Caragana sinica (Buc'hoz) Rehd. (jin ji er) polysaccharide regulates the immune function and intestinal microbiota of cyclophosphamide (CTX) induced immunosuppressed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Caragana sinica (Buc'hoz) Rehd. is a plant widely grown in Yunnan, China, for both medicinal and edible purposes. The "National Compilation of Chinese Herbal Medicine" describes its nature as "slightly temperate and sweet". Caragana sinica is usually medicated with whole herbs, the main function is to replenish the kidneys and stop bleeding. Caragana sinica was used in folk medicine in Chuxiong, Yunnan, to treat deficiency colds, fatigue, fever, cough, hypertension, and other diseases. AIM OF THE STUDY: This article investigates the structural characteristics of Caragana sinica polysaccharide (CSP) and explores its immune-regulatory activity and molecular biological mechanisms in cyclophosphamide-induced immunosuppressed mice, as well as its effects on intestinal bacteria. METHODS: With the water-extraction and alcohol-precipitation method, Caragana sinica polysaccharide were extracted, obtaining CSP by purification. A variety of methods and techniques have been used to analyze the chemical properties and structural characteristics of CSP. Immunosuppressive mice model was established through intraperitoneal injection of cyclophosphamide (CTX) to study the immune-regulatory effects and mechanisms of CSP. RESULTS: The data indicated that CSP is a neutral heteropolysaccharide mainly composed of arabinose and galactose. This article uses immunosuppressive mice induced by cyclophosphamide (CTX) as the model. The results showed that CSP can promote the immune function of CTX treated immunosuppressed mice and regulate the diversity and composition of intestinal microbiota. CSP can increase macrophage phagocytosis, NK cell killing activity, and lymphocyte proliferation activity. It can also repair the index and morphological damage of the thymus and spleen. And by binding to the TLR4 receptor, MyD88 was activated and interacted with TRAF6 to promote the transfer of NF-κB into the nucleus. Thereby promoting cytokine release and increasing the production of IL-1ß, IL-6, IL-10, TNF-α, IgA, and IgG in the serum. CSP also effectively alleviated the liver damage caused by CTX through antioxidant activity. Furthermore, CSP can dramatically affect the intestinal microbiota and the body's immunity by boosting the relative presence of Bacteroides and Verrucamicrobiota. CONCLUSIONS: Research results indicated that CSP can regulate the immune function of mice, providing a basis for developing CSP as a potential immune modulator and functional food.

Skin Wound Healing and Anti-Wrinkle-Promoting In Vitro Biological Activities of Caragana sinica Flower Absolute and Its Chemical Composition.

Caragana sinica (CS; family Legume) was used as a medicinal material to treat neuralgia and arthritis in folk remedies and has been shown to have antioxidant, neuroprotective, and anti-apoptotic effects. However, CS is unknown for its biological activities related to skin. The present study explored the effects of CS flower absolute (CSFAb) on skin repair responses, viz., wound healing and anti-wrinkle-related responses using keratinocytes. CSFAb was extracted using hexane, and its composition was analyzed by GC/MS. The effects of CSFAb on human keratinocytes (HaCaT cells) were evaluated using Boyden chamber, sprouting, water-soluble tetrazolium salt, 5-bromo-2'-deoxyuridine incorporation, ELISA, zymography, and immunoblotting assays. GC/MS detected 46 components in CSFAb. In addition, in HaCaT cells, CSFAb increased the proliferation, migration, and sprout outgrowth and the phosphorylation of ERK1/2, JNK, p38 MAPK, and AKT, and also increased collagen type I and IV synthesis, reduced TNF-α-increased MMP-2 and MMP-9 activities, and upregulated hyaluronic acid (HA) and HA synthase-2 levels. These effects of CSFAb on wound healing and anti-wrinkle-related responses in keratinocytes suggest its potential use for skin repair and care preparations.

Hypoglycemic oligostilbenes from the stems of Caragana sinica.

