Chemical constituents and coagulation activity of Syringa oblata Lindl flowers

The leaves and bark of Syringa oblata Lindl are used as folk medicine which has heat-clearing, detoxifying, dampness-removing and jaundice-relieving effects. There are many studies about leaves of S. oblata because of its abundant resource, however, less reports about the components of S. oblata flowers. The previous studies on S. oblate flowers were mainly focused on the volatile components and its traditional pharmacological activity. Thus, this study aimed to investigate the nonvolatile chemical constituents and the coagulation activity of S. oblate flowers. The chemical constituents of S. oblate flowers were isolated with various column chromatographies and coagulation activity of the major constituents was investigated by assaying the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) on plasma of rabbit in vitro. Fifteen known compounds (namely compound 1-15) were isolated from S. oblata flowers. Compound 6, 10, 11 and 14 were isolated from Syringa genus for the first time. Compound 1, 2, 4, 5, 8 and 9 were isolated from the plant for the first time. The results of coagulation activity showed that water part of S. oblate flowers, lauric acid and kaempferol-rutinose significantly shorten PT (P < 0.001), TT (P < 0.001) and APTT (P < 0.001) compared with blank group, thus revealed that water extract of S. oblate flowers, lauric acid and kaempferol-rutinose possessed the procoagulant activity, but the effects were not better than that of Yunnan Baiyao as positive control.


Introduction
Syringa oblata Lindl, a medicinal plant which has the characteristics of trees or shrubs of the Oleaceae family, is native to north China. S. oblate tastes bitter, and has quality of cold. Chinese Materia Medica records that the leaves and bark of S. oblata have been used as folk medicine, which have heat-clearing, detoxifying, dampness -removing and jaundice-relieving effect [1].
These references indicate that triterpenes, phenethyl alcohol, phenylpropanoid and iridoid compounds are the main components in S. oblate. However, the flavonoids, organic acids and other constituents have been less reported.
In view of the fact that the S. oblate has a wide range of biological activities, including antibacterial, anti-inflammatory, antiviral, anti-tussive and expectorant effect, liver protection and cholagogue etc. [11]. The previous studies on S. oblate flowers were mainly focused on the volatile components and its traditional pharmacological activity. Thus, this study aimed to investigate the nonvolatile chemical constituents and the coagulation activity of S. oblate flowers.

Chemicals and material
The chemicals and material were similar to our previous research [12].

Plant material
Syringa oblata flowers were collected in April 2015 from the Kaifeng region of Henan Province, China and identified by Professor Changqin Li. A voucher specimen was deposited in National R & D Center for Edible Fungus Processing Technology, Henan University.

Animal
The male rabbit (approximately 20 months old, weight from 2.0 to 2.5 kg) was provided by Kaifeng Key Laboratory of Functional Components in Health Food (2016-02) to evaluate anticoagulant effect in vitro.

Ethics information
The study obtained ethical clearance from the Ethics Committee of College of Medical, Henan University (NO: 2016-36). The rabbits were treated as per the guidelines on the care and use of animals for scientific purposes.

Extraction and isolation
The extracted method was similar to our previous research [12]. The air-dried flowers of S. oblata (1.4 kg) were extracted with 70% ethanol to yield the crude extract (So. TE 378 g). The extract (378 g) was dissolved in MeOH-H 2 O (v:v = 3:1, 500 mL), and then mixed with D101 macroporous adsorbent resin. TE was separated by macroporous resin column chromatography, eluted with 20%, 40%, 60%, and 90% ethanol. After evaporation of the solvent, 235 g of water part, 27 g of 20% ethanol part, 61 g of 40% ethanol part, 20 g of 60% ethanol part and 35 g of 90% ethanol part were obtained.

The coagulation activity of Syringa oblata Lindl flowers in vitro
Blood samples were drawn from Rabbit's Auricular vein without anaesthesia. The method was similar to our previous research [12]. APTT, PT,TT and FIB were determined.
For all the tests mentioned above, blank solvent (dimethyl sulphoxide: Tween 80: normal saline = 2:1:17) was used as negative control, while the drugs of breviscapine (13.3 mg/mL) and Yunnan baiyao (5 mg/mL) used in the clinics were used as positive control. All the samples were dissolved in blank solvent. The concentrations of compounds were 5 mg/mL and all the extract samples were 15 mg/mL. PT, APTT, TT and FIB tests were conducted with Semi-Automated Coagulation Analyzer (CPC Diagnostics Pvt. Ltd, India).

