key: cord-0976445-cv81bq0a authors: YAO, Zhi-Hong; LIU, Ming-Yan; DAI, Yi; ZHANG, Yi; QIN, Zi-Fei; TU, Feng-Juan; YAO, Xin-Sheng title: Metabolism of Epimedium-derived Flavonoid Glycosides in Intestinal Flora of Rabbits and Its Inhibition by Gluconolactone date: 2011-11-30 journal: Chinese Journal of Natural Medicines DOI: 10.3724/sp.j.1009.2011.00461 sha: 867752d917b2bc3c4baa4ce3192dc1aa6d656938 doc_id: 976445 cord_uid: cv81bq0a Abstract Aim The metabolism of Epimedium-derived flavonoid glycosides (EF, with icariin as the main component) in rabbit intestinal flora and its inhibition by gluconolactone were investigated in this paper to help reveal the metabolic pathway of EF in rabbits and to identify the in vivo bioactive components of EF in the prevention of steroid-associated osteonecrosis. Methods EF were incubated at 37 °C anaerobically with rabbit intestinal flora, and then water-saturated ethyl acetate was used for sample extraction at different time points. Furthermore, gluconolactone was added at different concentrations (8, 12 and 16 mg·mL−1) to study its inhibition of the metabolism of EF in rabbit intestinal flora. The separation was performed on a ODS column by gradient elution with acetonitrile-water (including 0.1% formic acid respectively) as mobile phase at detection wavelength of 335 nm. Results EF were metabolized to icariside II in 2 h, and then to icaritin when incubated for 8 h; gluconolactone showed the inhibition of EF metabolism in rabbit intestinal flora in a concentration-dependent manner. Conclusion EF were found to be metabolized rapidly by hydrolysis of rabbit intestinal flora to produce icariside II and icaritin; and the total inhibition was achieved by gluconolactone at a concentration of 16 mg·mL−1. Traditionally, Herba Epimedii has been used as Chinese herbal medicine for invigorating the kidney, strengthening sinew and bone, dispelling wind and eliminating dampness [1] . Modern pharmacological research indicated that icariin was beneficial for vasodilatation [2] , immunoregulation [3] and anti-osteoporosis [4] . Recent studies also showed that oral administration of Epimedium-derived flavonoid glycosides (EF, with icariin as the main component) could exert beneficial effect on the prevention of steroid-associated osteonecrosis in rabbit model with inhibition of both intravascular-thrombosis and extravascular-lipid-deposition [5] . Further studies in vitro showed that icaritin, but not EF, displayed direct effects on the protection of vein endothelial cell and inhibition of lipid deposition in a dose-dependent manner [6] . These suggested that EF might be used as prodrug and exert effects by being metabolized to their aglycone with in vivo bioactivity of preventing anti-steroid-associated osteonecrosis. Since intestinal flora can hydrolyze glycoside drugs [7] [8] , and the metabolism of orally administered glycoside drugs in intestinal flora may affect their bioavailability and in vivo bioactivities, it is necessary to study the metabolism of EF in intestinal flora. Some investigation on the metabolism of icariin in intestinal flora of rats [9] and humans [10] had been carried out. However, the metabolism of EF in rabbit intestinal flora is still difficult to be estimated because of the differences of metabolic abilities of intestinal flora among different animal species. Therefore, this paper marks the first time that the metabolism of EF in intestinal flora of rabbits is studied. Gluconolactone is a type of specific inhibitor of β-glycosidase, which has been extensively used for limiting the hydrolysis of flavonoid glycosides [11] . Therefore, the inhibition of the metabolism of EF in rabbit intestinal flora by gluconolactone at different concentrations was also investigated in this paper. The present work might help to reveal the metabolic pathway of EF in rabbits as well as the in vivo bioactive components of EF in the prevention of steroid-associated osteonecrosis. Epimedium-derived flavonoid glycosides (purity of icariin ≥ 83%,epimedoside A ≤ 