, rhynchophylline, hirsuteine and hirsutine were screened through the correlation analysis between metabolites and anti-hypertension activities. And the relaxation effects of the six compounds on the mesenteric artery have verified. The results indicated that metabolomics combined with correlation analysis could be effective strategies to rapid explore the active compounds from TCM.

An enhanced strategy integrating offline two-dimensional separation with data independent acquisition mode and deconvolution: Characterization of metabolites of Uncaria rhynchophylla in rat plasma as a case.

The importance to clarify the drug metabolites is beyond doubt in view of their potential efficacy and safety. However, due to the complex matrix interference, relatively low content and the co-eluting effect, it is of a great challenge to comprehensively and systematically characterize the metabolites in vivo, especially for the traditional Chinese medicines (TCMs) due to the numerous types of components. In the present study, a comprehensive off-line two-dimensional separation system combining with data independent acquisition (DIA) mode and multi-dimensional data deconvolution method was established for chromatographic separation, data acquisition and data procession of indole alkaloids in rat plasma after intragastrically administrated with the extract of Uncaria rhynchophylla at the dose of 1 g/kg. The orthogonality of the off-line 2D separation system consisting of HILIC for first-dimensional separation and the PRLC for second-dimensional separation was valuated with the "asterisk" equations, and the results showed that off-line 2D separation system had passable orthogonality (A0 = 53.3%). Furthermore, the DIA mode was applied to capture MS/MS spectra in view of its advantage in acquiring MS data, and an effective multi-dimensional deconvolution method integrating the calculation of chemical formula, the extraction of diagnostic ion, the filter of ring double bond (RDB) and the judgement of neutral loss was established to parse the spectra for the complicated DIA data for comprehensive analysis of metabolites in rat plasma. Ultimately, a total of 127 indole alkaloids were tentatively characterized, and the main metabolic pathways were inferred as demethylation, dehydrogenation, hydroxylation and deglycosylation. The off-line two-dimensional separation system was applied for the comprehensive characterization of metabolites in vivo for the first time. This study suggested a new approach to enable the enrichment, separation and analysis of the low content components in vivo.

Fast determination of 16 circulating neurotransmitters and their metabolites in plasma samples of spontaneously hypertensive rats intervened with five different Uncaria.

This study aimed to establish a sensitive, reproducible, and rapid liquid chromatography method with tandem mass spectrometry detection to perform simultaneous quantitative analysis of 16 neurotransmitters and their metabolites in rat plasma, including levodopa, dopamine, norepinephrine, epinephrine, L-tryptophan, kynurenic acid, serotonin, melatonin, choline, acetylcholine, histamine, phenylethylamine, as well as excitatory (L-glutamic acid and L-aspartic acid) and inhibitory (γ-aminobutyric acid and L-glycine) neurotransmitters. These analytes were measured by ultra-high performance chromatography coupled with triple quadrupole mass spectrometry using a hydrophilic interaction chromatographic column (ethylene-bridged hybrid amide column). The internal standards of stable isotope labeling were used to improve the reliability of the results. Our method provided high linearity for all neurotransmitters (for all coefficients measured > 0.99), with inter- and intra-day accuracy from -14.82% to 17.49% and precision was between 0.89% and 17.70%. The method was subsequently verified in an animal study, where the intervention of five different Uncarias, the traditional Chinese medicine with hypotensive effects, was applied to the spontaneously hypertensive rats (SHRs). SHRs showed dysregulated plasma kynurenic acid, acetylcholine, and norepinephrine levels, and these neuroactive analytes were significantly restored by Uncaria treatment compared with the model group (SHR group). Compared with captopril, included as a positive control for its hypotensive effect, Uncaria had more effects on perturbing the levels of plasma neurotransmitters, which might indicate Uncaria's potential in treating symptoms related to the nervous system. These results suggested that the changes in the neurotransmitters and their metabolites in plasma may be related to the pathogenesis of hypertension. It also provided valuable information about the action mechanisms of Uncaria on its hypotensive effects.