Chemical constituents from the leaves and branches of Wikstroemia chamaedaphne with their activation of latent HIV activities.

A new compound, named coniferin B (1), and fourteen known compounds were purified and identified from the leaves and branches of Wikstroemia chamaedaphne Meisn. Their chemical structures were elucidated through analyzing spectroscopic and HRESIMS data. Compounds 2, 3, 5, 7-9, 11, and 13 were isolated from this plant for the first time. All compounds were assayed for cytotoxicity and activation of latent HIV activity on NH2 cells. The results showed that all compounds did not produce cytotoxicity at 10.0 µM and compounds 1, 9-11 showed weak activating activity with activation folds of 4.88, 7.14, 5.3, and 6.97, respectively.

Phillygenin Attenuated Colon Inflammation and Improved Intestinal Mucosal Barrier in DSS-induced Colitis Mice via TLR4/Src Mediated MAPK and NF-κB Signaling Pathways.

Ulcerative colitis (UC) is a chronic, relapsing, and nonspecific inflammatory bowel disease (IBD). Phillygenin (PHI), a natural bioactive ingredient, isolated from Forsythiae Fructus, exhibits anti-inflammatory, anti-oxidative, and hepatoprotective activities. However, few reports provide direct evidence on the efficacy of PHI in improving colitis mice. The present study elucidated that the symptoms of DSS-induced colitis mice were alleviated after PHI administration, including body weight loss, the disease activity index, colon length shortening, colonic pathological damage, splenomegaly, and hepatomegaly. PHI treatment improved the intestinal mucosal barrier by protecting goblet cells, promoting gene expressions of Clca1, Slc26a3, and Aqp8, increasing tight junction proteins (TJs), and reducing epithelial cell apoptosis. In addition, the levels of oxidative stress (MPO, SOD, and MDA) and inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-10) were reversed by PHI in colitis mice. According to transcriptome and network pharmacology analysis, inflammatory pathway might be an important mechanism for PHI to improve colitis. Western blotting displayed that the PHI inhibited the activation of tyrosine kinase Src mediated by TLR4, and then reduced the phosphorylation of downstream proteins p38, JNK, and NF-κB in colitis mice. In summary, our results suggested that PHI might be an appropriate and effective drug candidate to protect colitis.

Dimeric sesquiterpenoids and anti-inflammatory constituents of Sarcandra glabra.

Three novel dimeric sesquiterpenoids named sarglanoids A-C (1-3), two undescribed monomeric sesquiterpenoids named sarglanoids D (4) and E (5), and seven known compounds (6-12), were isolated and characterized from Sarcandra glabra. Compound 1 represents the first heterodimeric sesquiterpenoid composed of a eudesmane and an eremophilane moiety. Compound 2 possesses two eremophilane monomers featuring an undescribed dimerization pattern. Compound 3 is a symmetric eudesmane dimer with a rare 1,4-epoxy bridge. The structures of 1-5 were fully identified by spectroscopic methods and single-crystal X-ray diffraction experiments. Compounds 3 and 6 suppressed the LPS-triggered inflammatory responses in RAW 264.7 cells.

Discovering the Mechanisms of Wikstroelide E as a Potential HIV-Latency-Reversing Agent by Transcriptome Profiling.

The discovery of efficient and specific HIV-latency-reversing agents is critical for HIV therapy. Here, we developed wikstroelide E, a daphnane diterpene from the buds of Wikstroemia chamaedaphne, as a potential HIV-latency-reversing agent that is 2500-fold more potent than the drug prostratin. Based on transcriptome analysis, the underlying mechanism was that wikstroelide E regulated the MAPK, PI3K-Akt, JAK-Stat, TNF, and NF-κB signaling pathways. We clearly demonstrated that wikstroelide E reversed latent HIV infection by activating PKC-NF-κB signals, serving as a proxy for verifying the transcriptome data. Strikingly, the Tat protein contributes to the robust activation of latent HIV in wikstroelide-E-treated cells, producing an unexpected latency-reversing effect against latent HIV. This study provides the basis for the potential development of wikstroelide E as an effective HIV-latency-reversing agent.

