Isolation of Antithrombotic Phenolic Compounds from the Leaves of Crataegus pinnatifida

• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Four novel phenolic compounds (1–4) were isolated from the leaves of Crataegus pinnatifida, along with three known ones (5–7). Their structures were elucidated as: methyl 4-O-β-D-glucopyranosyl-3-[(2E,6E)-8-O-β-D-glucopyranosyl-3,7-dimethyl-2,6-octadienyl] benzoate (1), biphenyl-5-ol-3-O-β-D-glucoside (2), 3,4′-dimethoxy-biphenyl-5-ol-4-O-β-D-glucoside (3), (E)-6-(benzoyloxy)-1-hydroxyhex-3-en-2-O-β-D-glucoside (4), shanyenoside A (5), eriodectyol (6), and 2″-O-rhamnosyl vitexin (7), using a combination of mass spectroscopy, 1D and 2D NMR spectroscopy, and chemical analysis. The antithrombotic activity of the isolated compounds was investigated on the transgenic zebra fish system. Among them, eriodectyol (6) potently inhibited the production of thrombus.

Hawthorn, well-known as both a medicine and food material, is the common name of the plants in the genus Crataegus of the Rosaceae family, which are distributed in the Northern Hemisphere, mostly in China, Japan, South Korea, Europe, and North America. More than 1000 species of the genus have been described [1], [2]. Currently, hawthorn leaves, flowers, and both green (unripe) and red (ripe) berries are used to make herbal preparations to treat patients with severe heart diseases, such as congestive heart failure, high blood pressure, hypoxia, and hyperlipemia [3], [4]. Aqueous alcohol extracts of hawthorn fruits and leaves are used as dietary supplements and herbal medicines in the United States and Europe for treating heart failure degrees I–III according to the classification of the New York Heart Association (NYHA) (11) [5]. Fruits and leaves of hawthorn are rich in phenolic compounds, which are considered the key bioactive compounds of this plant, offering antioxidative, free radical scavenging, anti-inflammatory, vasorelaxing, and hypolipidemic effects [6], [7]. Previous investigations on the leaves of Crataegus pinnatifida Bge. by our group have resulted in the isolation and characterization of phenols, lignans, and terpenoids. In order to isolate the minor active constituents, we reinvestigated this plant and obtained four novel phenolic compounds (1–4) from the leaves of C. pinnatifida, along with three known ones (5–7) ([Fig. 1]).

Compound 1 was obtained as colourless needles, and its molecular formula was determined as C₃₀H₄₄O₁₄ by HRESIMS at m/z 651.2624 [M + Na]⁺ (calcd. 651.2623). The ¹H NMR spectrum ([Table 1]) showed the presence of two methyl groups attached to the quaternary carbon at δ 1.60 (3H, s), 1.68 (3H, s), a methoxyl group attached to the carbonyl group at δ 3.80 (3H, s), a 1,3,4-trisubstituted benzene indicated by the signals at δ 7.16 (1H, d, J = 8.4 Hz), 7.76 (1H, br.d, J = 8.4 Hz), 7.71 (1H, br.s, J = 8.4 Hz), two olefinic groups at δ 5.33 (1H, m), 5.40 (1H, m), and two anomeric proton signals at δ 4.08 (1H, d, J = 7.8 Hz) and 4.94 (1H, d, J = 7.8 Hz). The ¹³C NMR spectrum exhibited two methyls at δ 14.6, 16.6, one methoxyl at δ 52.6, two olefinic groups at δ 132.5, 127.8, 136.9, 122.4, six aromatic carbons at δ 159.6, 114.7, 129.4, 123.3, 130.8 × 2, and one carbonyl carbon at δ 166.8, in addition to the sugar part. Further analysis of 2D NMR spectra of 1 established the novel skeleton as in [Fig. 1]. The ¹³C NMR chemical shifts and spin-spin coupling constants (7.8, 7.8 Hz) of two anomeric protons allowed the identification of two β-glucopyranosyl moieties [8]. The absolute configuration of the sugar part in 1 was determined by acid hydrolysis. The glucose isolated from the acid hydrolysis of 1 gave a positive specific rotation [α]_D ²⁰ = +15.0 (c = 0.80, H₂O), indicating that it was D-glucose [9]. This fact was further confirmed by comparing its R_f value with the authentic sample. Both the glucose isolated and authentic sample exhibited the same R_f value (0.45) in the same condition. The glycosidic sites were established unambiguously by an HMBC experiment in which long-range correlations between H-1' (δ _H 4.08) and C-1 (δ _C 74.4), H-1'' (δ _H 4.94) and C-10 (δ _C 159.6) were observed ([Fig. 2]). In addition, the geometry of the 2,3- and 6,7-double bonds were both assigned to be E on the basis of high-field olefinic methyl resonances at δ _C 14.6 (C-15) and 16.6 (C-16) [10]. Consequently, the structure of compound 1 was established as methyl 4-O-β-D-glucopyranosyl-3-[(2E,6E)-8-O-β-D-glucopyranosyl-3,7-dimethyl-2,6-octadienyl]benzoate, and given the trivial name shanyenoside E.

