Antrodia cinnamomea exerts an anti-hepatoma effect by targeting PI3K/AKT-mediated cell cycle progression in vitro and in vivo.

Antrodia cinnamomea is extensively used as a traditional medicine to prevention and treatment of liver cancer. However, its comprehensive chemical fingerprint is uncertain, and the mechanisms, especially the potential therapeutic target for anti-hepatocellular carcinoma (HCC) are still unclear. Using UPLC‒Q-TOF/MS, 139 chemical components were identified in A. cinnamomea dropping pills (ACDPs). Based on these chemical components, network pharmacology demonstrated that the targets of active components were significantly enriched in the pathways in cancer, which were closely related with cell proliferation regulation. Next, HCC data was downloaded from Gene Expression Omnibus database (GEO). The Cancer Genome Atlas (TCGA) and DisGeNET were analyzed by bioinformatics, and 79 biomarkers were obtained. Furtherly, nine targets of ACDP active components were revealed, and they were significantly enriched in PI3K/AKT and cell cycle signaling pathways. The affinity between these targets and their corresponding active ingredients was predicted by molecular docking. Finally, in vivo and in vitro experiments showed that ACDPs could reduce the activity of PI3K/AKT signaling pathway and downregulate the expression of cell cycle-related proteins, contributing to the decreased growth of liver cancer. Altogether, PI3K/AKT-cell cycle appears as the significant central node in anti-liver cancer of A. Cinnamomea.

Effects and mechanisms of natural plant active compounds for the treatment of osteoclast-mediated bone destructive diseases.

Bone-destructive diseases, caused by overdifferentiation of osteoclasts, reduce bone mass and quality, and disrupt bone microstructure, thereby causes osteoporosis, Paget's disease, osteolytic bone metastases, and rheumatoid arthritis. Osteoclasts, the only multinucleated cells with bone resorption function, are derived from haematopoietic progenitors of the monocyte/macrophage lineage. The regulation of osteoclast differentiation is considered an effective target for the treatment of bone-destructive diseases. Natural plant-derived products have received increasing attention in recent years due to their good safety profile, the preference of natural compounds over synthetic drugs, and their potential therapeutic and preventive activity against osteoclast-mediated bone-destructive diseases. In this study, we reviewed the research progress of the potential antiosteoclast active compounds extracted from medicinal plants and their molecular mechanisms. Active compounds from natural plants that inhibit osteoclast differentiation and functions include flavonoids, terpenoids, quinones, glucosides, polyphenols, alkaloids, coumarins, lignans, and limonoids. They inhibit bone destruction by downregulating the expression of osteoclast-specific marker genes (CTSK, MMP-9, TRAP, OSCAR, DC-STAMP, V-ATPase d2, and integrin av3) and transcription factors (c-Fos, NFATc1, and c-Src), prevent the effects of local factors (ROS, LPS, and NO), and suppress the activation of various signalling pathways (MAPK, NF-κB, Akt, and Ca2+). Therefore, osteoclast-targeting natural products are of great value in the prevention and treatment of bone destructive diseases.

Inflammatory Response and Oxidative Stress as Mechanism of Reducing Hyperuricemia of Gardenia jasminoides-Poria cocos with Network Pharmacology.

Hyperuricemia (HUA) is a metabolic disease, closely related to oxidative stress and inflammatory responses, caused by reduced excretion or increased production of uric acid. However, the existing therapeutic drugs have many side effects. It is imperative to find a drug or an alternative medicine to effectively control HUA. It was reported that Gardenia jasminoides and Poria cocos could reduce the level of uric acid in hyperuricemic rats through the inhibition of xanthine oxidase (XOD) activity. But there were few studies on its mechanism. Therefore, the effective ingredients in G. jasminoides and P. cocoa extracts (GPE), the active target sites, and the further potential mechanisms were studied by LC-/MS/MS, molecular docking, and network pharmacology, combined with the validation of animal experiments. These results proved that GPE could significantly improve HUA induced by potassium oxazine with the characteristics of multicomponent, multitarget, and multichannel overall regulation. In general, GPE could reduce the level of uric acid and alleviate liver and kidney injury caused by inflammatory response and oxidative stress. The mechanism might be related to the TNF-α and IL-7 signaling pathway.