[Study on residues of heavy metals and harmful elements in Qingqiao and Laoqiao based on chemometrics].

Forsythiae Fructus is divided into Qingqiao and Laoqiao due to different harvesting periods. So far, the accumulation of heavy metals in the two types of Forsythiae Fructus has not been reported. In this study, the residual levels of copper(Cu), lead(Pb), chromium(Cr), arsenic(As), cadmium(Cd) and mercury(Hg) in 29 batches of Laoqiao and 60 batches of Qingqiao were determined by inductively coupled plasma mass spectrometry(ICP-MS). The samples were collected from Shanxi, Shaanxi, Henan, and Hebei Provinces. In addition, the diversity and correlation of harmful elements in Qingqiao and Laoqiao were analyzed by multivariate statistical method. Furthermore, principal component analysis(PCA) was used to analyze the harmful elements concentrations of Qingqiao and Laoqiao. The results showed that there was a significant difference on the residual levels of heavy metals and harmful elements between Qingqiao and Laoqiao. Among them, the content of Pb in Laoqiao is significantly higher than that in Qingqiao(P<0.01), while the content of Cu is significantly lower than that in Qingqiao. However, the difference in harmful elements among different producing areas of Forsythiae Fructus is not significant. PCA analysis showed that Qingqiao and Laoqiao were successfully grouped into two categories. This study suggests significant difference in the residual levels of heavy metals and harmful elements between Qingqiao and Laoqiao. Besides, Forsythiae Fructus has a certain enrichment of Pb in the fruit ripening stage(Laoqiao). This study provides a reference for the quality classification and safety of Forsythiae Fructus.

[Phenolic constituents from Wikstroemia chamaedaphne].

The phenolic constituents of Wikstroemia chamaedaphne were investigated by various column chromatographic methods including silica gel,Sephadex LH-20,ODS and preparative HPLC,and their chemical structures were identified by physico-chemical properties and spectral analyses. Thirteen phenolic compounds were isolated and elucidated,including five flavonoids: luteolin 7-O-ß-D-glucopyranoside(1),luteolin 4'-O-ß-D-glucopyranoside(2),kaempferol 3-O-ß-D-glucopyranoside(3),chrysoeriol 4'-O-ß-D-glucopyranoside(4),chrysoeriol(5); and eight lignans:(-)-secoisolariciresinol(6),acanthosessilin A(7),(-)-nortrachelogenin(8),(+)-isolariciresinol(9),sesamin(10),syringaresinol(11),(+)-epipinoresinol(12),and [3,3',4,4'-tetrahydro-6,6'-dimethoxy-3,3'-bi-2 H-benzopyran]-4,4'-diol(13). Compounds 1, 3, 5-8, 10, 11 and 13 were obtained from the plants of W. chamaedaphne for the first time,and compounds 1,5,7,10 and 13 were obtained from the Wikstroemia genus for the first time.

In Vitro Anticoagulant Activity and Active Components of Safflower Injection.

Safflower injection is well-known as a traditional Chinese medicine used to improve the blood circulation. In this study, seven safflower injection samples from different companies were evaluated for their in vitro anticoagulant activity by measuring their activated partial thromboplastin time (APTT) and prothrombin time (PT) against human plasma. The screening results suggested that the safflower injections exhibited a significant prolonging influence on APTT (p < 0.05 vs. the control group), but not on prolonging PT (p > 0.05 vs. the control group). The safflower injection was separated into four fractions, and among them, fraction four demonstrated the most anticoagulant activity, with an APTT of 95.4 ± 1.4 s at a concentration of 4.0 µg/µL (p < 0.01 vs. control group). In addition, three active components, p-hydroxybenzaldehyde, p-hydroxy-cinnamic acid, and (8Z)-decaene-4,6-diyne-1-O-ß-d-glucopyranoside were isolated from fraction four with Sephadex LH-20 and C18 column chromatography. All three active components showed significant prolonging of APTT (p < 0.05 vs. control group). Among them, p-hydroxy-cinnamic acid exhibited the most activity (p < 0.01 vs. control group). The results indicated that safflower injection strongly affects the intrinsic coagulation system, and we suggest that this might be the mechanism by which the safflower injection activates and promotes blood circulation.