Six new oligostilbenes, carastilphenols A-E (1-5) and (-)-hopeachinol B (6), with three reported oligostilbenes were obtained from the stems of Caragana sinica. The structures of compounds 1-6 were determined by comprehensive spectroscopy analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Thus, natural tetrastilbenes were determined as absolute configuration for the first time. Also, we did several pharmacological essays. In the antiviral tests, compounds 2, 4 and 6 showed moderate anti-coxsackie virus B3 type (CVB3) effect on Vero cells activities in vitro with IC50 values of 19.2 âˆ¼ 69.3 µM; and compounds 3 and 4 showed different levels of anti-respiratory syncytial virus (RSV) effect on Hep2 cells activities in vitro with IC50 values of 23.1 and 33.3 µM, respectively. As for hypoglycemic activity, compounds 6-9 (10 µM) showed the inhibition of α-glucosidase in vitro with IC50 values of 0.1 âˆ¼ 0.4 µM; and compound 7 showed significant inhibition (88.8%, 10 µM) of protein tyrosine phosphatase 1B (PTP1B) with IC50 value of 1.1 µM in vitro.

Effects of water extract and ethanol extract from the root of Caragana sinica on hyperuricemia in mice / 中国药房

OBJECTIVE To investigat e the effects of water extract （WCS）and ethanol extract （ECS）from the root of Caragana sinica on hyperuricemia （HUA）in mice. METHODS Kunming mice were randomly divided into normal control group ， model group ，allopurinol group （positive control ，5 mg/kg），benzbromarone group （positive control ，7.8 mg/kg），WCS low-dose ， medium-dose and high-dose groups （38，75，150 mg/kg），ECS low-dose ，medium-dose and high-dose groups （50，100，200 mg/kg）， with 10 mice in each group. Except for the normal control group ，the other mice were given potassium oxazinate intraperitoneally and hypoxanthine intragastrically for consecutive 7 d to establish HUA model. On the third day of modeling ，mice in each administration group were given corresponding drugs intragastrically ，and normal control group and model group were given equal volume of normal saline once a day for 5 consecutive days.The body weight of mice were weighted during administration ；one hour after the last administration ，the organ indexes of liver ，kidney and spleen were calculated ；the contents of serum uric acid （SUA）， blood urea nitrogen （BUN）and serum creatinine （SCR）；the activity of xanthine oxidase （XOD）in serum and liver tissue were determined. Relative mRNA and protein expressions of XOD in liver tissue ，relative expre ssions of GLUT9，URAT1 and OAT 1 in renal tissue were all detected ；and the pathological changes of renal tissue were observed. RESULTS There were no significant differences in liver index and spleen index in each group （P＞0.05）. Compared with normal control group ， except for allopurinol group ， there were no significant differences in the body weight and the contents of BUN and SCR in mice of other administration groups （P＞0.05）；the renal index and SUA content of mice in the m odel group and allopurinol group were significantly increased （P＜0.05）；in the model group ，the XOD activity in serum and liver tissue ，the relative mRNA and protein expression of XOD in liver tissue ，the relative expressions of GLUT 9 and URAT 1 protein in renal tissue were significantly increased （P＜0.05），and the relative expression of OAT 1 protein in renal tissue was significantly decreased （P＜ 0.05）. Compared with model group ，renal indexes of mice were decreased significantly in WCS and ECS groups （P＜0.05），and the pathological damage of renal tissue was significantly improved ；SUA content ，XOD activity in serum and liver tissue ，the relative mRNA and protein expression of XOD in liver tissue ，and the relative expression of URAT 1 protein in renal tissue were decreased significantly in administration groups （P＜0.05）. The relative expression of GLUT 9 protein in renal tissue of mice in benzbromarone group and ECS high-dose group decreased significantly （P＜0.05）；relative expression of OAT 1 protein in renal tissue of mice in benzbromarone group ，WCS low-dose and high-dose groups ，ECS low-dose group were increased significantly （P＜0.05）. CONCLUSIONS WCS and ECS can significantly decrease the contents of SUA in HUA model mice ，and improve pathological state of renal tissue ，the mechanism of which may be associated with inhibiting XOD activity and uric acid reabsorption，and down-regulating protein and mRNA expression of XOD.

Inhibition effect of Caragana sinica root extracts on Osteoarthritis through MAPKs, NF-κB signaling pathway.

