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.

A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3ß/ß-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3ß (p-GSK-3ß), thereby promoting ß-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic ß-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Chemical constituents of Wikstroemia alternifolia and their neuroprotective activities.

Phytochemical investigation on the plant of Wikstroemia alternifolia led to the isolation of 26 compounds including two new ones, wikstralternifols A and B (1 and 7). Their structures including the absolute configuration were elucidated by spectroscopic data together with analysis of experimental and calculated ECD data. All compounds were isolated from this plant for the first time, and their main structural types were lignans, sesquiterpenoids, and flavonoids. In the sodium nitroprusside-induced rat pheochromocytoma PC-12 cell model, the neuroprotective activities of the selected sesquiterpenoids (1 and 4) and lignans (7 - 14) were screened at the concentration of 10 µM, and 7 - 14 displayed better activities than the positive control edaravone.

Anti-HIV Tigliane-Type Diterpenoids from the Aerial Parts of Wikstroemia lichiangensis.

Tigliane-type diterpenoids have attracted much attention in drug discovery since they have been reported to exhibit remarkable biological effects, such as tumor-promoting, antineoplastic, and anti-HIV activities. In continuing our efforts to discover novel biologically important diterpenoids from Wikstroemia species, Wikstroemia lichiangensis was investigated phytochemically for the first time. As a result, four new (1-4) and one known (5) tigliane-type diterpenoid were isolated, and their structures were elucidated by spectroscopic data analysis. Tiglianes (1-5) showed potent anti-HIV activity against HIV-1 infection of MT4 lymphocytes with IC50 values of 1.1-65.4 nM.

Retusone A, a Guaiane-Type Sesquiterpene Dimer from Wikstroemia retusa and Its Inhibitory Effects on Histone Acetyltransferase HBO1 Expression.

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Three new compounds from the dried root bark of Wikstroemia indica and their cytotoxicity against HeLa cells.

Three new compounds (1-3) and twelve known compounds (4-15) were isolated from the n-butanol extraction of the 70% ethanol extract of the dried root barks of Wikstroemia indica. The structures of new compounds were identified by chemical evidence and extensive analysis of spectroscopic data (HR-ESI-MS, 1 D and 2 D NMR) and ECD calculations. All isolated compounds were evaluated for their cytotoxicity against HeLa cells using MTT assay and the results showed that daphnoretin (compound 5) owned the highest cytotoxicity against HeLa cells (IC50=28.89 µmol/L).

Inhibitory Effects of Luteolin 7-Methyl Ether Isolated from Wikstroemia ganpi on Tnf-A/Ifn-Γ Mixture-Induced Inflammation in Human Keratinocyte.

Plants of the genus Wikstroemia are traditionally used in China to treat various inflammatory diseases. The purpose of this study was to isolate the components of Wikstroemia ganpi (Siebold & Zucc.) Maxim., to evaluate their anti-atopic activities and to identify candidates with anti-atopic therapeutics. A total of 24 compounds were isolated by bioassay-guided separation, including one novel compound, which was tilianin 5-methyl ether. The anti-atopic activities of the isolated compounds were determined using TNF-α-treated RBL-2H3 cells and HaCaT cells. The mRNA expressions of IL-4, IL-6, GM-CSF, G-CSF and TRPV1 were reduced by luteolin 7-methyl ether. The study shows that the luteolin 7-methyl ether isolated from W. ganpi is a potential therapeutic agent for the treatment of atopic dermatitis.

Diarylpentanones from the root of Wikstroemia indica and their cytotoxic activity against human lung A549 cells.

One new diarylpentanone, 4(S)-hydroxy-1, 5-diphenyl -2(E)-en-1-pentanone (1), two diarylpentanones isolated from Wikstroemia indica for the first time (3 and 5) and two other known diarylpentanones were isolated from petroleum ether fraction of root of Wikstroemia indica. The structure of the new compound including absolute configuration was elucidated by extensive spectroscopic techniques, including 1D, 2D NMR, HR-ESI-MS and ECD spectroscopy. All isolated compounds were tested for their cytotoxic activity against cancer-derived cell lines A549. Compound 5 exhibited remarkable cytotoxic activity comparable to that of positive control cisplatin.

LC-MS Identification, Isolation, and Structural Elucidation of Anti-HIV Tigliane Diterpenoids from Wikstroemia lamatsoensis.

