Three new flavonoid glycosides from the aerial parts of Allium sativum L. and their anti-platelet aggregation assessment

Abstract Three new flavonoid glycosides, Dasuanxinoside F–H (1–3), were isolated from the aerial parts of Allium sativum, together with eight known compounds which were firstly reported in this plant, including three flavonoid glycosides (4–6) and five phenylethanoid glycosides (7–11). Their structures were identified by UV-vis, IR, 1D and 2D NMR spectra, as well as HR-ESI-MS analyses. The inhibitory effect of the isolated compounds on platelet aggregation induced by adenosine diphosphate (ADP) was evaluated in vitro. The results showed that most compounds displayed different degrees of inhibition. Among them, 2, 5, 8 and 9 exhibited the strongest activity on platelet aggregation. Graphical Abstract


Introduction
Allium sativum L. is a perennial herb of Liliaceae family, whose underground bulbs are well known as garlic, and used as traditional Chinese medicine (TCM), Dasuan. It is recorded in Chinese pharmacopoeia (2020 edt), with the functions of detoxification, detumescence, insecticide and dysentery control (Chinese Pharmacopoeia Commission 2020, 25-26). Scholars in the field of medicine and food (Bianchi 2015) have done a lot of research work on garlic and more than 130 compounds have been isolated from garlic bulbs since the 1970s, mainly including organic sulfur compounds (Tan et al. 2015;Woo et al. 2015;Ono et al. 2017;Kim et al. 2018;Nakamoto et al. 2018;Nohara et al. 2018), steroidal saponins (Matsuura et al. 1988;Lanzotti et al. 2012), flavonoids (Kim et al. 2005;Li et al. 2015), pyranone derivatives (Kodera et al. 2002), cerebrosides, phenols, vitamins, amino acids, polysaccharides, protein and volatile oil. Clinically, the bulbs of A. sativum are commonly used in the treatment of cardiovascular diseases, infections, digestive system and urinary system diseases, etc. (Karuppiah and Rajaram 2012;Ried and Fakler 2014;Bin et al. 2020;Mai et al. 2021). A. sativum is an indispensable dual-purpose plant for medicine and food. The huge market demand has led to the cultivation of garlic all over the world. However, most people only harvest bulbs, and a large number of aboveground parts are discarded directly. This has resulted in a huge waste of resources.
In this study, the chemical isolation and purification were carried out for the aerial parts of A. sativum, and three new (1-3) along with eight known (5-11) compounds were obtained. Their structures were identified by ultraviolet visible (UV-vis), infra-red (IR), nuclear magnetic resonance (NMR) spectra and high resolution electrospray ionisation mass spectrometry (HR-ESI-MS), as well as comparing the data with the literature. The anti-platelet aggregation activities of compounds were also studied in vitro. As a result, nine ingredients were active, revealing the pharmacodynamic material basis of the aerial parts of A. sativum on platelet aggregation. This study provided a potential for decreasing resources waste, further deeply exploiting and utilizing the aerial parts.

Anti-platelet aggregation activity of the isolated compounds
Isolated compounds 1-9 were evaluated for anti-platelet aggregation activity using aspirin as a positive control. The results showed that compounds 2, 5, 8 and 9 had strong anti-platelet aggregation activity, which was equivalent to positive drug. Compounds 1, 3 and 7 showed weak activity. Compounds 4 and 6 were almost inactive. The data were shown in Table S3 (Supplementary material).

Plant materials
The aerial parts of Allium sativum L. were collected from Heilongjiang province of China in July 2017 and identified by Zhenyue Wang of Heilongjiang University of Chinese Medicine. The voucher specimen (No. 20170575) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine, Heilongjiang, China.

Acid hydrolysis of 1-3
Acid hydrolysis experiments were carried out by using the methods in the literature . In short, the sugar residues were gained by hydrolyzing of compounds 1-3 (2.0 mg) with H 2 SO 4 (2 mol/L, 2.0 mL), and then treated with trimethylchlorosilane, respectively. Then the sugar derivants were further analyzed by GC. The results showed that the sugar derivatives of compounds 1-3 were D-glucose (t R ¼ 11.32 min).

Anti-platelet aggregation activity test
The platelet aggregation experiment were carried out by using the methods in the literature (Chen et al. 2011). Briefly, SD male rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mlÁkg À1 ), and blood was taken from abdominal aorta with syringe containing 3.8% sodium citrate (v/v 3.8% sodium citrate: Blood ¼ 1:9). After 30 minutes of static setting, blood samples were centrifuged for 8 minutes with 1000 rpm at room temperature, and then platelet-rich plasma (PRP) was absorbed from the upper layer. The remaining plasma was centrifuged for 10 minutes with 3000 rpm to obtain platelet-poor plasma (PPP), which was used as a reference solution in platelet aggregation assays. The platelets of PRP were adjusted to the proper number (2-2.5 Â 10 11 /L) with PPP for the aggregation assay. The 90 lL PRP and 5 lL sample solution (final concentration: 200 lmol.L À1 ) were added into 96-well plates and incubated on 37 C for 10 minutes. After incubation, 5 lL ADP (final concentration: 5 lmol.L À1 ) was added and 570 nm transmission was monitored with Microplate Reader for every 30 seconds until absorbance (A) became stable. The aggregation rate (AR) ¼ (A PRP -A sample )/(A PRP -A PPP ) Â 100%. The aggregation inhibition rate (AIR) ¼ [1 -(AR sample/ AR control )] Â 100%.

Conclusions
We studied the chemical components from the aerial parts of A. sativum and assessed their activity based on its clinical application of treating cardiovascular diseases.