Glaucocalyxin A suppresses osteoclastogenesis induced by RANKL and osteoporosis induced by ovariectomy by inhibiting the NF-κB and Akt pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Glaucocalyxin A (GLA), the most abundant active component of the aboveground sections of Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, possesses various pharmacological activities, such as antioxidant, antithrombosis, anticoagulation, antibacterial, antitumor, anti-inflammatory activities. According to previous studies, inflammation is closely associated with osteoclast differentiation and activity. Although GLA has demonstrated effective anti-inflammatory properties, its effects on osteoclast differentiation remain unclear. AIM OF THE STUDY: To examine the possible inhibitory effects of GLA and its molecular mechanisms in osteogenesis induced by RANKL as well as ovariectomy (OVX)-induced osteoporosis (OP) in mice. MATERIALS AND METHODS: Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and a bone resorption pit assay were applied for identifying the effects of GLA on the differentiation of osteoclasts and the function of bone resorption. The mRNA expression of the genes related to osteoclast differentiation was measured by quantitative PCR. Protein expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-fos and phosphorylation of inhibitor of nuclear factor kappa B (IκBα), protein kinase B (AKT), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 in RANKL-induced osteoclasts was determined using western blotting. The effect of GLA on OP was studied using a mouse model of OVX. RESULTS: At nontoxic concentrations ≤0.5 µM in vitro, GLA suppressed the formation of osteoclasts induced by RANKL with the decreased number and area size of TRAP-positive multinuclear osteoclasts, and the resorption of bone function by reducing F-actin ring number and bone resorption pit areas. It also reduced the expression of the genes specific for osteoclasts, which included genes encoding NFATc1, cathepsin K, c-fos, TRAP, vacuolar-type ATPase d2, and dendritic cell-specific transmembrane protein. Moreover, GLA repressed NF-κB and Akt pathway activation induced by RANKL. Micro-CT analysis of femur samples indicated decreased bone loss and greater trabecular bone density after GLA treatment, which showed that GLA played a protective role by inhibiting bone loss in OVX-induced OP mice in vivo. CONCLUSIONS: Our study is the first to show that GLA has significant therapeutic potential in OP, which is the disease of osteoclast increase caused by estrogen deficiency.

Vindoline Attenuates Osteoarthritis Progression Through Suppressing the NF-κB and ERK Pathways in Both Chondrocytes and Subchondral Osteoclasts.

Disruption of extracellular matrix (ECM) homeostasis and subchondral bone remodeling play significant roles in osteoarthritis (OA) pathogenesis. Vindoline (Vin), an indole alkaloid extracted from the medicinal plant Catharanthus roseus, possesses anti-inflammatory properties. According to previous studies, inflammation is closely associated with osteoclast differentiation and the disorders of the homeostasis between ECM. Although Vin has demonstrated effective anti-inflammatory properties, its effects on the progression of OA remain unclear. We hypothesized that Vin may suppress the progress of OA by suppressing osteoclastogenesis and stabilizing ECM of articular cartilage. Therefore, we investigated the effects and molecular mechanisms of Vin as a treatment for OA in vitro and in vivo. In the present study, we found that Vin significantly suppressed RANKL-induced osteoclast formation and obviously stabilized the disorders of the ECM homeostasis stimulated by IL-1ß in a dose-dependent manner. The mRNA expressions of osteoclast-specific genes were inhibited by Vin treatment. Vin also suppressed IL-1ß-induced mRNA expressions of catabolism and protected the mRNA expressions of anabolism. Moreover, Vin notably inhibited the activation of RANKL-induced and IL-1ß-induced NF-κB and ERK pathways. In vivo, Vin played a protective role by inhibiting osteoclast formation and stabilizing cartilage ECM in destabilization of the medial meniscus (DMM)-induced OA mice. Collectively, our observations provide a molecular-level basis for Vin's potential in the treatment of OA.

Natural Products: Review for Their Effects of Anti-HBV.

