Bmk9 and Uricase Nanoparticle Complex for the Treatment of Gouty Arthritis and Uric Acid Nephropathy.

Uric acid is the final product of purine metabolism, and excessive serum uric acid can cause gouty arthritis and uric acid nephropathy. Therefore, lowering the uric acid level and alleviating inflammation in the body are the key points to treating these diseases. A stable nanosuspension of peptide BmK9 was prepared by the precipitation-ultrasonication method. By combining uricase on the surface of a positively charged carrier, a complex consisting of neutral rod-shaped BmK9 and uricase nanoparticles (Nplex) was formed to achieve the delivery of BmK9 and uricase, respectively. The formulation of Nplex has a diameter of 180 nm and drug loading up to 200%, which releases BmK9 and uricase slowly and steadily in drug release tests in vitro. There was significantly improved pharmacokinetic behavior of the two drugs because Nplex prolonged the half-life and increased tissue accumulation. Histological assessments showed that the dual drug Nplex can reduce the inflammation response in acute gouty arthritis and chronic uric acid nephropathy in vivo. In the macrophage system, there was lower toxicity and increased beneficial effect on inflammation with Nplex than free BmK9 or uricase. Collectively, this novel formulation provides a dual drug delivery system that can treat gouty arthritis and uric acid nephropathy.

Identification of poly(ADP-ribose)polymerase 1 and 2 (PARP1/2) as targets of andrographolide using an integrated chemical biology approach.

Here, we describe the identification of PARP1/2 as direct binding proteins of andrographolide (Andro) using protein microarray, surface plasmon resonance (SPR), and enzyme activity assays. We then evaluated the proliferation inhibition, apoptosis, and cell migration effects of Andro on the MDA-MB-436 cell line in vitro. The final biological evaluation confirmed that Andro was a highly effective single agent in the MDA-MB-436 xenograft model and had a low hERG-mediated cardiac toxicity. Therefore, Andro represents the first natural product, non-amide member of a novel nanomolar-potency PARP1/2 inhibitor family.

Isolation and characterization of ent-pimarane diterpenoids from Sigesbeckia pubescens.

Five new ent-pimarane diterpenoids ent-16-nor-2-oxopimar-8(14)-ene-15,19-dial (1), ent-16-nor-2α,19-dihydroxypimar-8-en-15-al (2), 3-O-acetyldarutigenol (3), 19-O-acetylkirenol (4), ent-16-nor-3ß,15-dihydroxypimar-8(14)-ene (5) were isolated and characterized from the ethanol extract of Sigesbeckia pubescens. Their structures were elucidated on the basis of spectroscopic analysis. The absolute configuration of C-15 in compounds 3 and 4 was assigned using Snatzke's method. All these compounds were assessed for their anti-inflammatory potential by measuring the inhibitory effects on NO production in LPS-induced RAW 264.7 macrophage cells and compound 4 showed significantly inhibitory activity with IC50 value of 5.9 µM.

Synthesis and biological evaluation of panaxatriol derivatives against myocardial ischemia/reperfusion injury in the rat.

Panaxatriol (PT) is a natural product derived from ginseng that possesses cardioprotective effects in isolated rat hearts. To develop more potent therapeutic agents against myocardial ischemia/reperfusion (MI/R) injury from natural products, a novel series of heterocycle ring-fused panaxatriol derivatives were designed and synthesized. In vitro results showed that approximately half of them exhibited increased cytoprotective activity compared with PT in a cardiomyocyte model of oxygen-glucose deprivation and reperfusion (OGD/R) injury. Furthermore, the in vitro activity of the representative derivative, compound 18, was also confirmed in a rat model of MI/R injury. In vivo results showed that 18 can markedly reduce myocardial infarction size, decrease circulating cardiac troponin I (cTnI) leakage, and alleviate cardiac tissue damage in the rats. Therefore, these findings provide the basis for further development of novel anti-MI/R injury agents.

Synthesis and biological evaluation of Isosteviol derivatives as FXa inhibitors.

