| BackgroundIntestinal bacteria are of vital importance to human health,it can help to decompose the nutrients in human body, balance the immune system and produce the needed vitamin. However, the disorder of some intestinal bacteria may cause disease and even increase risk for illness. Studies have shown that intestinal bacteria also have a strong ability to metabolize the drugs. At present, it has become a new research area for finding the potential biological active substances from metabolites by intestinal bacteria. The method of administration of traditional Chinese medicine(TCM) is usually oral administration, and it will be inevitably influenced by the intestinal bacteria in body. Many natural products like glycoside ingredients have been converted to pharmacological active substances by intestinal bacteria. Because of the large molecular weight and polarity of these compounds, they stay for a long time in intestine and are susceptible to the affection of intestinal flora. So they will produce the secondary glucosides or aglycone by intestinal flora, and play their pharmacological effects. A large number of studies have shown that the metabolites of natural products by intestinal bacteria are really the active substances that can reach the target organs.Ardipusilloside-Ⅰ(ADS-Ⅰ) is a triterpenoid saponin extracted from Ardisia pusilla A.DC. It may cause hemolysis, thus makes it unsuitable for intravenous administration. Besides, ADS-Ⅰ has a poor intestinal absorption and the low bioavailability after oral administration, which limits the development of this compound like saponin as a new drug candidate. Previous studies have demonstrated that the potent anti-tumor activities of ADS-Ⅰ both in vitro and in vivo, and it had significant inhibitory effects on several solid tumors after oral administration. Our group had found that ADS-Ⅰ can hardly be absorbed by rats gastrointestinal and the substances that absorbed in rats were deglycosylated metabolites of ADS-Ⅰ. All these studies suggest that the metabolites of ADS-Ⅰ may be the key components for the inhibitory activity against the growth of tumor in animal experiments after oral administration.The research simulated the human intestinal flora metabolism system, and established the human intestinal membrane Caco-2 cell monolayers model. The separation, identification and gathering of ADS-Ⅰ metabolites were achieved by chromatography-mass spectrometry(LC-MS) and high performance liquid chromatography-nuclear magnetic resonance(HPLC-NMR). The antitumor activity of the two new compounds isolated from the metabolism system was tested in vitro, and the way of cell membrane of the new compounds was studied. The research explore the material base of ADS-Ⅰ for antitumor in body and provided new ideas and solutions for searching the new bioactive substances.Aims Isolation, identification and gathering of active metabolites of ADS-Ⅰ by human intestinal bacteria, and preliminary study of the antitumor activity and cell permeability of the metabolites in vitro.Methods 1. Biological transformation of ADS-Ⅰ by human intestinal bacteria in vitro. High performance liquid chromatography-evaporative light scattering detector method(HPLC-ELSD) and ultra-high performance liquid chromatography-electrospray ion-tandem mass spectrometry method(UHPLC-ESI-MS/MS) were used to determine the chemical structures of ADS-Ⅰ metabolites.2. The metabolites of ADS-Ⅰ were separated and purified by a Sephadex LH-20 chromatography column, and the chemical structures of the metabolites are identified by NMR and MS. MTT assay was utilized to investigated the growth inhibition of ADS-Ⅰ and its metabolites on human hepatoma cells(SMMC-7721) and human breast cancer cells(MCF-7).3. We established Caco-2 cell model, and evaluated the cell cytotoxicity of ADS-Ⅰ and its metabolites by MTT assay. It helped select the optimal drug concentration for penetration absorption. Followed study the transportation of the drug by the two-way experiments. The drug amount of transmembrane transportation was measured by UHPLC-MS. According to the formula of apparent permeability of drug, the absorption capacity of ADS-Ⅰ and its metabolites in the intestine were determined.Results1. It was found that four metabolites were produced and deglycosylation reactions are the main reactions when ADS-Ⅰ was incubated with human intestinal bacteria. The chemical structures of ADS-Ⅰ metabolites was initially deduced, including three secondary glucosides(M1, M2, M3) and one aglycone(M4).2. We obtained two metabolites(M1 and M2) by sephadex LH-20 chromatography column. We concluded that M1 was identified as 3-O-[α-L-rhamnose(1→2)-β-D-glucopyranose(1→3)]-α-L-arabinose- cyclamiretin A, and M2 was confirmed as 3-O-[β-D-glucopyranose(1→3)]-α-L-arabinose-cyclamiretin A by analyzing NMR data and MS data.3. MTT assay showed that the IC50 of ADS-Ⅰ, M1, M2 on SMMC-7721 cells cultured for 72 h were 5.48± 0.47, 9.65± 0.62, 40.91 ± 1.27μmol/L respectively. The IC50 of ADS-Ⅰ, M1, M2 on MCF-7 cells in cultured for 72 were 8.77± 0.57, 24.10± 0.83, 6.76 ± 0.49 μmol/L respectively. We thus found that the cytotoxic activity of M2 is higher than that of ADS-Ⅰ in MCF-7 cells.4. We used the Caco-2 cell model to study the absorption and transportation of ADS-Ⅰ and its metabolites. The Papp value of ADS-Ⅰ was 1.88 ± 0.21×10-6 cm·s-1 for AP to BL direction, and was 0.69 ± 0.15×10-6 cm·s-1 for BL to AP direction in 120 min. The Papp values of M1(10 μmol/L) in the direction of AP- BL and BL- AP were 4.30 ± 0.43×10-6 cm·s-1 and 1.76 ± 0.26×10-6 cm·s-1 respectively, and of M2(10 μmol/L) were 4.74 ± 0.47×10-6 cm· s-1and 2.12 ± 0.23×10-6 cm·s-1 respectively, and efflux ratio(ER) were all 0.3- 0.6.The data showed that M1 and M2 had a good intestinal permeability, but ADS-Ⅰ was a poorly absorbed compound and intestinal absorption is limited by oral administration.ConclusionsThis study based on the intestinal flora metabolism system is to study the metabolism of ADS-Ⅰ. Here, we show that main reaction in the metabolism of ADS-Ⅰ in human intestinal bacteria was deglycosylation, and it produced four metabolites. We isolated two new compounds from the metabolism system. The results showed that metabolites M1 and M2 all have the inhibitory activities on human liver cancer cells(SMMC- 7721) and on human breast cancer cells(MCF- 7). They have a good intestinal permeability and better intestinal absorption than ADS-Ⅰ. This study preliminarily expounded the metabolism of ADS-Ⅰ in human intestine bacteria, and the cell transportation way for ADS-Ⅰ metabolites. The results provided a scientific basis for intestinal transformation and absorption of ADS-Ⅰ and its metabolites in human. Moreover, it provided a new sight and method for finding the new antitumor active substances from natural products. |
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