Compound 2 was obtained as amorphous powder. The molecular formula was established as C₁₈H₂₀O₇ by HRESIMS, which showed a quasimolecular ion peak [M + Na]⁺ at m/z 371.1104 (calcd. 371.1101). The ¹H NMR spectrum ([Table 1]) showed three proton signals of a phenyl ring (1,3,5-substitution pattern) at δ 6.75 (1H, br.s), 6.46 (1H, br.s), and 6.68 (1H, br.s), five aromatic protons of a single phenyl ring (single substitution pattern) at δ 7.58 (1H, d, J = 8.4 Hz), 7.42 (2H, t, J = 8.4 Hz), 7.33 (2H, t, J = 8.4 Hz) and one anomeric proton signal at δ 4.86 (1H, d, J = 7.2 Hz). The ¹³C NMR spectrum exhibited twelve aromatic carbons of two phenyl rings at δ 142.8, 106.4, 159.7, 103.6, 159.3, 108.3, 140.9, 127.3 × 2, 128.3, 129.6 × 2, and a glucopyranosyl group at δ 101.2, 74.0, 77.8, 70.5, 77.6, 61.3, which revealed that 2 had a skeleton of biphenyl. Further demonstration of the planar skeleton structure of 2 was obtained from the HMBC experiment ([Fig. 2]). The correlation between the signal at δ 159.7 (C-3) and δ 4.86 (H-1′) indicated that the glucopyranosyl group was located at C-3. β-Stereochemistry of anomeric carbon was determined by the coupling constant (J = 7.2 Hz) of the anomeric proton [8], [11]. The absolute configuration of glucose was determined by acid hydrolysis and comparison with an authentic sample [9]. Therefore, the structure of 2 was identified as biphenyl-5-ol-3-O-β-D-glucoside and given the trivial name shanyenoside F.

Compound 3 was obtained as colourless needles. According to the HRESIMS, ¹H, and ¹³C NMR spectroscopic data, the molecular formula of 3 was determined to be C₂₀H₂₄O₉, [M + Na]⁺ at m/z 431.1314 (calcd. 431.1313). The ¹H NMR spectrum ([Table 1]) showed proton signals of two methoxyl groups at δ 3.78 (3H, s), 3.81 (3H, s), two aromatic proton signals of a phenyl ring (1,3,4,5-substitution pattern) at δ 6.68 (1H, br.s) and 6.69 (1H, br.s), four aromatic protons of a phenyl ring (1,4 substitution pattern) at δ 7.54 (2H, d, J = 8.4 Hz), 6.98 (2H, d, J = 8.4 Hz), and one anomeric proton signal at δ 4.60 (1H, d, J = 7.2 Hz). The ¹³C NMR spectrum exhibited twelve aromatic carbons of two phenyl rings at δ 137.9, 107.9, 153.8, 134.0, 151.4, 103.3, 133.2, 128.4 × 2, 159.5, 114.9 × 2, and a glucopyranosyl group at δ 105.9, 74.6, 77.9, 70.3, 76.9, 61.2, which suggested that 3 had the same skeleton as 2. Further demonstration of the planar skeleton structure of 3 was obtained from the HMBC experiment ([Fig. 2]). The correlation between the signal at δ 134.0 (C-4) and δ 4.60 (H-1′) indicated that the glucopyranosyl group was located at C-4. β-Stereochemistry of anomeric carbon was determined by the coupling constant (J = 7.2 Hz) of the anomeric proton [8], [11]. Furthermore, the absolute configuration of glucose was determined by acid hydrolysis and comparison with an authentic sample [9]. Therefore, the structure of 3 was identified as 3,4′-dimethoxy-biphenyl-5-ol-4-O-β-D-glucoside and given the trivial name shanyenoside G.

