Cnidium officinale polysaccharide enhanced RAW 264.7 cells activation and NK-92 cells cytotoxicity against colon cancer via NF-κB and MAPKs signaling pathways.

In this study, Cnidium officinale-derived polysaccharides were isolated and investigated for their immune enhancing and anticancer activities. The isolated crude and its fractions, such as F1 and F2, contain carbohydrates (51.3-63.1%), sulfates (5.4-5.8%), proteins (1.5-7.1%), and uronic acids (2.1-26.9%). The molecular weight (Mw) of the polysaccharides ranged from 59.9 to 429.0 × 103 g/mol. The immunostimulatory activity of the polysaccharides was tested on RAW 264.7 cells, and the results showed that the F2 treatment notably enhanced pro-inflammatory activity in RAW 264.7 cells by increasing NO production and the expression of various cytokines. Furthermore, the influence of polysaccharide treatment on natural killer cells (NK-92) anticancer activities was investigated using a colon cancer cell line (HCT-116). Crude polysaccharide and its fractions showed no direct cytotoxicity to NK-92 and HCT-116 cells. However, the treatment of F2 showed an enhancement of NK-92 cells cytotoxicity against HCT-116 cells by upregulating the mRNA expression of IFN-γ, TNF-α, NKGp44, and granzyme-B. The western blot results showed that the induced RAW 264.7 cells activation and NK-92 cells cytotoxicity occur via NF-κB and MAPK signaling pathways. Overall, C. officinale-derived polysaccharides show potential as immunotherapeutic agents capable of enhancing pro-inflammatory macrophage signaling and activating NK-92 cells; thus, they could be useful for biomedical applications.

Discovery of a traditional Chinese herbal combination for the treatment of atopic dermatitis: saposhnikoviae radix, astragali radix and cnidium monnieri.

Atopic dermatitis (AD) is a skin disease characterized by pruritus. The present study aimed to discover a herbal combination with anti-allergic and anti-inflammatory activities to treat AD. First, the anti-allergic and anti-inflammatory activities of herbs were evaluated by RBL-2H3 degranulation and HaCaT inflammatory models. Subsequently, the optimal proportion of herbs was determined by uniform design-response surface methodology. The effectiveness and synergistic mechanism was further verified. Cnidium monnieri (CM) suppressed ß-hexosaminidase (ß-HEX) release, saposhnikoviae radix (SR), astragali radix (AR), and CM inhibited the release of IL-8 and MCP-1. The optimal proportion of herbs was SR∶AR∶CM = 1: 2: 1. The in vivo experiments results indicated that the topical application of combination at high (2 ×) and low (1 ×) doses improved dermatitis score and epidermal thickness, and attenuated mast cell infiltration. Network pharmacology and molecular biology further clarified that the combination resisted AD by regulating the MAPK, JAK signaling pathways, and the downstream cytokines such as IL-6, IL-1ß, IL-8, IL-10, and MCP-1. Overall, the herbal combination could inhibit inflammation and allergy, improving AD-like symptoms. The present study discovers a promising herbal combination, worthy of further development as a therapeutic drug for AD.

Two O-Methyltransferases Mediate Multiple Methylation Steps in the Biosynthesis of Coumarins in Cnidium monnieri.

Coumarins with methoxy groups such as osthole (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4) are typical bioactive ingredients of many medicinal plants. The methylation steps remain widely unknown. Herein, we report the discovery of two methyltransferases in the biosynthesis of O-methyl coumarins in Cnidium monnieri by transcriptome mining, heterologous expression, and in vitro enzymatic assays. The results reveal that (i) CmOMT1 catalyzes the methylation of osthenol (8) as the final step in the biosynthesis of 1, (ii) CmOMT2 shows the highest efficiency and preference for methylating xanthotoxol (11) to form 2, and (iii) CmOMT1 and CmOMT2 also efficiently transform bergaptol (10) and 8-hydroxybergapten (13) into 3 or 4, suggesting the CmOMTs mediate multistep methylations in the biosynthesis of linear furanocoumarins in C. monnieri.