Hepatitis B is a global infectious disease, seriously endangering human health. Currently, there are mainly interferons and nucleoside analogues treatment of hepatitis B in the clinic, which have certain therapeutic effects on hepatitis B, but their side effects and drug resistance are increasingly prominent. Therefore, it is urgently needed to discover and develop new anti-HBV drugs, especially natural products, which have novel, high efficiency, and low toxicity anti-HBV compounds with novel antiviral mechanisms. In this manuscript, the natural products (polysaccharides and 165 compounds) with the activity of antihepatitis B virus are discussed according to their chemical classes, including 14 phenylpropanoids, 8 flavonoids,12 xanthones, 13 anthroquinones, 47 terpenoids, 6 alkaloids, 15 enediynes, 11 aromatics, 18 phenylalanine dipeptides compounds, and 13 others. In addition, the anti-HBV mechanism and targets of natural product were also discussed. The aim of this review is to report new discoveries about anti-HBV natural products and to provide reference for researchers.

Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS2 in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism.

Scheflera heptaphylla (L.)Frodin, a kind of Traditional Chinese Medicine, is commonly used in anti-inflammatory, analgesic, anti-viral, anti-tumor, and hemostasis. This study aimed to determine the anti-hepatoma components and its mechanism from the leaves of S. heptaphylla. The spectrum-effect relationships were analyzed by the method of Partial least squares, indicating that P1, P2, and P10 were positively correlated to inhibitory activity of Huh7 cells. Whereas others were negatively correlated. The technologies of component knock-out and UPLC-MS2 were used to determine compounds as 3,4-Dicaffeoylquinic acid (P6), 3,5-Dicaffeoylquinic acid (P7), 3α-Hydroxy-lup-20(29)-ene-23,28-dioic acid (P10, named Compound A). The results forecasted that Compound A had the best correlation with inhibitory activity. The effects of Compound A on the activities of human hepatoma cells (Huh7, SMMC-7721, HepG 2) and normal hepatocytes (L0-2, Chang liver) were evaluated. Cell apoptosis was observed with inverted microscope and flow cytometer. In addition, the proteins, related to apoptosis, were detected by Western blot. The results showed that Compound A (400 nM) could significantly inhibit the activity of three hepatoma cells (P < 0.001) with slight toxicity to normal hepatocytes, and the IC50 values were 285.3 and 315.1 nM, respectively, which were consistent with the prediction of spectrum-effect relationships. After treatment with Compound A, the number of hepatoma cells decreased significantly. And the apoptosis rate of Huh7 cells increased significantly (P < 0.001) in Compound A (200, 400 nM) groups, SMMC-7721 and HepG 2 were directly necrotic. Compound A groups could significantly improve the level of intracellular reactive oxygen species (ROS) (P < 0.05, P < 0.001) in Huh7 with no effect on normal hepatocytes. The content of apoptotic protein (Bax and Bim) in mitochondria was significantly increased in Compound A groups (P < 0.001). On the contrary, the content of anti-apoptotic protein (Bcl-xL and Mcl-1) decreased significantly (P < 0.001). These results demonstrated that Compound A was the main anti-hepatoma active component in the S. heptaphylla leaves. It achieved the effect of promoting apoptosis of Huh7 cells by regulating the levels of ROS and Bcl-2 family protein in mitochondrial apoptosis pathway.

Efficient Extraction of Anti-Inflammatory Active Ingredients from Schefflera octophylla Leaves Using Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with HPLC.

Schefflera octophylla (Lour.) Harms, a kind of traditional Chinese medicine (TCM), is commonly used for anti-inflammatory, analgesic, rheumatism, fever, and hemostasis therapy. In our previous studies, two major triterpenoids were isolated and identified from leaves of S. octophylla, and evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells; both of them displayed significant anti-inflammatory activities at their noncytotoxic concentrations. Therefore, it is very useful to establish an efficient and green extraction method to isolated the two major triterpenoids from leaves of S. octophylla. In this paper, ionic liquid based ultrasonic-assisted extraction (ILUAE) was successfully applied to extract the two major triterpenoids from leaves of S. octophylla. Four single factors (ionic liquids (ILs) concentration, solid-liquid ratio, centrifugal speed, mesh number), with a greater impact on extraction rate, were selected from a variety of influencing factors, and the optimal conditions were obtained by Box-Behnken response surface methodology (RSM). Under optimal conditions, the total extraction yield and extraction rate of two triterpenoids were 288.03 mg/g and 28.80%, respectively, which was 6.80% higher than that of 70% Ethanol (220 mg/g and 22%, respectively).