1.65%, hexandraside F< 0.91%, epimedin A<1.18%, epimedin B≤1.52%, epimedin C<4.1% and icariside II<1.84%) were provided by Beijing TongRenTang Health Pharmaceutical Co., Ltd. (Beijing, China). Icariin (purity>98%), icariside II (purity>98%) and icaritin (purity>98%) were purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China). Icariside I (purity≥98%) was purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China). Desmethylicaritin (purity>90%) was self-made by our laboratory. Gluconolactone (purity>99%) was purchased from Sigma (St. Louis, Mo, USA). Acetonitrile was of HPLC grade and purchased from Dikma Technologies Inc. (Beijing, China). Methanol was of HPLC grade and purchased from Yuwang Co., Ltd. (Shandong, China). Watsons water was purchased from Beijing Watsons Water Co., Ltd. (Beijing, China). Other reagents were of analytical grade. An Agilent 1200 HPLC system consisted of a degasser (G1322A), a quaternary pump (G1311A), an autosampler (G1329A), a thermostat column compartment (G2316A) and a multiple wavelength detector (G1365D) were used for HPLC analysis of samples. XW-80A micro-vortex apparatus (Shanghai Huxi Analytical Instrument Factory, China) was used to vortex samples. A high-speed bench centrifuge (TGL-16G-A, Shanghai Anting Scientific Instrument Factory, China) was used to centrifuge samples. KQ3200E ultrasonic wave purifier (Kunshan Ultrasonic Instruments Co., Ltd.) was used for sample ultrasonication. The intestinal incubation experiments were carried out in 2.5 L anaerobic incubation bags (MGC AnaeroPack·Anaero, Mitsubishi Gas Chemical Co., Inc., Chiyoda-ku, Japan) and a 2.5 L anaerobic incubation pot (Labmed AG025, LABMED BIOTECH Co., Ltd., Guangzhou, China). LAIHENG L-128 Nitrogen blowing instrument (Beijing Laiheng Scientific Co., Ltd., Beijing, China) was used for sample condensation. Four New-Zealand male rabbits with body weight of 3-4 kg were purchased from the Experimental Animal Center of Guangdong Province and kept in an animal room at constant temperature (23 ± 2) °C and humidity (55% ± 10%) with a 12 h of light per day and access to water and food ad libitum. [12] 37.5 mL of A solution (0.78% K 2 HPO 4 ), 37.5 mL of B solution (0.47% KH 2 PO 4 , 1.18%NaCl, 1.2% (NH 4 ) 2 SO 4 , 0.12% CaCl 2 , 0.25% MgSO 4 ·H 2 O), 50 mL of C solution (8% Na 2 CO 3 ), 0.5 g of L-cysteine, 2 mL of 25% L-ascorbic acid, 1 g of eurythrol, 1 g of tryptone and 1 g of nutrient agar were mixed together and diluted with distilled water to 1 L. Then the solution was adjusted to pH 7.5-8.0 with HCl (2 mol·L −1 ). [13] Fresh feces of rabbits were homogenized in normal saline solution at the ratio of 1 g to 4 mL immediately and then followed by filtration through gauze. 10 mL of the filtrate was mixed with 90 mL of anaerobic culture solutions to prepare 100 mL of intestinal flora cultural solution. 1 mg of EF was accurately weighed and first dissolved in 500 μL of methanol, then diluted with sufficient normal saline solution to create a final volume of 50 mL, yielding EF standard solution at a concentration of 20.0 μg·mL −1 . The samples were pretreated by liquid-liquid extraction thrice. In each round, 1mL of water-saturated ethyl acetate was added into 0.5 mL of sample incubated anaerobically at 37 °C, and then vortexed for 1min and centrifuged at 9 960 r·min −1 for 1 min. The supernatant obtained from the three rounds of liquid-liquid extraction was combined and dried under nitrogen stream at room temperature. The residue was re-dissolved in 150 μL of methanol by ultrasonic for 0.5 min and vortexed for 0.5 min. Then the supernatant was obtained by centrifuging at 9 960 r·min −1 for 1min and transferred into a 400 μL borosilicate glass insert placed inside a sample vial for HPLC analysis. Chromatographic separation was performed on a XB-C 18 column (4.6 mm × 250 mm i.d., 5 µm) coupled with a Phenomenex-C 18 guard column (13 mm × 4.6 mm i.d., 5 μm). Optimum separation was achieved with a binary mobile phase at a flow rate of 0.8 mL·min −1 . The column temperature was held