Compound 4 was isolated as amorphous powder, and its molecular formula was determined to be C₁₉H₂₆O₉, based on HRESIMS (experimental m/z [M + Na]⁺ at m/z 421.1467, calcd. 421.1469). The ¹H NMR spectrum ([Table 1]) showed the presence of five aromatic protons of a single phenyl ring (single substitution pattern) at δ 7.97 (2H, dd, J = 8.4, 1.2 Hz), 7.52 (2H, t, J = 8.4 Hz), 7.65 (1H, t, J = 8.4 Hz), and one trans olefin group at δ 5.47 (2H, m, J = 17.4 Hz), 5.54 (1H, m, J = 17.4 Hz). In the sugar part, one anomeric proton signal at δ 4.15 (1H, J = 7.8 Hz) and the ¹³C NMR signals of sugar showed the existence of a glucopyranosyl moiety ([Table 1]). This assignment was confirmed by detailed analysis of the HSQC and HMBC spectra. The HMBC correlation between the signal at δ 74.0 (C-2) and δ 4.15 (H-1′) indicated that the glucopyranosyl group was located at C-2. The β-form of glucose was indicated by the coupling constant of the anomeric proton signal at δ _H 4.15 (1H, d, J = 7.8 Hz) in the ¹H NMR data [10]. The absolute configuration of glucose was further determined by acid hydrolysis and comparison with an authentic sample [9]. Thus, the structure of compound 4 was established as (E)-6-(benzoyloxy)-1-hydroxyhex-3-en-2-O-β-D-glucoside and given the trivial name shanyenoside H.

By comparing physical and spectroscopic data with those from the literature, the three known constituents were identified as shanyenoside A (5) [12], eriodectyol (6) [13], and 2″-O-rhamnosyl vitexin (7) [14].

The small size (3–4 cm long), ease of care (easily kept and bred in the laboratory), and rapid generation time (produce 200 eggs per spawning and reach sexual maturity within 2–3 months) are but a few of the aspects making zebrafish a particularly useful model organism [15], [16], [17], [18], [19], [20]. All isolated compounds were tested in vivo for their antithrombotic activity on transgenic zebra fish system. Among them, compound 6 showed significant antithrombotic activity. Compared with the control group, the forming thrombosis time of the tested zebra fish was obviously prolonged, which indicated that compound 6 (eriodectyol) potently inhibited the production of thrombus (* p < 0.05) ([Fig. 3]).

Materials and Methods

The leaves of C. pinnatifida were collected from Liaoning province, China, in June 2007, and authenticated by Prof. Qi-Shi Sun, Department of Pharmacognosy, Shenyang Pharmaceutical University. A voucher specimen (No. CPLN0706) is kept in the Nature Products Laboratory of the Shenyang Pharmaceutical University, Shenyang, China.

