Bioactivity-based analysis and chemical characterization of cytotoxic compounds from a poisonous mushroom, Amanita spissacea, in human lung cancer cells in vitro.

As part of our systematic study on Korean toxic mushrooms, bioactivity-guided fractionation of the MeOH extract of Amanita spissacea (Amanitaceae) fruiting bodies and chemical investigation of its cytotoxic fractions led to the isolation of (9E)-8-oxo-9-octadecenoic acid (1), (10E)-9-oxo-10-octadecenoic acid (2), (9E)-8-oxo-9-octadecenoate methyl ester (3), (9Z)-9-octadecenoate-(2'S)-2',3'-dihydroxypropyl ester (4), (9Z)-9-octadecenoic acid (5), and palmitic acid (6). The structures of the isolates were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among the isolated compounds, compounds 1 and 2 exhibited the most potent cytotoxic activity in all human lung cancer cell lines examined, with IC50 values ranging from 255.7 to 321.0 µM and 250.2 to 322.5 µM, respectively. The cytotoxicity of these compounds was also found to be mediated by apoptosis associated with caspase-3 activation. These findings provide experimental evidence suggesting the potential of A. spissacea as a promising natural source for the discovery of novel anticancer drug candidates.

Megastigmane Derivatives from the Cladodes of Opuntia humifusa and Their Nitric Oxide Inhibitory Activities in Macrophages.

Opuntia humifusa, known as the eastern prickly pear cactus and locally called "Cheonnyuncho" in Korea, is cultivated widely on Jeju Island, Korea. Phytochemical analysis of the methanolic extract of the cladodes of O. humifusa, for which previous research is relatively limited, was performed under the guidance of LC/MS-based analysis. As a result, one new megastigmane (1) and four new megastigmane glucosides (2-5) were isolated along with 18 known compounds (6-23). The structures of the new compounds were established by 1D and 2D NMR and HRESIMS, and their absolute configurations were established by chemical reactions, quantum chemical electronic circular dichroism calculations, and DP4+ analysis using the gauge-including atomic orbital NMR chemical shift calculations as well as the application of Snatzke's method. The isolated compounds (1-23) were tested for NO production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 cells to investigate their anti-inflammatory effects. Compounds 10 and 11 exhibited significant inhibitory effects on LPS-induced NO production in a dose-dependent manner. The potential mechanistic pathway of 10 and 11 was also investigated using Western blotting, indicating that compounds 10 and 11 inhibit NO through iNOS expression.

Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages.

Hippophae rhamnoides L. (Elaeagnaceae; commonly known as "sea buckthorn" and "vitamin tree"), is a spiny deciduous shrub whose fruit is used in foods and traditional medicines. The H. rhamnoides fruit (berry) is rich in vitamin C, with a level exceeding that found in lemons and oranges. H. rhamnoides berries are usually washed and pressed to create pomace and juice. Today, the powder of the aqueous extract of H. rhamnoides berries are sold as a functional food in many countries. As part of our ongoing effort to identify bioactive constituents from natural resources, we aimed to isolate and identify those from the fruits of H. rhamnoides. Phytochemical analysis of the extract of H. rhamnoides fruits led to the isolation and identification of six compounds, namely, a citric acid derivative (1), a phenolic (2), flavonoids (3 and 4), and megastigmane compounds (5 and 6). Treatment with compounds 1-6 did not have any impact on the cell viability of RAW 264.7 mouse macrophages. However, pretreatment with these compounds suppressed lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse macrophages in a concentration-dependent manner. Among the isolated compounds, compound 1 was identified as the most active, with an IC50 of 39.76 ± 0.16 µM. This value was comparable to that of the NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor with an IC50 of 28.48 ± 0.05 µM. Western blot analysis demonstrated that compound 1 inhibited the LPS-induced expression of IKKα/ß (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), nuclear factor kappa-B (NF-κB) p65, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in RAW 264.7 cells. Furthermore, LPS-stimulated cytokine production was detected using a sandwich enzyme-linked immunosorbent assay. Compound 1 decreased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production in LPS-stimulated RAW 264.7 cells. In summary, the mechanism of action of 1 included the suppression of LPS-induced NO production in RAW 264.7 cells by inhibiting IKKα/ß, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α.

Anti-adipogenic Effect of ß-Carboline Alkaloids from Garlic (Allium sativum).