[Optimization of method for determination of Forsythia Fructus and discussion on the establishment of quality control standard for green (old) Forsythia Fructus].

In order to obtain the optimum method for content determination of Forsythia Fructus (FF), a variety methods for the sample preparation of FF were evaluated by the content determination methods of Chinese Pharmacopoeia. And an optimum method was screened and as follows: 30 times with 70% ethanol solution in ultrasonic extractor for half an hour. The method can achieve the best effect of simultaneously extracting forsythoside A and forsythin. Then, a HPLC method for simultaneous determination of forsythoside A and forsythin was established by methodology. The HPLC chromatographic conditions: the mobile phase consisted of acetonitrile (A)-0.4% acetic acid solution (B) with gradient elution [0-33 min，15%A，33-43 min，15%-25%A，43-60 min，25% A] was at the flow rate of 1 mL·min⁻¹, the column temperature was 25 °C, and the detection wavelength was 330 and 277 nm. Moreover, the contents of forsythoside A and forsythin for 10 Green Forsythia Fructus (GF) and 5 Old Forsythia Fructus (OF) were determined by this method and Chinese Pharmacopoeia. The result not only displayed that the established method is effective, rapid, and simple, but also showed that the contents of forsythoside A and forsythin for GF and OF were significantly different. Which implied that the forsythoside A and forsythin limit standard for GF and OF should be controled by different values. This studies provide an important basis for the establishment of the content determination of FF and the quality control standard for GF and OF.

[Biological activity and hydroxy safflor yellow A content of intermediates in preparation of Safflower Injection].

In order to investigate the effect of various production processes on the quality of Safflower Injection, the biological activities of the intermediates were evaluated by measuring activated partial thromboplastin time (APTT) and adenosine diphosphate (ADP) induced platelet aggregation in vitro. Intermediates were produced by key processes, such as extraction, concentration, twice alcohol precipitation, water sedimentation and two sterilizations during the production of Safflower Injection. The content of main chemical components in intermediates was determined by HPLC. The results showed that with the advance of the preparation process of Safflower Injection, the inhibition of ADP-induced platelet aggregation rate of each intermediate decreased gradually, and the trend of extending APTT activity decreased first and then increased. Meanwhile, the content of hydroxy safflor yellow A (HSYA) was gradually lowered, the content of p-hydroxy-cinnamic acid was increased, and new chemical component p-hydroxybenzaldehyde was produced. In conclusion, sterilization played a key role in the biological activity and HSYA content of Safflower injection.

Antimicrobial Diterpenoids of Wedelia trilobata (L.) Hitchc.

Continued interest in the metabolites of Wedelia trilobata (L.) Hitchc, a notoriously invasive weed in South China, led to the isolation of twenty-six ent-kaurane diterpenoids, including seven new ones 1-7. Their structures and relative configuration were elucidated on the basis of extensive spectroscopic analysis, including 1D- and 2D-NMR experiments. The antimicrobial activities of all isolated diterpenoids were evaluated against a panel of bacteria and fungi.

[Simultaneous determination of four coumaroylspermidine constituents in Carthamus tinctorius by HPLC-DAD].