Osteoarthritis (OA) is a common joint disease characterized by degradation and inflammation of cartilage extracellular matrix. We aimed to evaluate the protective effect of Caragana sinica root (CSR) on interleukin (IL)-1ß-stimulated rat chondrocytes and a monosodium iodoacetate (MIA)-induced model of OA. In vitro, cell viability of CSR-treated chondrocytes was measured by MTT assay. The mRNA expression of Matrix metallopeptidases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) and extracellular matrix (ECM) were analyzed by quantitative real-time PCR (qRT-PCR). Moreover, the protein expression of MAPK (phosphorylation of EKR, JNK, p38), inhibitory kappa B (IκBα) and nuclear factor-kappa B (NF-κB p65) was detected by western blot analysis. In vivo, the production of nitric oxide (NO) was detected by Griess reagent, while those of inflammatory mediators, MMPs and ECM were detected by ELISA. The degree of OA was evaluated by histopathological analyses, Osteoarthritis Research Society International (OARSI) score and micro-CT analysis. CSR significantly inhibited the expression of MMPs, ADAMTSs and the degradation of ECM in IL-1ß-stimulated chondrocytes. Furthermore, CSR significantly suppressed IL-1ß-stimulated of MAPKs, NF-κB signaling pathway. In vivo, CSR and Indomethacin inhibited the production of inflammatory mediators, MMPs and degradation of ECM in MIA-induced model of OA. In addition, CSR improved the severity of OA. Taken together, these results suggest CSR is a potential therapeutic active agent in the treatment of OA.

Neuraminidase Inhibitors from the Roots of Caragana sinica.

Two new oligostilbenes, caragasinins D and E, along with four known compounds, kobophenol A, α-viniferin, wistin, and 5-hydroxy-2-[(4-hydroxyphenyl)acetyl]-3-methoxybenzoic acid, were isolated from the roots of Caragana sinica. These compounds were spectroscopically analyzed for their structures and configurations and compared with existing data. The configurations of caragasinins D and E were elucidated by 1 H-NMR spectroscopy, CD spectroscopy, and time-dependent density-functional theory simulated ECD spectral data. All six compounds were evaluated for their inhibitory activity against neuraminidase (NA) from Clostridium perfringens. Among the tested compounds, 5-hydroxy-2-[(4-hydroxyphenyl)acetyl]-3-methoxybenzoic acid demonstrated statistically significant NA inhibitory activity, which was comparable to the positive control, mangiferin.

Study on in vitro Inhibitory Activity of the Extracts from 9 Kinds of TCM for Dredging Collaterals and Dispelling Wind on Xanthine Oxidase / 中国药房

OBJECTIVE：To study in vi tro inhibitory act ivities of 9 kinds of TCM for dredging collaterals and dispelling wind on xanthine oxidase （XO），and to screen TCM with outstanding activity. METHODS ：Using xanthine as substrate and xanthinase as reaction enzyme ，allopurinol as positive control ，with water extract and methanol extract （hereinafter referred to as “ethanol extract”）from the stem and leaves of Hedera nepalensis ，the whole plant of Piper wallichii ，the fruits of Rubus corchorifolius ，the root of Caragana sinica ，the root of Wisteria sinensis ，the root of Rubus crataegifolius ，the bark of Catalpa ovata ，the root of Campsis grandiflora ，the stem of P. hancei （hereinafter referred to by plant name ）and petroleum ether ，dichloromethane，ethyl acetate，n-butanol and water fraction of active extract as the objects ，inhibition rate of each sample to XO was detected by spectrophotometry；IC50values were calculated with Graphpad prism 6.0 software to screen active extract/fraction. Double reciprocal method was used to determine the type of enzyme inhibition. RESULTS ：Among 9 kinds of TCM and 18 kinds of the extracts ，the inhibitory rates to XO of 500 μg/mL extracts from each TCM（except for ethanol extract of P. wallichii ），250 μg/mL water extract and ethanol extract of H. nepalensis ，P. wallichii ，R. corchorifolius and P. hancei ，250 μg/mL water extract of C. ovata ，250 μ g/mL ethanol extract of C. sinica ，R. crataegifolius and C. grandiflora ，125 μ g/mL ethanol extract of C. sinica and R. crataegifolius，62.5 μg/mL ethanol extract of C. sinica were more than 50％. The IC 50 value of the ethanol extract from C. sinica was 43.43 μg/mL，which was lower than the extracts of other TCM ，and which was the active extract. The IC 50 values of petroleum ether，dichloromethane，ethyl acetate ，n-butanol and water fracti on of ethanol extract from C. sinica were ＞200，193.35，7.67， 14.80 and ＞200 μg/mL，respectively. The IC 50 value of ethyl XO was competitive-noncompetitive inhibition ， which was different from competi tive inhibition of positive control. CONCLUSIONS：The ethanol extracts of C. sinica ，R. crataegifolius ，P. wallichii ，R. corchorifolius ，P. hancei ，H. nepalensis and the water extracts of H. nepalensis ，P. wallichii ，C. ovata show certain inhibitory activity in vitro to XO ，especially ethanol extract of C. sinica . The ethyl acetate fraction of the ethanol extract of C. sinica has similar inhibitory activity to allopurinol but their inhibition types are different.

