Effect of Kumatakenin Isolated From Cloves on the Apoptosis of Cancer Cells and the Alternative Activation of Tumor-Associated Macrophages

The flower bud of <i>Syzygium aromaticum</i> (clove) has been used for centuries as a spice and herbal medicine. The biological activities of kumatakenin, a flavonoid that has recently been isolated from cloves, are poorly characterized. In the present study, the anticancer effects of kumatakenin in human ovarian cancer cells and tumor-associated macrophages (TAMs) were investigated. We found that kumatakenin exhibited significant cytotoxic activity in human ovarian cancer cells, SKOV3 and A2780. A propidium iodide and Annexin V-FITC staining assay showed that kumatakenin induces apoptosis in ovarian cancer cells. Kumatakenin treatment increased the activity of caspase-3, -8, and -9, and caspase inhibitors attenuated kumatakenin-induced SKOV3 cell death. In addition, kumatakenin was found to reduce the expressions of MCP-1 and RANTES, which are major determinants of macrophage recruitment at tumor sites in ovarian cancer cells. Moreover, kumatakenin inhibited the expression of M2 markers and cancer-promoting factors, including IL-10, MMP-2/-9, and VEGF, in macrophages stimulated by the ovarian cancer cells. In conclusion, these results suggest that kumatakenin shows anticancer activities by inducing apoptosis of ovarian cancer cells and inhibiting the alternative activation of TAM

Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock

Two chromone <i>C</i>-glucosides, biflorin (<b>1</b>) and isobiflorin (<b>2</b>), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of <b>1</b> and <b>2</b> on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in RAW 264.7 macrophages were evaluated, and <b>1</b> (IC₅₀ = 51.7 and 37.1 μM, respectively) was more potent than <b>2</b> (IC₅₀ > 60 and 46.0 μM). The suppression of NO and PGE₂ production by <b>1</b> correlated with inhibition of iNOS and COX-2 protein expression. Compound <b>1</b> reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound <b>1</b> inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, <b>1</b> reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, <b>1</b> reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound <b>1</b> showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of <b>1</b>

New Flavonol Glucuronides from the Flower Buds of <i>Syzygium aromaticum</i> (Clove)

Repeated chromatography of the EtOAc-soluble fraction from the 70% EtOH extract of the flower buds of <i>Syzygium aromaticum</i> (clove) led to the isolation and characterization of four new flavonol glucuronides, rhamnetin-3-<i>O</i>-β-d-glucuronide (<b>1</b>), rhamnazin-3-<i>O</i>-β-d-glucuronide (<b>2</b>), rhamnazin-3-<i>O</i>-β-d-glucuronide-6″-methyl ester (<b>3</b>), and rhamnocitrin-3-<i>O</i>-β-d-glucuronide-6″-methyl ester (<b>4</b>), together with 15 flavonoids (<b>5</b>–<b>19</b>) having previously known chemical structures. The structures of the new compounds <b>1</b>–<b>4</b> were determined by interpretation of spectroscopic data, particularly by 1D- and 2D-NMR studies. Six flavonoids (<b>6</b>, <b>7</b>, <b>9</b>, <b>14</b>, <b>18</b>, and <b>19</b>) were isolated from the flower buds of <i>S. aromaticum</i> for the first time in this study. The flavonoids were examined for their cytotoxicity against human ovarian cancer cells (A2780) using MTT assays. Among the isolates, pachypodol (<b>19</b>) showed the most potent cytotoxicity on A2780 cells with an IC₅₀ value of 8.02 μM

Iridoids from the Roots of <i>Patrinia scabra</i> and Their Inhibitory Potential on LPS-Induced Nitric Oxide Production

An activity-guided fractionation procedure of the 70% aqueous EtOH extract from the roots of <i>Patrinia scabra</i> led to the isolation and characterization of five new iridoids, patriscabrins A–E (<b>1</b>–<b>5</b>), along with 13 known compounds. The structures of <b>1</b>–<b>5</b> were determined by interpretation of spectroscopic data, particularly by 1D and 2D NMR, ECD, and VCD studies. Thereafter, isolates were evaluated for their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells. Of these, the new iridoids <b>2</b> and <b>5</b> and the known lignan patrineolignan B (<b>6</b>) exhibited IC₅₀ values of 14.7 to 17.8 μM

Anti-Microbial Activity of Aliphatic Alcohols from Chinese Black Cardamom (Amomum tsao-ko) against Mycobacterium tuberculosis H37Rv

The fruits of Amomun tsao-ko (Chinese black cardamom; Zingiberaceae) contain an abundance of essential oils, which have previously demonstrated significant antimicrobial activity. In our preliminary search for natural anti-tuberculosis agents, an acetone extract of A. tsao-ko (AAE) exhibited strong antibacterial activity against Mycobacterium tuberculosis H37Rv. Therefore, the aim of this study was to find the principal compounds in an AAE against M. tuberculosis. Nine aliphatic compounds (1–9) including a new compound (1, tsaokol B) and a new natural unsaturated aliphatic diester (6), together with three acyclic terpenoids (10–12), were isolated from an AAE by repetitive chromatography. The structures of the isolates were determined by spectroscopic data analysis. All isolates were evaluated for activity against M. tuberculosis H37Rv. Isolated compounds 1–6, and 11 had MICs ranging from 0.6–89 µg/mL. In contrast, compounds 7 to 10, and 12 had MICs that were >100 µg/mL. Tsaokol A (3) was the most active compound with MICs of 0.6 µg/mL and 1.4 µg/mL, respectively, against replicating and nonreplicating M. tuberculosis. These results are the first to illustrate the potency of tsaokol A (3) as a natural drug candidate with good selectivity for treating tuberculosis

Eupatilin diminishes the activation of NF-κB pathway in the post-ischemic brain of tMCAO-challenged mice.

