New Steroidal Glycosides Isolated as CD40L Inhibitors of Activated Platelets

Three new compounds were isolated from the dried bulbs of Allium macrostemon Bunge. Their structures were elucidated from their spectral data as (25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12β,22,26-tetraol-3-O-β-D-glucopyranos-yl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galactopyranoside (1), (25R)-26-O-β-D-glucopyranosyl-5α-furostane-3β,12α,22,26-tetraol-3-O-β-D-gluco- pyranosyl (1→2) [β-D-glucopyranosyl (1→3)]-β-D-glucopyranosyl (1→4)-β-D-galacto- pyranoside (2) and (25R)-26-O-β-D-glucopyranosyl-5β-furostane-3β,12α,22,26-tetraol-3-O-β-D-glucopyranosyl (1→2)-β-D-galactopyranoside (3), respectively. The inhibition effect of all compounds on CD40 ligand (CD40L) expression on the membrane of activated platelets stimulated by ADP was tested. Compounds 1 and 2 exhibited significant inhibitory activities in a dose dependent manner (P < 0.05), suggesting their potential application as CD40L inhibitors.


Introduction
Recent reports suggest that most anti-platelet agents can be used as anti-inflammatory drugs and platelet activation is sometimes critical in the development of inflammation [1]. As a member of the tumor necrosis factor-α family of proteins, CD40L has been identified as a proinflammatory mediator and risk factor for cardiovascular events on activated platelets [2]. It can bind and activate platelet α Ⅱ b β 3 in thrombosis and inflammation [3,4]. It is also able to activate endothelial cells to have a proinflammatory phenotype [5]. The stimulation of endothelial cells by platelets expressing CD40L contributes to inflammatory cell recruitment in atherosclerosis [6]. And the investigation on CD40L inhibitor would be significant and useful.
It was reported that, as the main constituents of dried bulbs of Allium macorstemon Bunge, steroidal glycosides could strongly inhibit the platelet aggregation of human beings [7]. But their activity on platelet inflammatory was never reported. This paper concerns the isolation and structure elucidation of three new steroidal glycosides from ethanol extract of the dried bulbs of Allium macorstemon Bunge.
Their inhibition on CD40L expression on the membrane of activated platelets was tested.
The inhibitory effect of compounds 1-3 of CD40 ligand (CD40L) expression on the membrane of activated platelets stimulated by ADP was measured by real time PCR. Compounds 1 and 2 exhibited significantly inhibit expressions of CD40L in ADP induced active platelets in a dose dependent manner (P < 0.05) (Figure 3) which suggested that they may be new type of CD40L inhibitors for curing platelet inflammatory related diseases.

General
Melting points were determined with a Yanaco MP-S 3 micro-melting point apparatus and are uncorrected. Optical rotations were obtained on a P-1020 digital polarimeter (JASCO Corporation). IR spectra were measured on a Shimadzu FT/IR-8400 spectrometer. 1D and 2D NMR spectra were taken on a Bruker AV-400 (400 MHz for 1 H-NMR) spectrometer in C 5 D 5 N solution. ESIMS spectra were acquired using a Bruker Esquire 2000 mass spectrometer. Column chromatography was carried out on Diaion HP-20 (Mitsubishi Kasei, Japan), silica gel (200-300 mesh, Qingdao Factory of Marine Chemical Industry, Qingdao, China) and ODS (40-63 µm, Merck). TLC analyses were taken on Silica gel 60F 254 (Qingdao Factory of Marine Chemical Industry, Qingdao, China) and the spots were detected spraying with Ehrlich reagent and heating. Preparative HPLC was performed using an ODS column (250 × 20 mm, 10 μm, Shimadzu Pak; Detector: RID). Quantitative real-time PCR was performed using the PE5700 PCR system (Biosystems, Foster, CA, USA). The Qiagen RNeasy minikit system (Qiagen, Valencia, CA, USA) was used to collect mRNA from ADP induced active platelets. The mRNA was converted to cDNA using the AMV Reverse Transcription System (Promega, Madison, Wi, USA).