Firstly, a series of Isosteviol derivatives were synthesized and evaluated for FXa inhibitory activity. Among these compounds, the inhibitory activity of compounds 22, 35 and 38 on FXa was better than that of Isosteviol. Secondly, surface plasmon resonance (SPR) assays were performed for selected compounds. Compounds 22, 35, 38 have similar kinetic signatures, and affinity values were at µM level. Thirdly, compounds 22 and 35 displayed moderate-to-high anticoagulation activity and showed similar sensitivity to PT and aPTT. These findings will provide new insight into the exploration of FXa inhibition.

Synthesis and biological evaluation of Vinpocetine derivatives.

A new series of Vinpocetine derivatives were synthesized and evaluated for their inhibitory activity on PDE1A in vitro. Seven compounds with higher inhibitory activity were selected for surface plasmon resonance (SPR) binding experiments. Compared with Vinpocetine, these high potency compounds presented a higher binding affinity with PDE1A, which was consistent with inhibitory activity. After further screening, compounds 5, 7, 21, 34 and Vinpocetine were selected to examine the vasorelaxant effects on endothelium-intact rat thoracic aortic rings. The study suggested that the effects of compounds 7 and 21 were the most significant with the maximum value of 93.46 ±â€¯0.77% and 92.90 ±â€¯0.78% (n = 5) at a concentration of 100 µM respectively. Based on these studies, compounds 7 and 21 were considered for further development as hit compounds.

Discovery of novel, potent, isosteviol-based antithrombotic agents.

Thrombosis is a pathological coagulation process and can lead to many serious thrombotic diseases. Here, we report a novel potent antithrombotic compound (6k) based on isosteviol with anticoagulant and antiplatelet activities. 6k selectively inhibited FXa (Ki = 0.015 µM) against a panel of serine proteases and showed excellent anticoagulant activity (significant prolongation of ex vivo PT and aPTT over the vehicle, p < 0.01). 6k also significantly inhibited ADP-induced platelet aggregation in rats relative to the vehicle (p < 0.01). Furthermore, 6k exhibited potent ex vivo and in vivo antithrombotic activity in rats relative to the vehicle (p < 0.01 and p < 0.0001, respectively). Novel structure 6k, with potent antithrombotic activity, is expected to lead a promising approach for the development of antithrombotic agents.

Semisynthesis of epoxy-pimarane diterpenoids from kirenol and their FXa inhibition activities.

Kirenol is one of the biologically active diterpenoids from Siegesbeckia pubescens. In terms of the high content and typical structure, many ent-diterpenoids separated from S. pubescens were presumed to be biologically related to kirenol. Among them, epoxy-pimarane diterpenoids are belonging to a special family of naturally occurring compounds that attracted our attentions on their putative biosynthesis pathway and biological activities. Here, we designed and synthesized two known 14,16-epoxy-pimarane diterpenoids (2 and 3) and five 8,15-epoxy-pimarane diterpenoids (4-8) from kirenol. Their absolute structures were determined by 1D and 2D NMR data and the absolute configurations of 4 were confirmed by X-ray crystallographic data. Their inhibition effects on factor Xa (FXa) were evaluated to assess the potentiality of epoxy-pimarane diterpenoids as FXa inhibitor agents.

Semisynthesis of ent-norstrobane diterpenoids as potential inhibitor for factor Xa.

A semisynthesis of two ent-strobane diterpenoids strobols C (7) and D (14) was accomplished via a Wagnar-Meerwein rearrangement. Compounds 7, 14, and the intermediate products were evaluated for their inhibition on factor Xa (FXa). Among all the compounds screened for FXa inhibitory activity, three compounds 6, 7, and 9 showed significant inhibitory activities with IC50 values of 1067 ±â€¯164, 81 ±â€¯11, 1023 ±â€¯89 nM, respectively. The inhibitory activity on FXa described in this study highlight the importance of structural modification based on natural products in the development of FXa inhibitors.

Synthesis and evaluation of panaxatriol derivatives as Na+, K+-ATPase inhibitors.