Statistical analysis
The results of coagulation activity were expressed as mean ± standard deviation. The data analysis was performed by SPSS19.0 software with single factor analysis of variance (ANOVA One-Way) to determine the significant difference. The difference between groups with P < 0.05 and P < 0.001 were regarded as significant and highly significant, respectively. Results were shown in Table 1.

Chemical constituents in S. oblate flowers
Fifteen known compounds (1-15) were isolated and identified from S. oblata flowers. The structures of compounds were shown in Fig. 1. The above spectral data were basically consistent with those reported previously [13] and thus, compound 1 was identified as quercetin.   The above data were basically consistent with those reported in the Ref. [14]. Thus, compound 2 was identified as kaempferol-3-O-α-l-rhamnosyl-(1 → 6)-β-dglucoside (kaempferol-rutinose).   The above data were basically consistent with those reported in the Ref. [18]. Thus, the compound 6 was identified as dictamnoside A. .88 (C-β). The above data were basically consistent with those reported in the Ref. [19]. Thus, the compound 7 was identified as Lilacoside.  17.77 (C-6′′′). The above data were basically consistent with those reported in the Ref. [21]. Thus, the compound 9 was identified as rutin.  C-16). The above data were basically consistent with those reported in the Ref. [22]. Thus, the compound 10 was identified as palmitic acid. .99 (C-4-9), 32.14 (C-10), 22.96 (C-11), 14.29 (C-12). The above data were basically consistent with those reported in the Ref. [23]. Thus, the compound 11 was identified as lauric acid.  3.76 (C-30). The above data were basically consistent with those reported in the Ref. [16]. Thus, the compound 12 was identified as oleanolic acid. The above data were basically consistent with those reported in the Ref. [24]. Thus, the compound 14 was identified as naringenin.
Compound 15 A white powder. The molecular formula was C 35 H 60 O 6 . EI-MS m/z: 578[M] + . It was compared with reference substance of β-daucosterol, no difference was seen between them in term of the TLC detection. Thus compound 15 was identified as β-daucosterol.

Coagulation time test in vitro
In Fig. 2, water part, lauric acid and kaempferol-rutinose could significantly shorten PT (P < 0.001) compared with the blank group. The 40% ethanol part, 90% ethanol part and So.TE had significant anticoagulant activity (P < 0.001 and 0.001 < P<0.01) compared with the blank group. The effects of water part, lauric acid and kaempferol-rutinose were not different with that of Yunnan Baiyao.
In the Fig. 3, all the samples except 60% ethanol part and dictamnoside A could significantly shorten TT Fig. 2 The effects of S. oblata extract and compounds on PT in vitro (P < 0.001 and 0.001 < P < 0.01) compared with the blank group. The procoagulant activity of 20% ethanol part was the best one (P < 0.001) compared with the Yunnan Baiyao.
In Fig. 4, water part, 20% ethanol part, 40% ethanol part, 90% ethanol part, So, TE, lauric acid and kaempferol-rutinose could significantly shorten APTT (P < 0.001) compared with the blank group. Water part, 20% ethanol part, 40% ethanol part, 90% ethanol part, So.TE, lauric acid and kaempferol-rutinose had procoagulant activity compared with the Yunnan Baiyao, and 20% ethanol part had a higher activity than that of Yunnan Baiyao, while the others were not better than that of Yunnan Baiyao.
In Fig. 5, water part, 20% ethanol part, 40% ethanol part, 90% ethanol part, So.TE and lauric acid all could significantly increase the FIB content (P < 0.001) compared with the blank group. The procoagulant activity of the positive control was the best one (P < 0.001) compared with the Yunnan Baiyao.
The previous studies on S. oblate flowers were mainly focused on the volatile components and its traditional pharmacological activity. In the present study we found that the S. oblate flowers had a significant procoagulant activity for the first time. Our researches showed that water part, lauric acid and kaempferol-rutinose all displayed a significant procoagulant activity, and that the procoagulant activity of water part, lauric acid, and kaempferol-rutinose were not better than that of Yunnan Baiyao, which was used as the positive control.

Conclusions
In the present study, fifteen compounds were isolated and identified from S. oblate flowers, including triterpenic acids, fatty acids and flavonone glycosides etc. Water extract of S. oblate flowers, lauric acid and kaempferolrutinose possessed the procoagulant activity.