Kobophenol A Isolated from Roots of Caragana sinica (Buc'hoz) Rehder Exhibits Anti-inflammatory Activity by Regulating NF-κB Nuclear Translocation in J774A.1 Cells.

Kobophenol A (KPA) is a biologically active natural compound isolated from the roots of Caragana sinica (Buc'hoz) Rehder (C. sinica). However, the anti-inflammatory effects of KPA have not been reported. This study aims to find out whether KPA isolated from roots of C. sinica can act as a potential substance on inflammation and analyze the molecular mechanism using the lipopolysaccharide (LPS)-stimulated J774 A.1 macrophage cell line. We showed that KPA treatment significantly suppressed the production of nitric oxide (NO) by inhibiting inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner without cytotoxicity. In the KPA also inhibited pro-inflammatory cytokine gene expression and production, such as interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in LPS-stimulated J774 A.1 cells. As continuing study on the mechanisms involved, we confirmed that these effects of KPA were related to the inhibition of nuclear factor-κB (NF-κB) pathway including the suppression of IκB kinase α/ß (IKKα/ß) phosphorylation and translocation of NF-κB into the nucleus. Taken together, the present study is the first to demonstrate that KPA isolated from C. sinica suppresses the expression of inflammatory mediators and cytokines by inhibiting NF-κB nuclear translocation in LPS-stimulated J774 A.1 macrophages. KPA may be a potential candidate for the treatment of inflammatory diseases in the future.

α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis.

Rationale: cAMP up-regulates microphthalmia-associated transcription factor subtype M (MITF-M) and tyrosinase (Tyro) in the generation of heavily pigmented melanosomes. Here, we communicate a therapeutic mechanism of hyperpigmented disorder by α-viniferin, an active constituent of Caragana sinica. Methods: We used cAMP-elevated melanocyte cultures or facial hyperpigmented patches for pigmentation assays, and applied immunoprecipitation, immunobloting, RT-PCR or reporter gene for elucidation of the antimelanogenic mechanism. Results:C. sinica or α-viniferin inhibited melanin production in α-melanocyte-stimulating hormone (α-MSH)-, histamine- or cell-permeable cAMP-activated melanocyte cultures. Moreover, topical application with C. sinica containing α-viniferin, a standard in quality control, decreased melanin index on facial melasma and freckles in patients. As a molecular basis, α-viniferin accelerated protein kinase A (PKA) inactivation via the reassociation between catalytic and regulatory subunits in cAMP-elevated melanocytes, a feedback loop in the melanogenic process. α-Viniferin resultantly inhibited cAMP/PKA-signaled phosphorylation of cAMP-responsive element-binding protein (CREB) coupled with dephosphorylation of cAMP-regulated transcriptional co-activator 1 (CRTC1), thus down-regulating expression of MITF-M or Tyro gene with decreased melanin pigmentation. Conclusion: This study assigned PKA inactivation, a feedback termination in cAMP-induced facultative melanogenesis, as a putative target of α-viniferin in the treatment of melanocyte-specific hyperpigmented disorder. Finally, C. sinica containing α-viniferin was approved as an antimelanogenic agent with topical application in skin hyperpigmentation.