Air-dried leaves (5.0 kg) of C. pinnatifida were extracted with 70 % ethanol (30 L) by reflux (95 °C, 2 h) three times. The ethanol extract was concentrated in vacuo to yield a brownish-dark crude extract (492 g) which was subjected to macroporous resin (0.25–0.84 mm, 10 kg) column chromatography (17 × 150 cm) eluted with EtOH/H₂O (v/v = 0 : 100, 30 : 70, 50 : 50, 95 : 5) to yield 4 main fractions: Frs. A–D, according to chemical monitoring by TLC. Fr. B (120 g) was further separated by column chromatography (10 × 80 cm) on Diaion HP-20 eluted with MeOH/H₂O (v/v = 60 : 40, 70 : 30, 80 : 20, 100 : 0) to provide subfractions B1–B4, among which Fr. B1 (15 g) was purified by silica gel (200–300 mesh, 80 g) column chromatography (5 × 50 cm) eluting with a CH₂Cl₂-MeOH gradient system (v/v = 30 : 1, 20 : 1, 10 : 1, 5 : 1, and 0 : 1) to yield compounds 1 (12 mg) and 7 (500 mg) which were further purified over Sephadex LH-20 column chromatography (2.5 × 70 cm) eluting with MeOH : H₂O (2 : 3). Subfraction B2 (20 g) was applied to MCI gel column chromatography (4 × 40 cm) eluting with MeOH : H₂O (v/v = 0 : 100 → 100 : 0) and further purified over Sephadex LH-20 column chromatography (2.5 × 100 cm) eluting with MeOH : H₂O (1 : 1), followed by preparative HPLC (65 % MeOH) to yield compounds 2 (t _R 31.6 min, 11 mg), 3 (t _R 38.5 min, 28 mg), and 5 (t _R 40.2 min, 58 mg). Fr. B3 (10 g) was then combined on the basis of TLC analysis and finally purified by reversed-phase C18 silica gel (70 µm, 50 g) column chromatography (4 × 40 cm) using MeOH/H₂O with a gradient solvent system of (v/v = 30 : 70 → 90 : 10) and HPLC (75 % MeOH) to obtain compounds 4 (t _R 20.2 min, 10 mg) and 6 (t _R 23.2 min, 12 mg). The purities of all isolates (> 95.0 %) were analysed by HPLC.

Shanyenoside E (1) was obtained as colourless needles. HRESIMS m/z 651.2624 [M + Na]⁺ (calcd. for C₃₀H₄₄O₁₄Na, 651.2623). For ¹H and ¹³C NMR spectroscopic data, see [Table 1].

Shanyenoside F (2) was obtained as amorphous powder. HRESIMS m/z 371.1104 [M + Na]⁺ (calcd. for C₁₈H₂₀O₇Na 371.1101). For ¹H and ¹³C NMR spectroscopic data, see [Table 1].

Shanyenoside G (3) was obtained as colourless needles. HRESIMS m/z 431.1314 [M + Na]⁺ (calcd. for C₂₀H₂₄O₉Na 431.1313). For ¹H and ¹³C NMR spectroscopic data, see [Table 1].

Shanyenoside H (4) was isolated as amorphous powder. HRESIMS m/z 421.1467 [M + Na]⁺ (calcd. for C₁₉H₂₆O₉Na, 421.1469). For ¹H and ¹³C NMR spectroscopic data, see [Table 1].

Supporting information

General experimental procedures, detailed extraction/isolation of compounds, NMR and HRESIMS spectra of novel compounds, as well as protocols for the in vivo antithrombotic assay on the transgenic zebra fish system are available as Supporting Information.

Acknowledgements

This research was supported by the China National Fundamental Fund of Personnel Training (No. J1103606) and the China State Key Laboratory Open Project of Bioactive Substance and Function of Natural Medicines. The authors wish to thank Professor Qi-Shi Sun (Shenyang Pharmaceutical University, Shenyang, Peopleʼs Republic of China) for identification of the plant material.

Conflict of Interest

All authors declare that there are no conflicts of interest.