Structurally diverse tigliane diterpenoids have drawn significant research interest for drug discovery over many decades. Using LC-MS-guided fractionation and separation, the first phytochemical investigation on Wikstroemia lamatsoensis led to the isolation of eight tiglianes (1-8), including two new compounds, wikstrocin D (1) and wikstrocin E (2). The new structures were elucidated based on extensive physicochemical and spectroscopic analyses. The characteristic ESIMS/MS fragmentations of tiglianes 1-8 were also summarized. Among the isolated tiglianes, three compounds (8, 5, and 7) showed the most potent anti-HIV activity, with IC50 values of 0.18, 3.8, and 12.8 nM, respectively.

Comprehensive investigation of pharmacodyamic material basis of Wikstroemia indica (L.) C. A. Mey. by serum pharmacochemistry and bivariate correlation analysis.

In this study, the theory of serum pharmacochemistry of traditional Chinese medicine was used to analyze the constituents absorbed into serum after oral administration of Wikstroemia indica (L.) C. A. Mey. by ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The micro-liquid dilution method was used to determine the minimum inhibitory concentration of the serum containing Wikstroemia indica. The bivariate correlation analysis method was used to study the spectral-efficiency relationship between the drug-containing serum and the antibacterial activity, and find the main antibacterial active components in serum containing Wikstroemia indica. A total of 26 serum migration components were identified or speculated in the samples, including 11 prototype components and 15 metabolites. Of which, syringic acid, caffeic acid, dihydrocaffeic acid, 4-hydroxybenzoic acid, hippuric acid, 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid, triumbelletin, (7R)-3-hydroxy-1-methyl-2-oxo-7-(prop-1-en-2-yl)-2,3,5,6,7,8- hexahydroazulene-4- carbaldehyde and (1S,3aS,8aS)-1,3,5-trihydroxy-1,4-dimethyl-7-(propan-2- ylidene) octahydroazulen-6(1H)-one were bacteriostatic active substances. It is the first time to study the constituents in serum containing Wikstroemia indica and reveal its antibacterial pharmacodyamic material basis. The above works provide scientific reference for the in-depth study of Wikstroemia indica.

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.

Anti-HIV Tigliane Diterpenoids from Wikstroemia scytophylla.

During a chemical investigation of Wikstroemia scytophylla, three new [wikstrocins A-C (1-3)] and three known tigliane diterpenoids (4-6) were isolated. The structures of the new compounds were elucidated from extensive physiochemical and spectroscopic analysis. The correlations between the ECD Cotton effects and B ring structures of tiglianes were also evaluated. The isolated compounds were assessed for their anti-HIV activity against HIV-1 infection of MT4 cells, and two compounds (4 and 6) showed potent anti-HIV activity with IC50 values of 3.8 and 12.8 nM, respectively.

Bioactive daphnane diterpenes from Wikstroemia chuii with their potential anti-inflammatory effects and anti-HIV activities.

A phytochemical investigation on the stems and leaves of Wikstroemia chuii resulted in the isolation of three new daphnane diterpenes, wikstroechuins A-C (1-3), together with eight known analogues (4-11). The structures of new daphnane diterpenes (1-3) were determined on the basis of extensive spectroscopic methods and the known daphnane diterpenes (4-11) were identified by comparing their observable spectroscopic data with those reported spectral data in the literature. The anti-inflammatory effects as well as anti-HIV activities in vitro of all isolated daphnane diterpenes 1-11 were assessed. As a consequence, daphnane diterpenes 1-11 displayed remarkable inhibitory activities on NO (nitric oxide) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells showing IC50 values in the range of 0.12 ± 0.03 to 10.58 ± 0.16 µM. Meanwhile, daphnane diterpenes 1-11 displayed significant anti-HIV-1 reverse transcriptase (RT) effects showing EC50 values ranging from 0.09509 to 8.62356 µM. These research results indicated that the discovery of these new daphnane diterpenes with remarkable anti-inflammatory and anti-HIV activities from W. chuii, especially these new ones, could be extremely meaningful to the discovery of new anti-inflammatory agents and anti-HIV drugs as well as their potential practical values in the health and pharmaceutical products.

Curcumin and wikstroflavone B, a new biflavonoid isolated from Wikstroemia indica, synergistically suppress the proliferation and metastasis of nasopharyngeal carcinoma cells via blocking FAK/STAT3 signaling pathway.