The HPLC-DAD method was established to simultaneously determine the contents of four coumaroylspermidine[ N1, N5, N10-(Z)-tri-p-coumaroylspermidine (1), N1, N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (2), N1(E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (3), and N1, N5, N10-(E)-tri-p-coumaroyl-spermidine (4) ] in Carthamus tinctorius. The method was performed on an Eclipse XDB-C18 column (4.6 mm×250 mm, 5 µm) eluted with 47% methanol in an isocratic program. The flow rate was 1 mL•min⁻¹; the injection volume was 10 µL, and the column temperature was 30 ℃. The detective wavelength was 270, 280, 290, and 300 nm, respectively. Four coumaroylspermidine constituents showed a good linearity in the range of 0.002 1-0.041 6 (r=0.999 5), 0.002 6-0.051 2 (r=0.999 7), 0.002 7-0.054 0 (r=0.999 8) g•L⁻¹, and 0.005 0-0.100 4 (r=0.999 8) g•L⁻¹, respectively. The average recoveries of these four coumaroylspermidine constituents were in the range of 98.61%-100.9% (RSD 2.3%-3.0%). In conclusion, the method is simple, rapid, and sensitive, which could be used as a quantitative determination method for the four coumaroylspermidine components in C.tinctorius.

Phragmalin limonoids from the stem barks of Chukrasia tabularis var. velutina.

Seven new phragmalin limonoids, chukvelutilides I-O (1-7), were isolated from the stem barks of Chukrasia tabularis var. velutina. Their structures were elucidated by extensive spectroscopic analysis. Among them, compound 1 showed moderate lethal activity against brine shrimp larvae, with an LC50 value of 84.1 µM.

A green and simple method for enrichment of major diterpenoids from the buds of Wikstroemia chamaedaphne with macroporous resins and their activation of latent human immunodeficiency virus activity.

In this study, a green and efficient enrichment method for the four majors active diterpenoid components: pimelotide C, pimelotide A, simplexin, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate in the buds of Wikstroemia chamaedaphne was established using macroporous resin chromatography. The adsorption and desorption rates of seven macroporous resins were compared using static tests. The D101 macroporous resin exhibited the best performance. Static and dynamic adsorption tests were performed to determine the enrichment and purification of important bioactive diterpenoids in the buds of W. chamaedaphne. Diterpenoid extracts were obtained by using D101 macroporous resin from the crude extracts of W. chamaedaphne. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated that most of the diterpenoids were enriched in diterpenoid extracts. These results confirmed that diterpenoids in the buds of W. chamaedaphne could be enriched using macroporous resin technology, and the enriched diterpenoid extracts showed more efficient activation of the latent human immunodeficiency virus. This study provides a novel strategy for discovering efficient and low-toxicity latency-reversing agents and a potential basis for the comprehensive development and clinical application of the buds of W. chamaedaphne.

Cytotoxic limonoids from Melia azedarach.

Five new compounds (1-5), including two limonoids, one triterpenoid, one steroid, and one sesquiterpenoid, along with nine known limonoids (6-14), were isolated from the bark of Melia azedarach. The structures of the new compounds were elucidated by 2D NMR spectroscopy and mass spectrometry. The isolated compounds as well as three acetylated derivatives of 9 were evaluated for their cytotoxicities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) by an MTT assay. Seven limonoids (1, 6, 7, 8, 9, 9b, and 9c) showed significant inhibitory activities against tested cell lines with IC50 values ranging from 0.003 to 0.555 µM, and their preliminary structure-activity relationships are also discussed.

ß-Carboline alkaloids from the leaves of Trigonostemon lii Y.T. Chang.

A phytochemical work on the alkaloid constituents from Trigonostemon lii Y.T. Chang was conducted to give six new ß-carboline alkaloids, trigonostemines A-F (1-6) and eight known ß-carboline alkaloids (7-14). Their structures were elucidated by extensive spectroscopic techniques including 2D NMR experiments and mass spectrometry. All of the compounds were evaluated for their cytotoxic activities against the HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines. Trigonostemines A and B (1 and 2) exhibited stronger inhibitory activities than the positive control (cisplatin) in some cell lines.

Cycloartane and friedelane triterpenoids from the leaves of Caloncoba glauca and their evaluation for inhibition of 11ß-hydroxysteroid dehydrogenases.