Panaxatriol, a triterpene bearing a steroid-like structure similar to cardiac glycosides, was presumed to share the same bioactivity with cardiac glycosides, and may be a potential Na+, K+-ATPase inhibitor. In this paper, a series of panaxatriol derivatives were synthesized and evaluated for Na+, K+-ATPase inhibitory activities. The results of biological tests showed that more than half of the synthesized derivatives presented increased inhibitory activities compared with panaxatriol. Of these compounds, 13a with a 3, 4-seco skeleton showed the most potent inhibitory activity, which was equal to that of the standard drug digoxin. To understand the binding mode of the most active compound, molecular docking study of 13a with Na+, K+-ATPase was conducted. Therefore, 13a may serve as a new lead compound for the development of novel Na+, K+-ATPase inhibitors.

The synthesis and anti-metastatic effects of optical isomers of ionone alkaloid 9-(N,N-dimethyl)-4,7-megastigmedien-3-one.

Ionone alkaloid 9-(N,N-dimethyl)-4,7-megastigmedien-3-one (compound 1) is a new anti-metastatic natural product. However, it was previously reported as optical isomers mixture. Herein, the optical isomers (6a-6d) of compound 1 were synthesized. The absolute configurations of 6a-6d were determined by ECD experiments and calculated spectra with time-dependent density functional theory (TDDFT). The anti-metastatic effects of the optical isomers were examined by transwell assay. These results revealed that compound 6a had potential anti-metastatic activity with an IC50 value of 0.512 ±â€¯0.093 µM.

Isolation and characterization of diterpene glycosides from Siegesbeckia pubescens.

Five new diterpenoid glycosides, siegesides A-E (1-5), along with the known compound darutoside (6) were isolated and characterized from the ethanol extract of Siegesbeckia pubescens. The structural elucidation of the isolates was accomplished by extensive HRESIMS and NMR analysis. Compounds 1 and 2 are epimers of 6. All isolates were evaluated for their inhibition on the migration of MB-MDA-231 breast cancer cells induced by the chemokine epithelial growth factor, and compound 2 showed superior inhibitory activities in comparison with the positive control drug with IC50 value of 0.27µM.

ent-Strobane and ent-Pimarane Diterpenoids from Siegesbeckia pubescens.

Two strobane diterpenoids, strobols A (1) and B (2), 15 new pimarane diterpenoids (3-6 and 8-18), and the known compounds kirenol (19), darutigenol (20), and ent-2ß,15,16,19-tetrahydroxypimar-8(14)-ene (7) were isolated from the aerial parts of Siegesbeckia pubescens Makino. The structures of the new compounds were established based on the interpretation of HRESIMS and NMR analysis. The configurations of 1, 6, and 17 were confirmed by X-ray crystallographic data. Compounds 3, 5, and 11 inhibited the migration of MB-MDA-231 breast cancer cells induced by the chemokine epithelial growth factor, with IC50 values of 4.26, 3.45, and 9.70 µM, respectively.

Dimeric Abietane Diterpenoids and Sesquiterpenoid Lactones from Teucrium viscidum.

A new abietane diterpenoid, teuvisone (2), a pair of new dimeric abietane diterpenoid stereoisomers, biteuvisones A (3) and B (4), and three new sesquiterpenoid lactones, teuvislactones A-C (6, 7, and 10), were isolated from the whole plants of Teucrium viscidum, along with four known terpenoids (1, 5, 8, and 9). The structures of the new compounds were elucidated by spectroscopic analysis, and the absolute configurations of 5-10 were determined by electronic circular dichroism analysis. The isolated compounds were evaluated for their cytotoxic effects against five human cancer cell lines and for their α-glucosidase inhibitory effects.

Kirenol, a compound from Herba Siegesbeckiae, induces apoptosis in human chronic myeloid leukemia K562 cells.

Kirenol is a biologically active substance isolated from Herba Siegesbeckiae. In the experiments, we explored a novel antitumor activity of kirenol. The data demonstrated that kirenol had strong cytotoxic effects to human chronic myeloid leukemia K562 cells, the 50% inhibitory concentration (IC50) for kirenol was 53.05 ig/ml, 18.19 pg/ml and 15.08 microg/ml for 24, 48 and 72 h, determined using the MTT assay. Further studies showed that kirenol treatment caused externalization of phosphatidylserine, accumulation of ROS (reactive oxygen species), alteration of mitochondrial membrane potential, release of cytochrome c, reduction in the level of the Bcl-2 protein and upregulation of Bax and tBid, kirenol induced cell apoptosis in a caspase-independent manner. Further studies indicated that kirenol treatment triggered the arrest of cell cycle S period which might resulted from the up-regulation of phosphorylation of p53 (Ser 6 and Ser 37) and expression of p21 protein. Our results indicated that kirenol possesses antitumor action on human chronic myeloid leukemia K562 cells in vitro. Kirenol may have therapeutic potential for the treatment of cancer that deserves further investigation.

Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance.

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2 mg/kg Kirenol or 2 mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs) and IFNγ⁺CD4⁺ and IL4⁺CD4⁺ T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4⁺CD25⁺Foxp3⁺ and IL4⁺CD4⁺ T cells, and reduced the number of IFNγ⁺CD4⁺ T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-ß1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.

Angiogenesis inhibition and cell cycle arrest induced by treatment with Pseudolarix acid B alone or combined with 5-fluorouracil.

Angiogenesis inhibitors combined with chemotherapeutic drugs have significant efficacy in the treatment of a variety of cancers. Pseudolarix acid B (PAB) is a traditional pregnancy-terminating agent, which has previously been shown to reduce tumor growth and angiogenesis. In this study, we used the high content screening assay to examine the effects of PAB on human umbilical vein endothelial cells (HUVECs). Two hepatocarcinoma 22-transplanted mouse models were used to determine PAB efficacy in combination with 5-fluorouracil (5-Fu). Our results suggested that PAB (0.156-1.250 µM) inhibited HUVECs motility in a concentration-dependent manner without obvious cytotoxicity in vitro. In vivo, PAB (25 mg/kg/day) promoted the anti-tumor efficacy of 5-Fu (5 mg/kg/2 days) in combination therapy, resulting in significantly higher tumor inhibition rates, lower microvessel density values, and prolonged survival times. It was also demonstrated that PAB acted by blocking the cell cycle at both the G(1)/S boundary and M phase, down-regulation of vascular endothelial growth factor, hypoxia-inducible factor 1α and cyclin E expression, and up-regulation of cdc2 expression. These observations provide the first evidence that PAB in combination with 5-Fu may be useful in cancer treatment.

Effects of kirenol on bovine type II collagen-induced rat lymphocytes in vivo and in vitro.

OBJECTIVE: To investigate the effect of kirenol on bovine type II collagen (CII)-specific lymphocytes in vivo and in vitro, and explore the mechanism of kirenol-induced immunosuppression in antigen-specific lymphocytes. METHODS: Twenty-four Wistar rats were randomized into control group, collagen-induced arthritis (CIA) model group, kirenol group (2 mg/kg), and prednisolone group (2 mg/kg). After CII injection, the rats in the latter two groups received intragastric administration of kirenol and prednisolone for 30 days, and the spleens and draining lymph nodes of the rats were harvested to prepare single cell suspensions for measurement of the cytokine levels using ELISA. In the in vitro experiment, the lymphocytes from the control rats, with or without 20 µg/ml CII treatment in the presence of 0-80 µg/ml kirenol, were evaluated for cell proliferation and apoptosis using [(3)H]-thymidine incorporation and flow cytometry, respectively. RESULTS: Compared with those in CIA group, IFN-γ and TNF-α production was significantly reduced in splenocyte culture supernatant of kirenol group (P<0.05 and P<0.01, respectively), and the level of IL-10 and IL-4 was up-regulated (P<0.05 and P<0.01, respectively); IFN-γ and TNF-α secretion by the cultured lymph node cells (LNCs) significantly decreased (P<0.05 and P<0.001, respectively) and IL-10 and IL-4 production increased (P<0.05, P<0.001) in kirenol group. In the in vitro experiment, kirenol treatment caused obvious suppression of CII-induced LNC proliferation and dose-dependently induced antigen-specific apoptosis of the splenocytes and LNCs. CONCLUSION: Kirenol treatment reduces pro-inflammatory cytokine secretion, increases anti-inflammatory cytokine production, inhibits cell proliferation and induces apoptosis of CII-specific lymphocytes in vitro, suggesting the potential of kirenol as an immunosuppressant.