at 35 °C. The mobile phase consisted of acetonitrile (B)-water (A) (V/V) (including 0.1% formic acid respectively). The gradient program was as follows: 0-10 min 32%-80% B; 10-18 min 80%-100% B; 18-22 min 100% B; 22-23 min 100%-32% B; 23-28 min 32% B. The detection wavelength and the reference wavelength were set at 335 nm and 400 nm, respectively. Finally, 10 μL of sample was injected automatically into the HPLC system. As reported, the metabolites of Epimedium in vivo included icariin (parent drug), icariside I, icariside II, desmethylicaritin and icaritin [14] [15] . Therefore, these five reference substances were selected and mixed to prepare the sample of mixed reference substances used for identification of the metabolites of EF in rabbit intestinal flora. 3.7 The study of EF metabolisms in rabbit intestinal flora 35 pieces of 2 mL of polypropylene centrifuge tubes were used, each of which was added 50 μL of EF standard solution (20.0 μg·mL −1 ) as well as 500 μL of intestinal flora cultural solution and incubated at 37 °C anaerobically. Five tubes were taken out at each time point of 0, 0.5, 1, 2, 4, 8, and 12 h. Then the samples were pre-treated immediately referring to pre-treatment of samples in 3.4 immediately and analyzed as HPLC conditions in 3.5 in order to study the metabolism of EF in intestinal flora of rabbits. In addition, EF were incubated in anaerobic culture solutions as negative control, and then subjected to the same procedures as for the rabbit intestinal flora described above to investigate whether EF were metabolized in anaerobic culture solutions. Different amounts of gluconolactone were added into three portions of rabbit intestinal flora fluid to obtain three different concentrations: 8 mg·mL −1 as low concentration, 12 mg·mL −1 as intermediate concentration and 16 mg·mL −1 as high concentration. For each rabbit intestinal flora cultural solution at one of the three prepared gluconolactone concentrations, 35 pieces of 2 mL of polypropylene centrifuge tubes were used, each of which was added 50 μL of EF standard solution (20.0 μg·mL −1 ) as well as 500 μL of corresponded rabbit intestinal flora cultural solution above and incubated at 37 °C anaerobically. Five tubes were taken out at each time point of 0, 0. 5, 1, 2, 4, 8, and 12 h. Then the samples were pre-treated immediately as pre-treatment of samples in 3.4 immediately and analyzed as HPLC conditions in 3.5 in order to study the inhibition of the metabolism of EF in rabbit intestinal flora by gluconolactone. In the experiment, EF were shown to be metabolized by rabbit intestinal flora quickly, as was evidenced by the complete elimination of icariin (the main component of EF) as parent drug after incubation for 2 h, and a metabolite, icariside II reached the maximum peak area simultaneously and then decreased gradually with further incubation. After incubation for 8 h, another metabolite, icaritin was generated and its peak area could increase gradually upon longer incubation time. The mean profiles and corresponding chromatograms are shown in Figs. 1 and 2 respectively. The metabolism of EF in anaerobic culture solutions was also investigated as negative control and the results showed that EF could not be metabolized in anaerobic culture solutions. The inhibition of the EF metabolism in rabbit intestinal flora with different concentrations of gluconolactone (8, 12, 16 mg·mL −1 ) was performed. Gluconolactone showed this kind of inhibition at low concentration (8 mg·mL −1 ) in rabbit intestinal flora, i.e. the amount of icariside II and icaritin decreased and the residue amount of icariin increased (Fig. 3A) . The enhanced inhibition of gluconolactone was shown at the intermediate concentration (12 mg·mL −1 ) in rabbit intestinal flora, i.e., icaritin formation was inhibited while the amount of icariside II decreased. And at each time point, the SD of icariside II was less than 1.0 (Fig. 3B) . Gluconolactone showed complete inhibition at high concentration (16 mg·mL −1 ) in rabbit intestinal flora; that