Five new triterpenoids, caloncobic acids A and B (1 and 2), caloncobalactones A and B (3 and 4), and glaucalactone (5), along with the known compounds 3ß,21ß-dihydroxy-30-nor-(D:A)-friedo-olean-20(29)-en-27-oic acid (6) and acetyltrichadenic acid B (7), were isolated from the leaves of Caloncoba glauca. The structures of 1-5 were elucidated using spectroscopic methods. Compounds 1-7 were evaluated for their inhibitory activities against two isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1 and 11ß-HSD2). Compounds 1 and 2 exhibited strong inhibitory activities against mouse (EC(50) 132 and 13 nM) and human (EC(50) 105 and 72 nM) 11ß-HSD1.

Trigohowilols A-G, degraded diterpenoids from the stems of Trigonostemon howii.

Two new degraded diterpenoids, trigohowilols A (1) and B (2), four new heterodimers, trigohowilols C-F (3-6), one new homodimer, trigohowilol G (7), and three known degraded diterpenoids (8-10) were isolated from the methanol extract of the stems of Trigonostemon howii. Compounds 1-7 were evaluated for their cytotoxic activity against five human tumor cell lines by an MTT assay, and trigohowilols E (5) and F (6) exhibited inhibitory activity with IC50 values ranging from 2.33 to 12.57 µM. Moreover, compounds 1-6 showed weak antimicrobial activities (MIC values: 6.25-25 µg/mL) against Staphylococcus aureus, Pseudomonas aeruginosa, MRSA 92(#), and MRSA 98(#) using a 2-fold dilution method.

Trigoflavidols A-C, degraded diterpenoids with antimicrobial activity, from Trigonostemon flavidus.

Trigoflavidols A (1) and B (2), tetranorditerpenoid dimers possessing a rearrangement skeleton with a spiroketal core moiety, and trigoflavidol C (3), a hexanorditerpenoid, have been isolated from Trigonostemon flavidus along with two known compounds. Compounds 1 and 2 showed moderate antimicrobial activities (MIC values: 3.12-6.25 µg/mL) against Staphylococcus aureus, 8(#)MRSA, and 82(#)MRSA, and 1, 2, and 5 showed weak activities (IC(50) values: 3.75-28.99 µM) against various human tumor cell lines.

Cycloartane triterpenoids from Cassia occidentalis.

A phytochemical investigation of the whole plants of Cassia occidentalis led to the isolation of two new cycloartane triterpenoids, cycloccidentalic acids A and B (1 and 2), and five new related saponins, cycloccidentalisides I-V (3-7), together with sixteen known compounds. The structures of the isolated compounds were elucidated through detailed spectroscopic analyses, including 1D and 2D NMR techniques, and chemical methods. Compounds 2 and 5 showed modest anti-HIV-1 activities with EC50 values of 2.23 µM and 4.36 µM, respectively, in comparison to the positive control.

Potential HIV latency-reversing agents with STAT1-activating activity from the leaves of Wikstroemia chamaedaphne.

Developing highly effective HIV latency-reversing agent is an inportmant approach for the treatment of AIDS via the "shock and kill" of latent HIV. In this study, two unreported modified daphnane-type diterpenes (chamaedaphnelide A and epi-chamaedaphnelide A) and one unreported tigliane-type diterpene (chamaedaphnelide B), along with four known daphnane-type diterpenes and one known tigliane-type diterpene were obtained from the leaves of Wikstroemia chamaedaphne. Chamaedaphnelide A and epi-chamaedaphnelide A represents the first A ring cleavage daphnane-type backbone. Chamaedaphnelide A, epi-chamaedaphnelide A, chamaedaphnelide B, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate showed HIV latency-reversing activity, especially chamaedaphnelide B and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate displayed equally potential to positive drugs prostratin with reversing latent HIV on more than 100-fold compared to unstimulated cells. Furthermore, the activation of STAT1 was involved in the HIV latency-reversing activity of these diterpenes, firstly demonstrating that daphnane- and tigliane-type diterpenes can rapidly activate STAT1 activity. Indeed, these results also supported that activating STAT1 activity is a pathway for reversing latent HIV.