Phytochemical constituents of Camellia osmantha fruit cores with antithrombotic activity

Abstract Camellia osmantha is a new species of the genus Camellia and is an economically important ornamental plant. Its activity and ingredients are less studied than other Camellia plants. This study investigated the antithrombotic effect and chemical components of C. osmantha fruit cores using platelet aggregation assays and coagulation function tests. The cores of C. osmantha fruits were extracted with ethanol to obtain a crude extract. The extract was dissolved in water and further eluted with different concentrations of methanol on an MCI resin column to obtain three fractions. These samples were used for antithrombotic activity tests and phytochemical analysis. The results showed that the extract and its fractions of C. osmantha have strong antithrombotic activity, significantly reducing the platelet aggregation rate and prolonging the thrombin time (TT). The total saponins, flavonoids, and polyphenols in the active fractions may be responsible for the antithrombotic activity. The chemical constituents were analyzed by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐QTOF/MS). Twenty‐three compounds were identified rapidly and accurately. Among them, ellagic acid, naringenin, and quercetin 3‐O‐glucuronide may be important antithrombotic constituents. Furthermore, interactions between these compounds and the P2Y1 receptor were investigated via molecular modeling, because the P2Y1 receptor is a key drug target of antiplatelet aggregative activity. The molecular docking results suggested that these compounds could combine tightly with the P2Y1R protein. Our results showed that C. osmantha fruit cores are rich in polyphenols, flavonoids, and saponins, which can be developed into a promising antithrombotic functional beverage for the prevention and treatment of cardiovascular and cerebrovascular diseases.

and are consumed as a beverage because of their specific efficacy in preventing cardiovascular disease. It has been reported that polyphenols, saponins, and catechins in Camellia plants have the potential to prevent cardiovascular and cerebrovascular diseases (Bansal et al., 2012). To date, no definitive evidence has been found to confirm that extracts of C. osmantha can inhibit the activation of coagulation factors and platelets, and prevent the conversion of fibrinogen into fibrin. Thus, the antithrombotic activity and the possible chemical compositions of C. osmantha fruit cores were determined by an activity-directed approach in the present study. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and platelet aggregation rate were determined to explore the anticoagulant effect of C. osmantha fruit cores. Possible antithrombotic chemical constituents were inferred by UPLC-QTOF/MS, and docking results were further verified by developing a pharmacophore model that clarified the key features required for an optimal P2Y1R affinity.

| Chemicals
The absorbance for determining the content of total flavonoids, total polyphenols, total polysaccharides, and total saponins was measured on a Shimadzu UV-Vis spectrophotometer (UV-1780, Shimadzu, Japan). Chemical composition analysis was performed on an Agilent 6545B Q-TOF LC/MS using a Supelcosil ABZ+PLUS column (150 mm × 4.6 mm, 3 μm). Platelet aggregation induced by adenosine diphosphate (ADP) was measured using a semiautomatic platelet aggregometer (LBY-NJ4, Beijing Precil Group, China).
PT, APTT, and TT were tested by a C-20004 semi-auto coagulation analyzer (Beijing Precil Group, China). Coagulation test reagents were purchased from Shanghai TaiYang Biotechnology, China. DNS (3, and Folin-Ciocalteu's phenol reagent were purchased from Beijing Solarbio Science & Technology Co. Ltd. ADP was obtained from Ark Pharm, Inc. (Libertyville, IL, USA). Rutin, glucose, gallic acid, and saponin were purchased from Energy Chemical Technology Co., Ltd (Shanghai, China). Vanillin, perchloric acid, acetic acid, aluminum nitrate, sodium hydroxide, and other reagents were of analytical grade.

| Plant material
Camellia osmantha fruit cores were collected during maturity (September 2019) in Nanning, Guangxi, and identified by Professor

Jin-Lin Ma of the Guangxi Zhuang Autonomous Region Forestry
Research Institute, one of the discoverers of C. osmantha. The samples were smashed and passed through an 80-mesh sieve after being dried in an oven at 60°C. The cores of C. osmantha fruit (200 g) were extracted three times with 80% analytical grade ethanol for 1 h with normal ultrasound-assisted extraction (300 w, 40 Hz) and filtered.
The filtrates were combined and dried under reduced pressure below 60°C to give a brown solid crude extract (CE, 2.15 g). The CE (1.8 g) was dissolved in water and was introduced into an MCI-gel CHP20P, and then eluted with different concentrations (0%, 50%, and 100%) of methanol to obtain fraction A (Fr-A, 0.6 g), fraction B (Fr-B, 0.48 g), and fraction C (Fr-C, 0.35 g). All samples including CE and three fractions were dried and stored at 4°C before performing the platelet aggregation assay, coagulation function test, and phytochemical analysis.

| Animals
Wistar rats, weighing 220-250 g, were obtained from the Animal Experiment Center of Youjiang Medical University for Nationalities, Baise, China. The animals were placed in an environment with a relative humidity of 40%-70% at a temperature of 25°C, and fed freely for 2 days with food and water before use.

