Melting point was determined on an Electrothermal 9100 melting point apparatus and was uncorrected. The ¹H-NMR spectra (δ ppm, J in Hz) was recorded in DMSO-d₆ on a Brucker AM-500 spectrometer (500 MHz), while ¹³C-NMR spectra was recorded in the same solvent on a Brucker AM-500 spectrometer at 125 MHz with tetramethylsilane (TMS) as an internal standard.

The heartwood of A. tegimentosum was collected in Pyongchang-gun, Gangwon province in Korea and dried. This plant was identified by Dr. Won-Bae Kim of Highland Agriculture Institute, Rural Development Administration of Korea. Heartwoods of A. tegmentosum (2 kg) were extracted with MeOH (each, 3.0 L) for 5 h three times under reflux. The extracted solution was filtered and concentrated under reduced pressure to give a viscous MeOH extract, which was freeze-dried to yield a powdery MeOH extract. A portion (118.7 g) of the MeOH extract was suspended in 800 ml distilled water and partitioned three times with CHCl₃ (each, 800 ml). The CHCl₃-soluble portion was dried in vacuo to yield the CHCl₃ fraction. The H₂O layer was successively fractionated with BuOH (each, 800 ml), and the BuOH-soluble portion was dried in vacuo to yield a BuOH fraction. Since BuOH fraction exhibited the most potent anti-nociceptive andti-gastropathic activity, it was chromatographed for isolation.

Ten grams of the BuOH fraction was subjected to silica gel column (SiO₂, Art No. 7734, Merck, Germany, 280 g, 5 × 55 cm) chromatography and gradiently eluted with CHCl₃-MeOH {from 1 L of 9:1 (v/v) to 2.5L of 6:4 (v/v)}. The eluate was collected to give seven fractions (I – VII) and then monitored by checking TLC. Fraction II was dried in vacuo and crystallized from MeOH to yield a white amorphous powder (2.5 g, compound 1, Fig.1). Fractions VI and VII were crystallized from MeOH to yield a white amorphous powder that was identified as sucrose by TLC, ¹H-NMR and ¹³C-NMR.

Fig. (1) Chemical structure of salidroside.

Compound 1 – Colorless powder, mp 220(C; ¹H-NMR(500 MHz, DMSO-d₆) (: 4.17 (1H, d, J=7.5 Hz, H-1′), 6.67 (2H, d, J=8.5 Hz, H-2.6), 7.04 (2H, d, J=3,5 Hz); ¹³C-NMR (125.5 MHz, DMSO-d₆) (: 35.3 (C-7), 61.5(C-6′), 70.3 (C-8), 70.5(C-4′), 73.8 (C-2′), 77.1 (C-3′), 77.3 (C-5′, 103.3 (C-1′), 115.4 (C-2,5), 129.2 (C-1), 130.2 (C-3,5), 155.9 (C-4); FAB-MS m/z 323.2 [M + Na]⁺.

Male ICR mice were purchased from Daehan Bio Link Co. and allowed to adapt to laboratory conditions (temperature: 20 ± 2(C, relative humidity: 40-60%, light/dark cycle: 12 h) for two weeks. Mice weighing 25 ± 2 g were used for experiments. Number of mice was 9 per group. For twenty-four hours before the experiment, the animals were offered only water. Considering that enzyme activities can vary throughout each day, the animals were sacrificed at a fixed time (10:00 A.M.-12:00 P.M.). All experiments were approved by the University of Kyungsung Animal Care and Use Committee. All procedures were conducted in accordance with the “Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health.

Acetic Acid-Induced Writhing Method:The MeOH extract of A. tegmentosum heartwood, its fractions (100 or 200 mg/kg) or compound 1 (10 or 20 mg/kg) were orally administered to the mice daily for one week, and the mice were then injected intraperitoneally (i.p.) with 10 mg/kg of 0.8% acetic acid (9 mice/experimental group) [12Jung HJ, Choi J, Nam JH, Park HJ. Anti-ulcerogenic effects of effects of the flavonoid-rich fraction from the extract of Orostachys japonicus J Med Food 2007; 10: 702-6.
[http://dx.doi.org/10.1089/jmf.2006.223] [PMID: 18158844] ]. The number of abdominal contractions in a 20-min period was then counted. Aspirin (100 mg/kg, p.o.) was used as a positive control. A significant reduction in the number of abdominal contractions compared to the control was considered a positive analgesic response.

