Content determination of eight phenols in Ilex hainanensis harvested at different time by HPLC-DAD / 中国中药杂志

This study intends to develop a high performance liquid chromatography-diode array detection(HPLC-DAD) method for simultaneous determination of chlorogenic acid, 2-hydroxymethyl-3-hydroxyl-1-butene-4-O-β-D-(6″-O-caffeoyl)-glucopyranoside, pubescenoside B, huazhongilexone-7-O-β-D-glucopyranoside, rutin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C in Ilex hainanensis. The HPLC conditions are as follows: Waters XBridge C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase of 0.5% formic acid in water(A)-acetonitrile(B), gradient elution(0-8 min, 5%-12% B; 8-18 min, 12%-18% B; 18-30 min, 18%-25% B; 30-40 min, 25%-30% B; 40-42 min, 30%-80% B; 42-45 min, 80% B) at the flow rate of 0.8 mL·min~(-1), detection wavelengths of 282, 324, and 360 nm, column temperature of 25 ℃, and injection volume of 5 μL. The content of the 8 phenols in 8 samples was 0.30-6.29, 0.29-3.27, 0.15-10.4, 0.51-5.85, 0.49-9.02, 0.51-4.68, 1.93-13.4, and 0.87-5.95 mg·g~(-1), respectively. Moreover, the content of phenols in the samples collected in October was higher than that of samples harvested in other months. The established method is accurate and sensitive for the determination of phenols in I. hainanensis, which is useful for the quality improvement of this herbal medicine.

Inhibitory effect of BF523 from Ilex hainanensis on ox LDL-induced foam cells formation / 中国中药杂志

Cardio-cerebral vascular disease induced by atherosclerosis is a serious cause of human health. The pathogenesis of AS is very complex,and the oxidized low-density lipoprotein( ox LDL) induced foam cells formation is considered to be the most important cytological change in AS. Based on the definition of " TCM chemical biology",we clarified the chemical composition of Ilex hainanensis,the effective substances of I. hainanensis on the activity of anti-AS were screened. Then we found that saponin BF523 had the good inhibitory effect on foam cell formation. In this research,we studied the BF523 as the research object to clarify the molecular target of the active compound of I. hainanensis by foam cell formation model. The results showed that BF523 significantly inhibited the oxidation of ox LDL-induced macrophage foaming and decreased the lipid content in macrophages. BF523 had inhibited the phagocytosis of ox LDL in macrophages by reducing the mRNA and protein levels of scavenger receptor CD36,thereby inhibiting the occurrence and development of AS. These findings not only clarified the mechanism of the inhibition of foam cell formation by saponin BF523,but also provided a useful exploration for the enrichment of the theory of " TCM chemical biology".

Determination of three pair of triterpenoid isomers in leaf of Ilex hainanensis by HPLC-ELSD / 中国中药杂志

The present study is to develop an HPLC-ELSD method for simultaneous determination of three pairs of triterpenoid isomers, Ilexsaponin A₁, Ilexhainanoside D, Ilexgenin A, 3β, 19α-dihydroxyolean-12-ene-24, 28-dioic acid (ilexhainanin E) ursolic acid and oleanic acid in the leaf of Ilex hainanensis, which could provide evidence to the quality control of this herb. The six constituents were measured on a Waters XBridge C₁₈ column (4.6 mm×250 mm, 5 μm), with a mobile phase consisting of methanol (A)- 0.5% formic acid in water (B) at a flow rate of 1.0 mL·min⁻¹ (0-18 min，70%-85% A，18-20 min，85%-95% A；20-35 min，95% A). The carrier gas was N₂, and the pressure was 2.8 L·min⁻¹. The drift tube in this experiment were set at 70 °C. The injection volume was 10 μL. The contents of the six triterpenoids in 6 samples were 3.7-8.5, 10.3 -22.1, 2.8-5.9, 7.8-14.1, 2.6-3.8 and 8.8-11.9 mg·g⁻¹, respectively. The established method is proved to be accurate and sensitive for the determination of triterpenoids in Ilicis Hainanensis Folium, and may be used for the quality improvement of this herb.

Ethanol extract of Ilex hainanensis Merr. exhibits anti-melanoma activity by induction of G/S cell-cycle arrest and apoptosis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate anti-melanoma effect of ethanol extract of Ilex hainanensis Merr. (IME) and elucidate its underlying mechanism.</p><p><b>METHODS</b>Thirty-six tumor-bearing mice were randomized into 6 groups (n=6) as follows: model group, IME 25, 50, 100, and 200 mg/kg groups and dacarbazine (DTIC) 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25, 50, 100 or 200 mg/kg per day, respectively. The mice in the DTIC group were intraperitoneally injected with DTIC 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The cell viability was evaluated by 3-(4,5-dime-thylthylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene and protein expressions of nuclear factor κB-p65 (NF-κB-p65), Bcl-2, B-cell lymphomaextra large (Bcl-xL) and Bax were detected by quantitative real-time polymerase chain reaction and Western blot analyses. Caspases-3, -8, and -9 activities were detected using the colorimetric method. In addition, a B16-F10 melanoma xenograft mouse model was used to evaluate the anti-cancer activity of IME in vivo. Furthermore, a survival experiment of tumor-bearing mice was also performed to evaluate the possible toxicity of IME.</p><p><b>RESULTS</b>IME significantly inhibited the proliferation of B16-F10 cells (P<0.01). Flow cytometric analysis showed that IME induced G/S cell cycle arrest and apoptosis (both P<0.01). IME inhibited activation of NF-κB, decreased the gene and protein expressions of Bcl-2, Bcl-xL, and increased the gene and protein expressions of Bax (all P<0.01). In addition, IME induced the activation of Caspases-3, -8, and -9 in B16-F10 cells. The study in vivo showed that IME significantly reduced tumor volume (P<0.01), and the inhibitory rate came up to 68.62%. IME also induced large areas of necrosis and intra-tumoral apoptosis that correlated with a reduction in tumor volume. Survival experiment showed that treatment with IME for 14 days significantly prolonged survival time and 20% of mice in the IME 200 mg/kg group were still alive until the 50th day. Notably, IME showed no apparent side-effects during the treatment period.</p><p><b>CONCLUSION</b>IME exhibited significant anti-melanoma activity in vitro and in vivo, suggesting that IME might be a promising effective candidate with lower toxic for malignant melanoma therapy.</p>