| Platelet aggregation assay
Platelet aggregation assays were performed according to the methods reported in the literature (Gao et al., 2014). Wistar rats were anesthetized with 1% pentobarbital. Blood samples collected from the abdominal aorta were placed in a centrifuge tube (3.8%, w/v) with sodium citrate at a ratio of blood:anticoagulant = 9:1. The supernatant (platelet-enriched plasma, PRP) was obtained by centrifugation at 160 g for 10 min at 25°C. The remaining blood sample was continuously centrifuged at 2000 g for 10 min to obtain plateletpoor plasma (PPP). The platelet aggregation assay was performed by adding different extracts (1 mg/ml) to clean test cups and by inducing ADP at a final concentration of 5 μM. The maximum aggregation rate (MAR) within 6 min was observed by using physiological saline (9 mg/ml) as the negative group and aspirin (66 μg/ml) as the positive control. The aggregation inhibition rate (AIR) was calculated as follows: EAR, Experimental group aggregation rate; NCAR, No-treatment control group aggregation rate.

| Coagulation function test
To determine the coagulation function of C. osmantha fruit cores in rats, PT, APTT, and TT assays were used to evaluate the coagulation effect. Fresh blood mixed with an anticoagulant in a 9:1 ratio collection from the rat aorta was centrifuged at 633 g for 15 min to obtain plasma. The CE (0.2 μl, 1 mg/ml) was added to 0.4 μl of plasma to obtain mixed plasma. The assay was performed according to the kit requirements. Briefly, 0.6 μl of plasma was incubated at 37°C for 3 min, and then a prewarmed PT reagent (0.6 μl) at 37°C was added to record the PT. Plasma (0.6 μl) was added to 0.6 μl of prewarmed APTT reagent at 37°C and incubated for 5 min at 37°C, and then prewarmed 0.6 μl of CaCl 2 (0.025 mol/L) was added. The clotting time was recorded for TT after the prewarmed plasma was mixed with the TT reagent at 37°C. Three fractions were measured according to the above method. Each sample was measured three times.
2.6 | Determination of total flavonoid, polyphenol, polysaccharide, and saponin contents 2.6.1 | Total flavonoids The total flavonoid content (TFC) was determined using a method described in the literature (Li et al., 2012). Four samples (10 mg/ ml), including CE or Frs-A to C, were placed in a 10-ml tube and then 0.15 ml of 5% NaNO 2 was added. After 6 min, 0.15 ml of 10% Al(NO 3 ) 3 was added. Then, 2 ml of 4% NaOH was added another 6 min later, and the total volume adjusted to 5 ml with distilled water.
The absorbance of the mixture was measured through a UV-1780 spectrophotometer at 510 nm. Each test was repeated three times and the results were averaged.

| Total polyphenols
The total polyphenol content (TPPC) of the C. osmantha fruit core extracts was measured by a colorimetric assay primarily based on procedures described by Ordóñez-Santos with a few modifications (Ordonez-Santos et al., 2017). Briefly, 0.5 ml of CE or Frs-A to C (10 mg/ml) was mixed with 1 ml of Folin-Ciocalteu's phenol reagent. After 3 min, 1 ml of saturated sodium carbonate solution was added to the mixture and adjusted to 10 ml with distilled water. The reaction was kept in the dark for 60 min before the absorbance was read with a UV-1780 spectrophotometer at 760 nm. Gallic acid was used to construct the standard curve (0.01-0.4 mmol/l).

| Total polysaccharides
Total polysaccharide content (TPSC) was measured by a sulfuric acid-phenol spectrophotometric assay. A total of 2.0 ml of CE or Frs-A to C (10 mg/ml) was placed into a stoppered tube, followed by a 5% phenol solution (1.0 ml). Then, 5.0 ml of concentrated sulfuric acid was added immediately, incubated for 30 min in a 40°C water bath, removed from the tube, and placed in a cold-water bath for 5 min. The absorbance of each solution was measured at a wavelength of 490 nm. The reference solution was prepared with 2.0 ml of double-distilled water. A standard curve was drawn with the concentration of the glucose diluent (4-20 μg/ml) plotted on the abscissa and the absorbance was plotted on the ordinate.

| Total saponin
The total saponin content (TSC) was measured by a vanillinperchloric acid spectrophotometric assay (Xiao et al., 2014). The solution of CE or Frs-A to C (10 mg/ml) was prepared in methanol (0.2 ml of aliquot) and was added to the colorimetric tube. After methanol solvent removal at 80°C, 0.5 ml of vanillin-acetic acid solution (5 mg/ml) and 1.5 ml of perchloric acid were added. The reaction mixture was incubated at 70°C for 15 min and then cooled and diluted with acetic acid to 10 ml. After 10 min, the absorbance of the diluted solution was measured at 540 nm with a UV-1780 spectrophotometer, which was normalized against a solution of the reagents without the sample. The standard curve based on saponin (11.43-68.58 μg/ml) was quantified. All samples were tested three times.