Garlic (Allium sativum L.) is utilized worldwide for culinary and medicinal use and has diverse health benefits. As part of our ongoing research to identify bioactive components from natural resources, phytochemical analysis of the methanolic extract of garlic led to the isolation and characterization of six compounds: Three eugenol diglycosides (1-3) and three ß-carboline alkaloids (4-6). In particular, the absolute configurations of ß-carboline alkaloids (5 and 6) were established by gauge-including atomic orbital nuclear magnetic resonance chemical shift calculations, followed by DP4+ analysis. Here, we evaluated the effects of compounds 1-6 on 3T3-L1 preadipocyte adipogenesis and lipid metabolism. 3T3-L1 adipocyte differentiation was evaluated using Oil Red O staining; the expression of adipogenic genes was detected using RT-qPCR. Among compounds 1-6, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (6) inhibited 3T3-L1 preadipocyte adipogenesis and reduced the expression of adipogenic genes (Fabp4, PPARγ, C/EBPß, Adipsin, and Adipoq). Moreover, it markedly decreased the actylation of α-tubulin, which is crucial for cytoskeletal remodeling during adipogenesis. Anti-adipogenic effects were observed upon treatment with compound 6, not only during the entire process, but also on the first two days of adipogenesis. Additionally, treatment with compound 6 regulated the expression of genes involved in adipocyte lipid metabolism, decreasing the lipogenic gene (SREBP1) and increasing lipolytic genes (ATGL and HSL). We provide experimental evidence of the health benefits of using (1R,3S)-1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid obtained from garlic to prevent excessive adipogenesis in obesity.

(3ß,16α)-3,16-Dihydroxypregn-5-en-20-one from the Twigs of Euonymus alatus (Thunb.) Sieb. Exerts Anti-Inflammatory Effects in LPS-Stimulated RAW-264.7 Macrophages.

To discover new pharmacologically active natural products, here, we performed the phytochemical analysis of a Korean medicinal plant. Euonymus alatus (Thunb.) Sieb. is a traditional medicinal plant that has been used as a remedy for various diseases in Asian countries. In particular, the cork cambium on the twigs of E. alatus has been used to treat dysmenorrhea, tumors, diabetes, and wound. Phytochemical analysis of the methanolic extract of E. alatus twigs led to the isolation of a sterol, which was identified as (3ß,16α)-3,16-dihydroxypregn-5-en-20-one (1) by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry. The stereochemistry of 1 was established with nuclear Overhauser effect spectroscopy (NOESY) analysis and comparison of electronic circular dichroism (ECD) data. To the best of our knowledge, the isolation of compound 1 from nature is first reported here, as well as the complete and revised NMR data assignment of 1. In lipopolysaccharide (LPS)-stimulated RAW-264.7 macrophages, compound 1 significantly inhibited nitric oxide (NO) production at an IC50 value of 12.54 ± 0.05 µM as well as the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the pre-treatment with compound 1 attenuated the LPS-induced phosphorylation of nuclear factor kappa B (NF-κB) p65 through the inhibition of the phosphorylation of IκB kinase alpha (IKKα), IKKß, and inhibitor of kappa B alpha (IκBα). Compound 1 also inhibited the LPS-induced phosphorylation of p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Taken together, compound 1 may serve as an anti-inflammatory constituent of E. alatus twigs and its anti-inflammatory property is thought to be associated with the inhibition of NO production via suppression of iNOS and COX-2 expression through inhibition of IKKα/ß, I-κBα and NF-κB p65 activation and downregulation of p38, JNK, and ERK mitogen-activated protein kinase signal pathways in RAW 264.7 macrophages. These findings also provide experimental evidence that compound 1 identified from E. alatus twigs could be a candidate for an anti-inflammatory agent.

Withaninsams A and B: Phenylpropanoid Esters from the Roots of Indian Ginseng (Withania somnifera).

Withania somnifera (L.) Dunal (Solanaceae), known as Indian ginseng or ashwagandha, has been used in Indian Ayurveda for the treatment of a variety of disorders, such as diabetes and reproductive and nervous system disorders. It is particularly used as a general health tonic, analgesic, and sedative. As part of continuing projects to discover unique bioactive natural products from medicinal plants, phytochemical investigation of the roots of W. somnifera combined with a liquid chromatography-mass spectrometry (LC/MS)-based analysis has led to the isolation of two novel phenylpropanoid esters, Withaninsams A (1) and B (2), as an inseparable mixture, along with three known phenolic compounds (3, 4, and 6) and a pyrazole alkaloid (5). The structures of the new compounds were elucidated using a combination of spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HR-ESIMS). Withaninsams A (1) and B (2) are phenylpropanoid esters that contain a side chain, 4-methyl-1,4-pentanediol unit. To the best of our knowledge, the present study is the first to report on phenylpropanoid esters with 4-methyl-1,4-pentanediol unit. The anti-inflammatory activity of the isolated compounds (1-6) was evaluated by determining their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, where compound 3 inhibited LPS-induced NO production (IC50 = 33.3 µM) and TNF-α production, a pro-inflammatory cytokine (IC50 = 40.9 µM). The anti-inflammatory mechanism through the inhibition of transcriptional iNOS protein expression was confirmed by western blotting experiments for the active compound 3, which showed decreased iNOS protein expression.