Caragasinin C: a new oligostilbene from the roots of Caragana sinica.

A new oligostilbene, caragasinin C (1), and seven known compounds, betulinic acid (2), 4-hydroxybenzaldehyde (3), (‒)-medicarpin (4), wistin (5), (2E,4S)-4-hydroxy-2-nonenoic acid (6), pallidol (7), and (+)-α-viniferin (8), were isolated from the roots of Caragana sinica. The structure of caragasinin C was established on the basis of spectroscopic techniques, including HRESIMS, 1D and 2D-NMR.

Content Determination of Four Triterpenes in Caragana sinica by HPLC and α-glucosidase Activity Assay in Vitro / 中国药学杂志

OBJECTIVE: To establish HPLC methods for the determination of betulinic acid, oleanolic acid, ursolic acid, and lupeol in Caragana sinica roots, and investigate the α-glucosidase activity of the four compounds in vitro. METHODS: An Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) was applied with methanol-water-phosphoric acid (85:15:0.1) as the mobile phase at a flow rate of 0.6 mL·min-1 to separate betulinic acid, ursolic acid, and oleanolic acid. An Inert Sustain C18 column (4.6 mm × 150 mm, 5 μm) was applied with acetonitrile-water (70:30) as the mobile phase at a flow rate of 0.8 mL·min-1 to separate lupeol. RESULTS: Good linearities were achieved for betulinic acid, oleanolic acid, ursolic acid, and lupeol within the range of 0.56-5.58, 0.42-4.20, 0.19-1.92, and 6.10-61.00 μg, respectively. The average recoveries were 99.79%, 98.51%, 98.05%, and 99.47%, and the RSDs were 1.53%, 1.74%, 1.78%, and 1.94%, respectively. CONCLUSION: The developed methods are accurate and can be used for the quality control of Caragana sinica. In addition to betulinic acid, the other three compounds show good α-glucosidase inhibitory activity and are expected to be developed as new hypoglycemic drugs.

Extract of Caragana sinica as a potential therapeutic option for increasing alpha-secretase gene expression.

BACKGROUND: Alzheimer's disease represents one of the main neurological disorders in the aging population. Treatment options so far are only of symptomatic nature and efforts in developing disease modifying drugs by targeting amyloid beta peptide-generating enzymes remain fruitless in the majority of human studies. During the last years, an alternative approach emerged to target the physiological alpha-secretase ADAM10, which is not only able to prevent formation of toxic amyloid beta peptides but also provides a neuroprotective fragment of the amyloid precursor protein - sAPPalpha. PURPOSE: To identify novel alpha-secretase enhancers from a library of 313 extracts of medicinal plants indigenous to Korea, a screening approach was used and hits were further evaluated for their therapeutic value. METHODS: The extract library was screened for selective enhancers of ADAM10 gene expression using a luciferase-based promoter reporter gene assay in the human neuroblastoma cell line SH-SY5Y. Candidate extracts were then tested in wild type mice for acute behavioral effects using an open field paradigm. Brain and liver tissue from treated mice was biochemically analyzed for ADAM10 gene expression in vivo. An in vitro blood-brain barrier model and an in vitro ATPase assay were used to unravel transport properties of bioactive compounds from extract candidates. Finally, fractionation of the most promising extract was performed to identify biologically active components. RESULTS: The extract of Caragana sinica (Buc'hoz) Rehder was identified as the best candidate from our screening approach. We were able to demonstrate that the extract is acutely applicable in mice without obvious side effects and induces ADAM10 gene expression in peripheral tissue. A hindered passage across the blood-brain barrier was detected explaining lack of cerebral induction of ADAM10 gene expression in treated mice. By fractionating C. sinica extract we identified alpha-viniferin as one of the biologically active components. CONCLUSION: The extract of C. sinica and alpha-viniferin as one of its bioactive constituents might serve as novel therapeutic options for treating Alzheimer's disease by increasing ADAM10 gene expression. The identification of alpha-viniferin represents a promising starting point to achieve blood-brain barrier penetrance in the future.