| UPLC-ESI-QTOF-MS analysis
Chemical compositions of the C. osmantha fruit cores were determined on an Agilent 6545B Q-TOF LC/MS according to our previous experimental procedures (Ouyang et al., 2020).

| Molecular docking
The structure buildings of ellagic acid, naringenin, and querce- Autodock vina was used for semiflexible docking, and the best affinity conformation was selected as the final docking conformation.
The active site was defined as including all atoms within a 6.5 Å radius of the cocrystallized ligand, and the default parameters were used.

| In vitro aggregation inhibition assay of CE and each fraction
Platelet aggregation function is the cardinal biological parameter used to evaluate antithrombotic activity. AIRs of CE and three fractions are shown in Figure 1a. The AIR of CE was 32.07%, higher than that of aspirin (p = .064), a well-known platelet aggregation inhibitor with an AIR at 29.80%. Among the fractions, Fr-C exhibited the best aggregation inhibition effect with an AIR of 37.61%, followed by Fr-B with an AIR of 36.04%. Moreover, Fr-B and Fr-C had significantly higher AIRs than CE and fraction A. Thus, Fr-B and Fr-C have significant inhibitory effects on platelets and may be active fractions for antithrombotic applications.

| Coagulation effects of CE and each fraction
Routine coagulation assays are important ways to evaluate the antithrombotic activities of compounds. APTT and PT reflect the status of the endogenous coagulation and exogenous coagulation systems, respectively . TT reflects the ability to convert fibrinogen to fibrin. As shown in Figure 1b, no significant differences were found between the extracts and the control group for PT and APTT, indicating that the anticoagulation effect may not be related to either the endogenous or exogenous coagulation pathways.   (Basu & Lucas, 2007). To explore the composition of Fr-B and Fr-C, total saponins, flavonoids, polyphenols, and polysaccharides were determined by UV-Vis spectrophotometric methods. As shown in Figure 2, Fr-A showed a higher polysaccharide content than CE and other fractions, while Fr-A had the weakest antiplatelet aggregation ability found in Figure 1a, suggesting that the antithrombotic activity of C. osmantha fruit cores may not be caused by polysaccharides. The contents of flavonoids and polyphenols in Fr-B were much higher than those in the other fractions, while its content of total polysaccharides was lower than that in Fr-A. Especially, the content of total polyphenols in Fr-B was 36.23 ± 2.18% gallic acid equivalents (GAE), significantly higher than that reported by Anesini et al. (Anesini et al., 2008). Similarly, the contents of polyphenols and flavonoids in Fr-C were higher than those in CE and Fr-A. Fr-B and Fr-C showed considerable antiplatelet aggregation ability, but their chemical compositions were not consistent. These results suggested that polyphenols and flavonoids in Fr-B, as well as saponins and flavonoids in Fr-C, may be the main contributors to the anticoagulant activity of the C. osmantha fruit cores.
Moreover, Spearman correlation analysis performed for the content of each fraction and AIR indicated a statistically significant correlation between total saponins and AIR (Table 1)

| HPLC-ESI-QTOF-MS analysis
The specific chemical compounds of Fr-B and Fr-C were further analyzed by UPLC-QTOF-MS. According to the retention time, the precursor m/z of the positive and negative ions, the isotope ratio, and the reference compounds confirmed from the Camellia genus, 23 compounds were initially identified that contained polyphenols, flavonoids, triterpenes, organic acids, etc. (Table 2). Although MCIgel was used as the separation material for isolation, 18 compounds were still found in both Fr-B and Fr-C. UV-Vis analysis of most peaks confirmed that these compounds had maximum absorption only at 278 nm with a distinct shoulder at 283 nm, but had no absorption in the visible region, suggesting they might be polyphenols (Uchida et al., 2016). A small part of the peaks also has a maximum absorption at 330-340 nm, which suggests that they may be flavonoids.
These findings are consistent with a previous analysis on the content of total polyphenols in Fr-B and Fr-C. anticoagulant effects. Ellagic acid can treat pathological arrhythmias, ventricular hypertrophy, and lipid peroxidation in rats with myocardial infarction caused by isoproterenol (Kannan & Quine, 2013)

| Docking analysis
The P2Y1 receptor (P2Y1R) facilitates platelet aggregation and is thus an important potential antithrombotic drug target. The P2Y1R protein structure contains a binding site for the receptor antagonist MRS2500 in its seven-transmembrane bundle, which also provides suitable pockets for numerous other ligands and nucleotide agonists of P2Y1R.

F I G U R E 4 Amino acids of 4XNW
interacting with ellagic acid (a), naringenin (b), and quercetin 3-O-glucuronide (c). Hydrogen bonds are shown as yellow dots and π-π bond is shown as pink dots

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicts of interest for this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.