<p>Mice were challenged with tMCAO and eupatilin (Eup, 10 mg/kg, <i>p</i>.<i>o</i>.) was administered immediately after reperfusion. (<b>A and B</b>) Effects of eupatilin (Eup) on NF-κB activation pathway were determined by Western blot in tMCAO-challenged brains 22 h after reperfusion. Changes in p-IKKα/β, p-IκBα, and IκBα protein levels in the hemisphere with ischemic challenge. (<b>A</b>) Representative Western blots. (<b>B</b>) Quantification. n = 6 per group. **<i>P</i><0.01 versus vehicle-treated tMCAO (tMCAO+veh). (<b>C and D</b>) Localization of NF-kB p65 was determined by double immunolabeling in tMCAO-challenged brains 22 h after reperfusion. (<b>C</b>) Representative images of NF-kB p65-immunopositive cells and double-immunopositive cells (NF-kB p65, brown; Iba1, red fluorescence). (<b>D</b>) Representative images of NF-kB p65-immunopositive cells and double-immunopositive cells (NF-kB p65, brown; GFAP, red fluorescence). Arrowheads indicate double-immunopositive cells. Scale bar, 50 μm.</p

Eupatilin reduces inflammatory responses in LPS-stimulated microglia.

<p>Effects of eupatilin on levels of nitrite (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>), and PGE₂ (<b>D</b>) were determined in the conditioned medium from LPS (100 ng/ml, 24 h)-stimulated BV2 microglia in the presence or absence of eupatilin at different concentrations (5, 10, and 20 μM). L-NMMA (20 μM) was used as a positive control. n = 3 per group. **<i>P</i><0.01 and ***<i>P</i><0.001, versus the vehicle-treated LPS group (LPS+veh).</p

Eupatilin reduces microglial activation in the post-ischemic brain 1 day after tMCAO challenge.

<p>Mice were challenged with tMCAO and eupatilin (Eup, 10 mg/kg, <i>p</i>.<i>o</i>.) was administered immediately after reperfusion. Effects of eupatilin on microglial activation were determined in tMCAO-challenged brain 22 h after reperfusion by immunohistochemistry against Iba1. (<b>A</b>) Representative images for Iba1-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Diagram boxes in top panels display brain areas where the images in lower panels were acquired. Dashed lines indicate the lesion site. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels). (<b>B</b>) Quantification of Iba1-immunopositive cells in both regions. n = 4 per group. **<i>P</i><0.01 versus the vehicle-treated tMCAO group (tMCAO+veh).</p

Eupatilin reduces lipid peroxidation in the post-ischemic brain of tMCAO-challenged mice.

<p>Mice were challenged with tMCAO and eupatilin (Eup, 10 mg/kg, <i>p</i>.<i>o</i>.) was administered immediately after reperfusion. (<b>A</b>) Effects of eupatilin on lipid peroxidation were determined by immunohistochemistry using an antibody against 4-HNE in tMCAO-challenged brains 22 h after reperfusion. Representative images of 4-HNE-immunopositive cells in periischemic (‘P’) and ischemic core (‘C’) regions. Diagram boxes in top panels display brain areas where the images in lower panels were acquired. Dashed lines indicate the lesion site. Scale bars: 200 μm (top panels), 50 μm (middle and bottom panels). (<b>B</b>) Effects of eupatilin (Eup) on 4-HNE production were determined by Western blot in tMCAO-challenged brains 1 day after reperfusion. Changes in 4-HNE and α-tubulin protein levels in the hemisphere with ischemic challenge were shown as representative Western blots and quantification. n = 6 per group. **<i>P</i><0.01 versus the vehicle-treated tMCAO group (tMCAO+veh).</p

Eupatilin reduces brain damage in tMCAO-challenged mice.

<p>Mice were challenged with tMCAO and eupatilin (Eup: 1, 3, and 10 mg/kg, <i>p</i>.<i>o</i>.) was given to mice immediately after tMCAO. Alternatively, 10 mg/kg eupatilin was given to mice 5 hours after MCAO induction. Effects of eupatilin on brain infarct volume (<b>A and B</b>), neurological function (<b>C</b>), and neural cell death (<b>D</b>) were assessed 22 h after reperfusion. Edaravone (Eda, 3 mg/kg, <i>p</i>.<i>o</i>.) was used as a positive control. (<b>A</b>) Representative images of TTC-stained brain slices indicating brain infarction. (<b>B</b>) Quantification of infarct volume. (<b>C</b>) Neurological score reflecting neurological functions. n = 10~15 per group. **<i>P</i><0.01 and ***<i>P</i><0.001 versus the vehicle-treated tMCAO group (tMCAO+veh). (<b>D</b>) Effects of eupatilin (Eup, 10 mg/kg, <i>p</i>.<i>o</i>.) administration immediately after tMCAO on neural cell death. Representative images of FJB-stained sections. Diagram boxes display the cerebral area where the images in middle and bottom panels were acquired. Dashed lines indicate the lesion site. Scale bars, 200 μm (top panels) and 50 μm (middle and bottom panels).</p