Toosendanin induces the apoptosis of human Ewing's sarcoma cells via the mitochondrial apoptotic pathway.

Toosendanin, a triterpenoid extracted from the root bark of Melia toosendan, has its origin from traditional Chinese medicine and has been used as a non­polluting and pesticide­free plant insecticide in China for fruit and vegetable production. In recent years, toosendanin has been found to inhibit tumor cell proliferation and promote tumor cell apoptosis. Ewing's sarcoma (ES) is the second most common primary malignant bone and soft tissue tumor in children and adolescents. Although the overall prognosis of ES has improved, the 5­year survival rate has not significantly increased. To analyze the role of toosendanin on ES progression, CCK­8 viability assay, flow cytometry, Hoechst 33258 staining and western blotting were performed. The present results suggested that toosendanin suppressed cell viability and induced apoptosis in human SK­ES­1 cells compared with DMSO treatment. In addition, in the present study, toosendanin was found to upregulate the expression of Bax and downregulate the expression of Bcl­2, altering the Bax/Bcl­2 ratio. Additionally, toosendanin promoted the release of cytochrome c, resulting in the activation of the mitochondrial apoptotic pathway, thus inducing the activation of caspase­9 and caspase­3, and the cleavage of PARP. Our results demonstrated that toosendanin inhibited the growth of ES cells in a dose­dependent manner and triggered mitochondrial apoptotic pathway to induce apoptosis. Therefore, toosendanin can potentially be utilized as an anticancer botanical drug for the treatment of ES.

Bergapten suppresses RANKL-induced osteoclastogenesis and ovariectomy-induced osteoporosis via suppression of NF-κB and JNK signaling pathways.

Bergapten (BP), derived from Cnidium monnieri (L.) Cusson, is an ingredient widely used in traditional Chinese medicine and has important biological and pharmacological activities. However, the effect of BP on ovariectomy-induced osteoporosis and the underlying mechanism are not entirely clear. In this study, we investigated the effects of BP on ovariectomy-induced osteoporosis and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vivo and in vitro, and explored the underlying mechanism. We found that BP treatment exerted beneficial effects on ovariectomy-induced osteoporosis in vivo. Further, BP attenuated osteoclastogenesis in bone marrow macrophages (BMMs) and RAW264.7 cells without any cytotoxicity. Additionally, BP specifically inhibited RANKL-induced NF-κB and JNK signaling,but did not suppress p38 and ERK. At the mRNA level, BP inhibits the OC-associated transcription factor NFATc1 and c-fos, thereby affecting the expression of OC differentiation-related genes. Moreover, BP disrupted the formation of F-actin rings, which are important for bone-resorbing activity, and impairs OC bone resorption. Therefore, BP may be a useful alternative therapy for post-menopausal osteoporosis.

Andrographolide prevents human breast cancer-induced osteoclastic bone loss via attenuated RANKL signaling.

Bone metastasis is a common and serious complication in advanced cancers such as breast cancer, prostate cancer, and multiple myeloma. Agents that prevent bone loss could be used to develop an alternative therapy for bone metastasis. RANKL, a member of the tumor necrosis factor superfamily, has been shown to play a significant role in cancer-associated bone loss. In this study, we examined the efficacy of the natural compound andrographolide (AP), a diterpenoid lactone isolated from the traditional Chinese and Indian medicinal plant Andrographis paniculata, in reducing breast cancer-induced osteolysis. AP prevented human breast cancer-induced bone loss by suppressing RANKL-mediated and human breast cancer cell-induced osteoclast differentiation. Molecular analysis revealed that AP prevented osteoclast function by inhibiting RANKL-induced NF-κB and ERK signaling pathway in lower dose (20 µM), as well as inducing apoptosis at higher dose (40 µM). Thus, AP is a potent inhibitor of breast cancer-induced bone metastasis.