Hot Plate Method: Mice (9 mice/experimental group) were administered the MeOH extract, its fractions (100 or 200 mg/kg daily), or compound 1 (10 or 20 mg/kg daily) for 1 week. One h after the final administration, the mice were placed on a hot-plate (Ugo Basile, Comerio, Italy) maintained at 70°C. The time until each animal licked a fore or hind paw or jumped off the plate was defined as the reaction time [12Jung HJ, Choi J, Nam JH, Park HJ. Anti-ulcerogenic effects of effects of the flavonoid-rich fraction from the extract of Orostachys japonicus J Med Food 2007; 10: 702-6.
[http://dx.doi.org/10.1089/jmf.2006.223] [PMID: 18158844] ]. Morphine (10 mg/kg, p.o.) was used as a reference drug.

HCl/Ethanol-Induced Gastric Lesions in Mice: After oral administration of the test solution, the mice were fasted for 24 hours prior to the experiment. The mice were then given an oral dose of 0.2 mL of 0.3 M HCl in 60% ethanol. After 24 hours the mice were sacrificed, and their stomachs were opened along the greater curvature and fixed in 2% formalin solution for 10 minutes. After the greater curvature was incised, the extent of gastric damage in the glandular region was defined as the ulcerative lesion index [13Mizui T, Dodeuchi M. Effect of polyamines on acidified ethanol-induced gastric lesions in rats Jpn J Pharmacol 1983; 33: 939-45.
[http://dx.doi.org/10.1254/jjp.33.939] [PMID: 6580476] ].

NSAID-Induced Gastric Lesions in Mice:Test solutions including the MeOH extract, its fractions (100 and 200 mg/kg per day), compound 1 (10 and 20 mg/kg per day) and the cimetidine control (100 mg/kg per day) were orally administered to the mice for 2 weeks. The animals were treated with indomethacin (30 mg/kg, s.c.) and bethanechol (5 mg/kg, i.p.), fasted for 1 hour, and then sacrificed. The stomachs were opened along the greater curvature and fixed in 2% formalin solution for 10 minutes. After the greater curvature was incised, the extent of gastric damage in the glandular region was evaluated according to the ulcerative lesion index [14Rainsford KD. A synergistic interaction between aspirin, or other non-steroidal anti-inflammatory drugs, and stress which produces severe gastric mucosal damage in rats and pigs Agents Actions 1975; 5: 553-8.
[http://dx.doi.org/10.1007/BF01972694] [PMID: 1220559] ].

Test solutions were administered as previously described and the mice were fasted for 24 hours. Each mouse was then anesthetized with ether. The abdomen of each anesthetized mouse was opened, and the pylorus was ligated; the abdomen was then closed after the sample solutions had been placed in the duodenal tract. Four hours after sealing-up their abdomens, the mice were anesthetized with ether, the stomachs were excised, and gastric juices were collected. These were centrifuged at 2,500 × g, and then the gastric juice volumes and pH values were measured and total acid output was calculated. The total acid output was determined by titration versus 0.05 N NaOH using phenolphthalein as an indicator [15Dai S, Ogle CW. A simple method for the production of peptic ulceration in the rat Life Sci 1973; 12: 505-12.
[http://dx.doi.org/10.1016/0024-3205(73)90344-5] ].

Results are expressed as means ± SD (n=9). Statistical analysis was performed with Duncan’s multiple range tests. Differences were considered significant at p < 0.05.

The analgesic and anti-gastropathic effects of the MeOH extract of A. tegmentosum heartwood were evaluated in mice since A. tegmentosum has been used to prevent or treat alcohol poisoning in the folkloric medicinal society of Korea. Excessive drinking often causes headache, abdominal pain and gastric disorders. In addition, phytochemical isolation was also performed to identify the biologically active substance in the heartwood.