BACKGROUND: Curcumin (CUR) is a natural diarylheptanoid with marked anti-tumor activities. Recent investigations demonstrate that CUR combines with some other phytochemicals exerts advantages over its single application manifested as lower toxicity, higher efficacy or more significant reversal of multidrug resistance. PURPOSE: This study aimed to elucidate a new biflavonoid (wikstroflavone B, WFB) isolated from Wikstroemia indica and to assess the synergistic inhibition of combined CUR and WFB (CUR/WFB) on human nasopharyngeal carcinoma (NPC) cell lines proliferation and metastasis. METHODS: WFB was obtained through sequential chromatographic methods including silica gel, Sephadex LH-20 and preparative HPLC. Its structure was determined by HRESIMS, 1D and 2D NMR spectroscopic analysis. The absolute configuration of WFB was assigned through comparison of experimental and calculated optical rotation (OR) values. Changes in cellular viability, migration and invasion were assessed by MTT, colony formation, wound healing and Transwell assays. The nature of synergistic interaction of CUR/WFB was determined through the combination index (CI) method under the median-effect analysis. Expression levels of indicated mRNAs and proteins were measured by qRT-PCR and Western blotting assays, respectively. RESULTS: WFB was isolated and structural elucidated. Compared with CUR or WFB used alone, CUR/WFB treatment inhibited more effectively on the cell viability, colony formation, cell migration and invasion. Both CI and dose reduction index (DRI) values indicated the significant synergistic effects existed between CUR and WFB. Besides, CUR/WFB showed the marked modulation on the genes involved in cell proliferation (survivin, cyclin D1, p53 and p21) and metastasis (MMP-2, MMP-9 and FAK). CUR/WFB treatment was also found to restrain the phosphorylation of FAK and STAT3 proteins. When pretreatment with a FAK inhibitor, the cell viability and metastasis were significantly attenuated. CONCLUSION: The results indicate that WFB can synergistically increase the inhibitory effects of CUR on NPC cells proliferation and metastasis, and these findings may afford a rational approach for developing the antitumor medications.

Guaiane type of sesquiterpene with NO inhibitory activity from the root of Wikstroemia indica.

Roots of Wikstroemia indica is widely used in China as a folk medicine in treatment of many diseases. However, active compounds of guaiane type of sesquiterpene remain largely unknown. In the present work, five new guaiane of type sesquiterpene compounds wikstronone A-E, along with one known guaiane type of sesquiterpene compound were isolated from the petroleum and CH2Cl2 fraction of roots of Wikstroemia indica. Structures of these compounds including absolute configuration were determined by extensive NMR and CD spectroscopic analysis. The inhibitory activity of all isolated compounds were assayed against LPS-induced NO production in RAW 264.7 macrophages by Griess reagent. Among all compounds, wikstronone A (1) showed remarkable NO inhibitory activity comparable to that of positive control Indometacin. Wikstronone D and E (4 and 5) showed weak NO inhibitory activity. The isolated guaiane type of sesquiterpene may be potent NO synthetase inhibitors.

Suppression of DNCB-Induced Atopic Skin Lesions in Mice by Wikstroemia indica Extract.

Wikstroemia indica (L.) C.A. Mey. is used in traditional Chinese medicine to treat inflammatory diseases such as arthritis and bronchitis. In this study, we aimed to investigate the effects of an ethanolic extract of W. indica on cutaneous inflammation in mice with 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD). Dermal administration of W. indica ethanolic extract to DNCB-sensitized hairless mice with dermatitis, for two weeks, reduced erythema, scaling, and edema. Skin hydration was improved and transepidermal water loss was reduced at a W. indica concentration of 1%. Furthermore, W. indica also significantly reduced serum IgE and IL-4 concentrations in our mouse model. These results suggest that W. indica has potential as a topical treatment for AD and as an adjunctive agent to control AD.

Chamaejasmine Isolated from Wikstroemia dolichantha Diels Suppresses 2,4-Dinitrofluoro-benzene-Induced Atopic Dermatitis in SKH-1 Hairless Mice.