Lobatamunsolides A-C, Norlignans from the Roots of Pueraria lobata and their Nitric Oxide Inhibitory Activities in Macrophages.

Phytochemical investigation of the methanol (MeOH) extract of Pueraria lobata roots, known as "kudzu", combined with liquid chromatography/mass spectrometry (LC/MS)-based analysis, resulted in the identification of four norlignans (1-4), including three new norlignans, lobatamunsolides A-C (1-3), and five known isoflavonoids (5-9). The structures of the new compounds were elucidated by a combination of spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and high resolution (HR)-electrospray ionization mass spectrometry (ESIMS), and their absolute configurations were determined by chemical reaction and quantum chemical electronic circular dichroism (ECD) calculations. The isolated compounds (1-9) were evaluated for their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Compound 9 displayed the strongest NO inhibitory effect and compound 2 showed a weak effect. The potential mechanism of the effect of compound 9 was investigated by analysis of its molecular docking on the active site of inducible nitric oxide synthase (iNOS), which showed the potential interactions of compound 9 with key amino acid residues and the heme cofactor of iNOS. The mechanism as the inhibition of transcriptional iNOS protein expression was confirmed by western blotting experiments.

Dual effects of isoflavonoids from Pueraria lobata roots on estrogenic activity and anti-proliferation of MCF-7 human breast carcinoma cells.

Pueraria lobata root (PLR), well known as Kudzu root, has recently become commercially available in Western dietary supplements for menopausal symptoms. The scientific basis for its action has been attributed to the action of phytoestrogens. This study aimed to investigate the estrogen-like activity of isoflavonoids isolated from P. lobata root and their safety with respect to their effect on breast cancer cell proliferation. In an E-screen assay, crude MeOH extract of PLR significantly increased the proliferation of MCF-7 cells in a concentration-dependent manner. Among the four fractions obtained by solvent fractionation of MeOH extract, the n-BuOH fraction had significant estrogen-like activities at all concentrations tested. Phytochemical analysis of the n-BuOH fraction led to the isolation of 10 isoflavones (1-10), among which genistein (10) had significant estrogen-like activities at all concentrations tested. These activities were significantly enhanced by treatment with genistein and 17ß-estradiol compared with 17ß-estradiol alone, and this effect was mediated by decreased expression of estrogen receptor (ER)α and phospho-ERα in MCF-7 cells. In a cell cytotoxicity assay, genistein (10) exhibited significant cytotoxicity in both ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. This cytotoxicity was characterized by the induction of apoptotic cells stained with annexin V conjugated with Alexa Fluor 488 and involved activation of mitochondria-independent and -dependent apoptosis pathways in MCF-7 cells. Our results demonstrated that genistein (10) has estrogen-like effects dependent on ER pathway activation and anti-proliferative effects mediated by the apoptosis pathway rather than the ER pathway in MCF-7 breast cancer cells.

Biological Evaluation of a New Lignan from the Roots of Rice (Oryza sativa).

LC/MS-based phytochemical analysis of an EtOH extract of the roots of rice (Oryza sativa; Gramineae), which takes a crucial role in the stable crop population in Asia, resulted in the isolation of a new lignan, oryzativol C (1), as a minor component. The chemical structure of compound 1 was unambiguously confirmed using spectroscopic evidence (including 1D- and 2D-NMR data), HR-ESI-MS, and CD data analysis. Considering the traditional medicinal efficacy of O. sativa and its importance as a food crop, compound 1 was evaluated for effects on breast cancer cell lines (MDA-MB-231) and on glucose-stimulated insulin secretion in an INS-1 pancreatic ß-cell line. Compound 1 showed mild cytotoxicity toward the MDA-MB-231. Furthermore, compound 1 stimulated insulin secretion in INS-1 pancreatic ß-cells without inducing cytotoxicity. These results indicate that compound 1 is an active ingredient of O. sativa that offers health benefits including inhibition of breast cancer cell proliferation and hyperglycemia control.