is, neither metabolite (icariside II or icaritin) was formed nor icariin was metabolized. And at each time point, the SD of icariin was less than 2.0 (Fig. 3C ). In brief, at certain concentrations, gluconolactone showed inhibition of the metabolism of EF in rabbit intestinal flora in a concentration-dependent manner, and 16 mg·mL −1 of gluconolactone could totally inhibit the metabolism of EF in rabbit intestinal flora. On the basis of the above results, in intestinal flora of rabbits, EF (with icariin as the main component) were thought to be metabolized by hydrolysis to first produce icariside II after removal of glucose, and then icariside II was metabolized to produce icaritin after further removal of rhamnose. Fig. 4 is the corresponding flow diagram. For both the metabolism of EF in rabbit intestinal flora in our experiment and the metabolism of icariin in human intestinal flora reported [10] , the same final metabolite, icaritin was produced. Considering that icaritin, but not EF, displayed direct protective effects dose-dependently on steroid-induced cell damage model for in vitro bioactivity evaluation [6] , EF could reduce the risk of steroid-associated osteoncrosis in rabbit model [5] , and Epimedium extract could reduce the probability of steroid-associated osteoncrosis for SARS patients in clinical evidence-based medicine [16] [17] , icaritin might be implied as the exact in vivo bioactive component of EF in the prevention of steroid-associated osteonecrosis. Gluconolactone was found to concentration-dependently inhibit the metabolism of EF in rabbit intestinal flora in the study. It showed that gluconolactone may be used as a potential tool medicine to investigate whether EF could reduce the risk of steroid-associated osteoncrosis in rabbit or not when its in vivo metabolism was inhibited by gluconolactone and accordingly confirm whether EF might act as a prodrug in the prevention of steroid-associated osteonecrosis. In summary, some partial research has been carried out in our study for the aim of revealing the in vivo bioactive component of EF in the prevention of steroid-associated osteonecrosis; nevertheless, further investigation on the in vivo metabolism and bioactivity evaluation of EF using rabbit as model should be launched to help achieve the above objective. Effects of Epimedium icariine on rabbit and dog cerebral blood flow Immunoregulatory effects of the Herba Epimediia glycoside icariin Current status of the anti-osteoporosis mechanism of icariin Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition Constitutional flavonoids derived from epimedium dose-dependently reduce incidence of steroid-associated osteonecrosis not via direct action by themselves on potential cellular targets Metabolism of drug by intestinal bacteria Metabolism of icariin and icariside ii by rat intestinal bacteria in vitro Metabolism of icariin by intestinal bacteria. Part. I. the transformation of icariin by intestinal flora Absorption and metabolism of flavonoids in the CACO-2 cell culture model and a perused rat intestinal model Characterization of metabolites of worenine in rat biological samples using liquid chromatography-tandem mass spectrometry A purgative action of barbaloin is induced by Eubacterium sp. strain BAR, a human intestinal anaerobe, capable of transforming barbaloin to aloeemodin anthrone Sensitive and rapid method to quantify icaritin and desmethylicaritin in human serum using gas chromatography-mass spectrometry Simple and sensitive liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera Report on the investigation of lower extremity osteonecrosis with magnetic resonance imaging in recovered severe acute respiratory syndrome in Guangzhou Osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids 12 和 16 mg·mL −1 )的葡萄糖酸内酯对淫羊藿总黄酮苷 经家兔肠菌代谢的抑制情况进行考察。采用 ODS 柱和乙腈-水(各含 0.1%的甲酸)的流动相梯度洗脱, 检测波长为 335 nm。结果： 淫羊藿总黄酮苷在家兔肠菌液中温孵 2 h 时被代谢成淫羊藿次苷 II, 在温孵 8 h 时被代谢成淫羊藿素 当葡萄糖酸内酯的浓度为 16 mg·mL −1 Thanks are given to Prof. SONG Li-Yan for her kind help in revising this paper.