Plants of the genus Wikstroemia have long been used as traditional medicines to treat diseases like pneumonia, rheumatism, and bronchitis. This study was designed to determine the effect of chamaejasmine, a biflavonoid present in W. dolichantha, on atopic dermatitis (AD)-like skin lesions in a 2,4-dinitrochlorobenzene (DNCB)-induced murine model of AD. Initially, we examined the anti-allergic activities of ten flavonoids from W. dolichantha by measuring ß-hexosaminidase release from RBL-2H3 cells. Subsequently, an SKH-1 hairless mouse model of AD was developed based on the topical application of DNCB. Chamaejasmine (0.5%) or pimecrolimus (1%, positive control) were applied to dorsal skins of DNCB-sensitized AD mice for two weeks. Serum IL-4 and IgE levels were determined using enzyme-linked immunosorbent assay kits and transepidermal water loss (TEWL) and skin hydration were measured using a Tewameter TM210 and a SKIN-O-MAT, respectively. Of the ten flavonoids isolated from W. dolichantha, chamaejasmine most potently inhibited DNP-specific IgE-induced degranulation in RBL-2H3 cells. Topical administration of chamaejasmine attenuated the clinical symptoms of DNCB-induced dermatitis (i.e., itching, dryness, erythema, and edema). Histological analyses demonstrated that dermal thickness and mast cell infiltration in dermis were significantly reduced by chamaejasmine. In addition, 0.5% chamaejasmine inhibited DNCB-induced increases in total IL-4 and IgE levels in serum, improved skin barrier function, and increased epidermis moisture. Our findings suggest chamaejasmine might be an effective therapeutic agent for the treatment of atopic diseases.

[Chemical profiling from water extract of Wikstroemia indica by UPLC-Q-TOF-MS/MS].

In this study,a method using ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) was established to identify complicated chemical constituents of Wikstroemia indica. Chromatographic separation was performed on an AcclaimTMRSLC 120-C18 column( 2. 1 mm×100 mm,2. 2 µm) using gradient elution with 0. 2% ammonium formate buffer salt solution( A)-0. 2% ammonium formate buffer salt solution methanol( B) as mobile phase. The column temperature was maintained at 30 ℃. The analytes were determined by positive and negative ion modes with electro-spray ionization source. A total of 52 compounds( including eleven coumarins,thirteen flavonoids,ten lignans,two amides,four phenolic acids,six sesquiterpenes and six other compounds) were identified or tentatively characterized from the water extract of W. indica by comparing their retention times and MS spectra with those of authentic standards or literature datas. Three compounds were found for the first time from W.indica namely isomer of indicanone,ß-hydroxypropiovanillone and epiprocurcumenol. Furthermore,the fragmentation rules of some compounds were speculated and summarized. In addition,the cleavage pathways of guaiane sesquiterpenes were described for the first time,which can provide reference for studying the fragmentation pathways of similar compounds. This study provides an easy way to identify chemical constituents of traditional Chinese medicine and a basis for the further study on chemical fundamentals of W. indica.

Effect of Topically Applied Wikstroemia dolichantha Diels on the Development of Atopic Dermatitis-Like Skin Symptoms in Mice.

Plants of the genus Wikstroemia are traditionally used to treat inflammatory diseases like bronchitis and rheumatoid arthritis. In the present study, the anti-atopic effects of an EtOH extract of Wikstroemia dolichantha (WDE) on oxazolone- and DNCB (2,4-dinitrochlorobenzene)-induced dermatitis in mice were investigated. Both ears of BALB/c mice were exposed to oxazolone, and dorsal skins of SKH-1 hairless mice were sensitized with DNCB to induce acute eczematous atopic skin lesions. 1% WDE was applied daily to oxazolone- and DNCB-induced AD mice for two or three weeks, respectively. Total IL-4 and IgE concentrations in serum, transepidermal water loss (TEWL) and skin hydration were assessed. High-performance liquid chromatography/mass spectrometry (HPLC/MS) was used to determine the composition of WDE. Dermal application of 1% WDE grossly and histopathologically improved oxazolone- and DNCB-induced AD skin symptoms. Epidermal thickness and mast cell infiltration were significantly lower in animals treated with WDE than in vehicle controls. Furthermore, in addition to reducing DNCB-induced increases in serum IL-4 (interleukin 4) and IgE (immunoglobulin E) levels, WDE also decreased TEWL and increased skin hydration (indicative of improved skin barrier function). The four flavonoids taxifolin, aromadendrin, padmatin and chamaejasmine were tentatively identified in WDE by HPLC-DAD/QTOF-MS. The above results show WDE protected against oxazolone- and DNCB-induced AD in mice by down-regulating the TH2-associated cytokine IL-4 and improving skin barrier function and suggest WDE might be useful for the management of atopic dermatitis.