Plant material
The bulbs of Allium Macrostemon Bunge were purchased from Liaoning Province of China and identified by Professor Qi-Shi Sun (Department of Pharmacognosy, Shenyang Pharmaceutical University). The voucher specimen has been deposited at the Department of Natural Product Chemistry, Shenyang Pharmaceutical University, Shenyang (110016), P. R. China.

Extraction and isolation
The dried bulbs of Allium Macrotemon Bunge (5 kg) were extracted twice with 60% ethanol (10 L) for 2 h each time. The ethanol extraction was concentrated under reduced pressure, suspended in water and then passed through Diaion HP-20 column using EtOH/H 2 O gradient system (0-100%). The 60% EtOH eluted fraction (90 g), which was subjected to silica gel column chromatography with   Table 1; 13 C-NMR, see Table 2.  Table 1; 13 C-NMR, see Table 2.  Table 1; 13 C-NMR, see Table 2.

Acid hydrolysis of saponins
Each glycoside (5 mg) was heated in an ampoule with 15% aq. HCl (5 mL) at 110 ºC for 2 h. The aglycone was extracted with dichloromethane three times and the aqueous residue was evaporated under reduced pressure. Then, pyridine (1 mL) and NH 2 OH·HCl (2 mg) were added to the residue, and the mixture was heated at 100 ºC for 1 h. After cooling, Ac 2 O (0.5 mL) was added and the mixtures were heated at 100 ºC for 1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC-MS using standard aldononitrile peracetates as reference samples. D-glucose and D-galactose were identified in a ratio of 4:1 for compound 1 and 2 and in a ratio of 2:1 for compound 3.

Platelets preparation
After obtaining consent, blood samples were collected from healthy volunteers (five men and five women; age range, 17-45 years) and 3.8% sodium citrate (nine parts blood, one part citrate) was added as an anticoagulant. All subjects avowed that they had not taken any anti-platelet drug at least 2 weeks prior to the extraction. Platelet-rich plasma (PRP) was prepared by centrifugation at 125×g for 10 min at room temperature. Platelet count was adjusted to 5 × 10 9 platelets/mL. PRP (400 µL) was added to the wells of a tissue-culture plate (24-well plate; well size 16 × 16 mm; Nunc) and pretreated with compounds (5, 20, 80, 320 µM) or AR-C67085MX (10 µM) at 37 ºC for 10 min, and then stimulated with 20 µM ADP for 5 min.

Real-time reverse-transcriptase-polymerase chain reaction
The mRNA was collected from ADP induced active platelets following the manufacturers' recommended protocol. The mRNA was converted to cDNA and cDNA samples were kept at -20 ºC until needed. The CD40L and housekeeping gene β-actin primers for PCR analysis were based on the human platelet sequences and designed using the Primer 5 software. The forward and reverse primers were as follows: CD40L mRNA: 5′ACATACAACCAAACTTCTCCC 3′ and 5′AGATGTTGTTTTACTGCTGGC 3′, (799 bp) β-actin mRNA: 5'-AAGGATTCCTATGTGGGC-3' and 5'-CATCTCTTGCTCGAAGTC-3, (362 bp) The reaction mixture contained TaqMan universal PCR Master Mix (25 μL), 10 μM (2 μL) of each primer, TaqMan probe (2.5 μL) and cDNA (2 μL). It was adjusted to 22.5 μL with DNase/RNase free water. Conditions of the real-time-PCR were as follows: 50 ºC for 2 min, 95 ºC for 15 min, and for 40 cycles, 95 ºC for 15 s and 60 ºC for 1 min. The Ct value is defined as the number of fractional cycles in which the fluorescence emitted by the reaction mixture exceeds a preset threshold and marks the beginning of an exponential growth of the fluorescence signal. The results were analyzed with PE 5700 software.

Statistical analysis
All results were expressed as the mean±standard error (SE). Analyses were performed with SPSS software Version 13.0 (SPSS, Chicago, IL). ANOVA was used to determine if significant variation existed between group means. Two-side p values less than 0.05 were considered to be significantly.