Ameliorative effects of total coumarins from the fructus of Cnidium monnieri (L.) Cuss. on 2,4-dinitrochlorobenzene-induced atopic dermatitis in rats.

Atopic dermatitis (AD), which is characterized by intense pruritus and serious inflammation, is a chronic skin disease. Modern studies have testified that the total coumarins from the fructus of Cnidium monnieri (TCFC) possess evident biological activities based on their coumarin compounds. The purpose of this manuscript is to investigate the effects of topical use of TCFC on immune response, inflammation, and skin barrier function in rats with 2,4-dinitrochlorobenzene (DNCB)-induced AD. Results indicated that the skin lesion scores of rats were obviously reduced after the management of TCFC, and the spleen and thymus indices also were markedly repressed. TCFC significantly inhibited the overproduction of TNF-α, interferon-γ, interleukin (IL)-4, IL-13, thymic stromal lymphopoietin, and immunoglobulin E; the epidermal thickness and number of mast cells were notably decreased. The western blot experiment was conducted to determine the effects of TCFC on the mitogen-activated protein kinases signaling pathway. Results indicated that phosphorylation of extracellular signal-regulated kinases, p38, and c-Jun amino-terminal kinases was significantly blocked by TCFC. In addition, TCFC could upregulate the expression of filaggrin in dorsal skin, which means that TCFC showed a protective effect on skin barrier disruption. Furthermore, TCFC downregulated the levels of IL-1ß, IL-4, IL-31, and TSLP mRNA and upregulated the expression of filaggrin mRNA in the dorsal skin of rats. Our research demonstrated the ameliorative effects of TCFC on AD-like rats by inhibiting immune response and inflammation and recovering skin barrier function.

Osthole induces cell cycle arrest and apoptosis in head and neck squamous cell carcinoma by suppressing the PI3K/AKT signaling pathway.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common lethal tumors with a high recurrence rate and low survival rate. Therefore, an urgent need exists for novel and effective treatment strategies for HNSCC patients. METHODS: Osthole, a natural ingredient extracted from Cnidium monnieri (L.) 'Cusson', has multiple pharmacological effects including antineoplastic activity. Regrettably, the antineoplastic effect of osthole in HNSCC cells remains undefined. We utilize in vitro assays to assess the anti-proliferative effects of osthole in HNSCC cells and tumorigenesis assays using FaDu cells in murine HNSCC models to assess in vivo function. Moreover, the possible molecular mechanisms of Osthole on HNSCC cells was also investigated. RESULTS: Our findings show that the anti-proliferation effect of osthole might function through induction of cell cycle arrest (G2/M phase) and apoptosis in HNSCC. Osthole could also down-regulating the protein level of cell cycle and apoptosis related proteins, such as Bcl-2, PARP1, Survivin, CyclinB1 and Cdc2, while up-regulating expression of Cleaved Caspase3/9, Cleaved PARP1 and Bax. Similarly, osthole suppressed the in vivo growth of FaDu cells in a subcutaneous tumor model. In terms of mechanism, our data show that osthole can suppress the PI3K/AKT pathway. CONCLUSIONS: In the current study, our in vitro and in vivo assay showed the suppressive effect of Osthole on HNSCC cells through induce cell cycle arrest (G2/M phase) and apoptosis. Moreover, the action mechanisms of Osthole on proliferation related signaling pathways was disclosed. Our present study suggests that osthole might be used as an effective therapeutic agent for patients with HNSCC.

Light elicited growth, antioxidant enzymes activities and production of medicinal compounds in callus culture of Cnidium officinale Makino.

Cnidium officinale Makino is an important medicinal plant of oriental clinics and is considered as the main source of phthalides, polyphenols, and flavonoids. However, there is no available report regarding the effect of different light colors on the secondary metabolites composition of C. officinale. In this study different light (dark, white, blue, red and red: blue) conditions were arranged to raise callus on MS medium containing 0.5 mg·L-1 of each 2,4-D and BAP. Callus grown in dark condition showed maximum (2.0 g) fresh weight with lower total phenolic and flavonoids contents. Also, in dark condition callus faced higher catalase (CAT) and guaiacol peroxidase (GPX) activities to avoid free radicals. Mix (red: blue) light condition favored the synthesis of phenolics and flavonoids in callus at the cost of higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes expression. However, DPPH free radical scavenging activity was less variable among the samples from the different light conditions. Interestingly, the HPLC profile showed higher (28.3 µg·g-1 DW) phthalide accumulation in dark grown-cultures. Compared to other light conditions, 3-butyledinephthalide accumulation was higher (0.43 µg·g-1 DW) in white light-grown callus. These findings suggest that light conditions play an important role in the regulation of in vitro callus growth and synthesis of important medicinal compounds of C. officinale.