• References

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Correspondence

• References

• 1 Liu PZ, Yang BR, Kallio HK. Characterization of phenolic compounds in Chinese hawthorn (Crataegus pinnatifida Bge. var. major) fruit by high performance liquid chromatography–electrospray ionization mass spectrometry. Food Chem 2010; 121: 1188-1197
  CrossrefPubMedSearch in Google Scholar
• 2 Min BS, Huong HT, Kim JH, Jun HJ, Na MK, Nam NH, Lee HK, Bae K, Kang SS. Furo-1,2-naphthoquinones from Crataegus pinnatifida with ICAM-1 expression inhibition activity. Planta Med 2004; 70: 1166-1169
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Liu TX, Cao YN, Zhao MM. Extraction optimization, purification and antioxidant activity of procyanidins from hawthorn (C. pinnatifida Bge. var. major) fruits. Food Chem 2010; 119: 1656-1662
  CrossrefPubMedSearch in Google Scholar
• 4 Ding XP, Wang XT, Chen LL, Qi J, Xu T, Yu BY. Quality and antioxidant activity detection of Crataegus leaves using on-line high-performance liquid chromatography with diode array detector coupled to chemiluminescence detection. Food Chem 2010; 120: 929-933
  CrossrefPubMedSearch in Google Scholar
• 5 Liu PZ, Kallio HK, Lü DG, Zhou CS, Ou S, Yang B. Acids, sugars, and sugar alcohols in Chinese hawthorn (Crataegus spp.) fruits. J Agric Food Chem 2010; 58: 1012-1019
  CrossrefPubMedSearch in Google Scholar
• 6 Liu PZ, Kallio HK, Yang B. Phenolic compounds in hawthorn (Crataegus grayana) fruits and leaves and changes during fruit ripening. J Agric Food Chem 2011; 59: 11141-11149
  CrossrefPubMedSearch in Google Scholar
• 7 Park Y, Choi S, Hwang S, Lee K. Changes of phenolics and antioxidant activity during hawthorn (Crataegus pinnatifida Bunge) fruit ripening. Planta Med 2010; 76: 341
  PubMedSearch in Google Scholar
• 8 Sun YJ, Li ZL, Chen H, Liu XQ, Zhou W, Hua HM. Three new cytotoxic aryltetralin lignans from Sinopodophyllum emodi . Bioorg Med Chem Lett 2011; 21: 3794-3797
  CrossrefPubMedSearch in Google Scholar
• 9 Mondol MA, Kim JH, Lee HS, Lee YJ, Shin HJ. Macrolactin W, a new antibacterial macrolide from a marine Bacillus sp. Bioorg Med Chem Lett 2011; 21: 3832-3835
  CrossrefPubMedSearch in Google Scholar
• 10 Wu H, Nakamura H, Kobayashi J, Kobayashi M, Ohizumi Y, Hirata Y. Structures of agelasines, diterpenes having a 9-methyl-adeninium chromophore isolated from the Okinawan marine sponge Agelas nakamurai Hoshino. Bull Chem Soc Jpn 1986; 59: 2495-2504
  CrossrefPubMedSearch in Google Scholar
• 11 Agrawal PK. NMR Spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 1992; 31: 3307-3330
  CrossrefPubMedSearch in Google Scholar
• 12 Chen J, Song SJ, He J, Xu SX. A new biphenyl glucoside from the leaves of Crataegus pinnatifida . J Shenyang Pharm Univ 2006; 23: 430-431
  PubMedSearch in Google Scholar
• 13 Liang YH, Ye M, Han J, Wang BR, Guo DA. Lignans and flavonoids from rhizome of Drynaria fortunei . Chin Tradit Herbal Drugs 2011; 42: 25-30
  PubMedSearch in Google Scholar
• 14 Sun JY, Yang SB, Xie HX, Li GH, Qiu HX. Studies on chemical constituents from fruit of Crataegus pinnatifida . Chin Tradit Herbal Drugs 2002; 33: 483-486
  PubMedSearch in Google Scholar
• 15 Finley KR, Zon LI. Zebra fish and stem cell research. In: Lanza R, editor Handbook of stem cells, Vol. 2. Amsterdam: Elsevier; 2004: 677-683
  CrossrefSearch in Google Scholar
• 16 Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharm Biochem Behav 2000; 67: 773-782
  CrossrefPubMedSearch in Google Scholar
• 17 Jagadeeswaran P, Gregory M, Day K, Cykowski M, Thattaliyath B. Zebrafish: a genetic model for hemostasis and thrombosis. J Thromb Haemost 2005; 3: 46-53
  CrossrefPubMedSearch in Google Scholar
• 18 OʼConnor MN, Salles II, Cvejic A, Watkins NA, Walker A, Garner SF, Jones CI, Macaulay IC, Steward M, Zwaginga JJ, Bray SL, Dudbridge F, Bono BD, Goodall AH, Deckmyn H, Stemple DL, Ouwehand WH. Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood 2009; 113: 4754-4762
  CrossrefPubMedSearch in Google Scholar
• 19 Zon LI, Peterson RT. In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 2005; 4: 35-44
  CrossrefPubMedSearch in Google Scholar
• 20 Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990; 60: 269-280
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material