Effect of explant type and plant growth regulators on callus induction, growth and secondary metabolites production in Cnidium officinale Makino.

This study reports the effect of explant type and plant growth regulators (PGRs) on callus induction in Cnidium officinale. Compared to stem, root explant showed maximum percent callus formation of 75% on Murashige and Skoog (MS) medium containing 2.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 µM benzyladenine (BA). At 30th day of callus culture on the said medium, callus fresh weight was sevenfold higher than other tested PGRs treatments. It was noted that MS medium supplemented with 27.1 µM 2,4-D showed the highest 0.30 mg g-1 DW of total phenols, while total flavonoids content reached to a maximum of 0.05 mg g-1 DW on the MS medium supplemented with 4.5 µM 2,4-D and 2.2 µM BA. Conversely, maximum (83.9%) DPPH free radical scavenging activity was observed in calli grown on the MS medium supplemented with 2.3 µM 2,4-D and 2.2 µM BA. The high-performance liquid chromatography (HPLC) analysis revealed higher phthalide content in callus than intact roots of in vitro plants. While 3-butylidenephthalide content in callus was comparable to the intact shoots and roots of in vitro grown C. officinale. The concentrations of 2,4-D played a significant role in the production of phthalide and 3-butylidenephthalide. Additional measures are recommended to further enhance their production in vitro.

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis.

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE2 production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS.

Investigation of the biotransformation of osthole by liquid chromatography/tandem mass spectrometry.

Osthole is an active ingredient and one of the major coumarin compounds that were identified in the genus Cnidium moonnieri (L.) Cussion, the fruit of which was used as traditional Chinese medicine to treat male impotence, ringworm infection and blood stasis conventionally. Recent studies revealed that osthole has diverse pharmacological effects, such as improving male sexual dysfunction, anti-diabetes, and anti-hypertentions. The inhibition of thrombosis and platelet aggregation and protection of central nerve were also observed. On the other hand, the metabolism of osthole has not yet been investigated thoroughly. Herein the biotransformation of osthole in rat was investigated after oral administration of osthole by using efficient and sensitive ultra-performance liquid chromatography-tandem quadrupole-time of flight mass spectrometry (UPLC-QTOF/MS). Eighteen osthole metabolites and the parent drug were detected and identified in rat urine. Fourteen metabolites of osthole were identified and characterized for the first time. Structures of metabolites of osthole were elucidated by comparing fragment pattern under MS/MS scan and change of molecular weight with those of osthole. The main phase I metabolic pathways were summed as 7-demethylation, 8-dehydrogenation, hydroxylation on coumarin and 3,4-epoxide. Sulfate conjugates were detected as phase II metabolites of osthole.

Metabolic profiling and identification of the genetic varieties and agricultural origin of Cnidium officinale and Ligusticum chuanxiong.

Quality control methods for Cnidium officinale and Ligusticum chuanxiong are lacking because their quality is influenced by multiple factors. Thus, there is a need to develop a multifactorial method for measuring quality that is both standardized and practical. Here, we report a profiling method based on gas chromatography-mass spectrometry (GC-MS) to discriminate among the genetic varieties and agricultural origins of C. officinale and L. chuanxiong. Our metabolome analysis identified 68 metabolites, 13 of which were newly identified in our samples. The S-plot of the OPLS discriminant analysis enabled us to determine significant biomarkers. Using only double-compound biomarkers, the samples were successfully classified into distinct groups defined by genetic variety and cultivation origin. This method will simplify the process of searching for quality control markers that can be used to determine genetic variety and agricultural origin.