As shown in Table 1, the MeOH extract of A. tegmentosum heartwoods alleviated pain and had gastropathic action, indicative of why it has been used to treat alcohol poisoning. Among the MeOH extract and its CHCl₃-, BuOH- and H₂O fractions, the BuOH fraction exhibited the most potent effect. Administration of 100 or 200 mg/kg of BuOH fraction significantly lengthened the jumping latency in the hot plate test from 10.3 ± 3.17 seconds for the control group to 15.2 ± 2.41 seconds or 17.9 ± 2.38 seconds, respectively. Control mice had a writhing number of 66.4 ± 2.41 compared to 53.4 ± 2.70 at the 100 mg/kg dose and 45.4 ± 2.88 at the 200 mg/kg dose for the BuOH fraction-treated group. Thus, the A. tegmentosum extracts exhibited central and peripheral analgesic effects in the hot plate test and the writhing test.

Table 1

Anti-Nociceptive Effect of the MeOH Extract of A. tegmentosum Heartwood and Its Fractions in Hot-Plate Test and Acetic Acid-Induced Writhing Syndrome in Mice

Ulcerative indices were determined in mice with HCl/ethanol- and indomethacin/bethanechol-induced ulcers by measuring ulcerative lesion diameter. As shown in Table 2, treatment with 0.3 M HCl and 60% EtOH caused gastric ulcers (diameter 27.4 ± 2.10 mm); however, pretreatment with the MeOH extract (100 and 200 mg/kg, p.o.) for 2 weeks reduced the ulcerative index compared to the control group. Indomethacin, a non-steroidal anti-inflammatory drug, and bethanechol, a cholinergic drug, were also administered to mice to induce gastric ulcers. Treatment with indomethacin/bethanechol caused gastric ulcers (diameter 14.7 ± 0.93 mm), whereas pretreatment with the MeOH extract reduced the ulcerative index compared to the control. Among the CHCl₃-, BuOH- and H₂O fractions, the BuOH fraction had the most potent anti-gastropathic activity. HCl/EtOH caused gastric ulcers (diameter 27.4 ± 2.10 mm), while treatment with the BuOH fraction decreased the diameter to 21.7 ± 3.43 mm and 18.9 ± 2.11 mm at 100 and 200 mg/kg doses, respectively. Furthermore, in the indomethacin/bethanechol-induced gastric ulcer, treatment with the BuOH fraction reduced the ulcerative index from 14.7 ± 0.93 mm in the control group to 10.8 ± 0.39 mm and 9.4 ± 0.50 mm at the 100 and 200 mg/kg doses, respectively.

Table 2

Effect of the MeOH Extract of A. tegmentosum Heartwood and Its Fractions on HCl-Ethanol- and Indomethacin- Bethanechol-Induced Gastric Ulcers in Mice

The volume of gastric juice was measured using the method described by Dai and Ogle [15Dai S, Ogle CW. A simple method for the production of peptic ulceration in the rat Life Sci 1973; 12: 505-12.
[http://dx.doi.org/10.1016/0024-3205(73)90344-5] ]. The pH values and total acid output were also measured. The effects of the MeOH extract and its fractions on gastric secretion in pylorus-ligated mice are shown in Table 3. Oral administration of the MeOH extract resulted in an increase of pH and decreases of gastric juice volume and total acid output in the pylorus-ligated mice at the 100 and 200 mg/kg doses. The BuOH fraction exhibited the most potent effects among the MeOH extract and tested fractions, suggesting that the bioactive compound was contained in the BuOH fraction.

Table 3

Effect of the MeOH Extract of A. tegmentosum Heartwood and Its Fractions on the Biochemical Parameters of Gastric Juice Obtained from Pylorus-Ligated Mice

Phytochemical isolation of the BuOH fraction led to the isolation of compound 1, which was identified as salidroside by comparing its spectroscopic data with the literature [11Park KM, Yang MC, Lee KH, Kim KR, Choi SU, Lee KR. Cytotoxic phenolic constituents of Acer tegmentosum maxim Arch Pharm Res 2006; 29: 1086-90.
[http://dx.doi.org/10.1007/BF02969296] [PMID: 17225455] ] (Fig. 1). As shown in Fig. (2), salidroside exhibited significant analgesic and anti-gastropathic effects at 10 and 20 mg/kg dosages (p.o.). Oral administration of morphine (positive control, 10 mg/kg, p.o.) lengthened the reaction time in the hot plate test by 176%, while 10 and 20 mg/kg dosages of salidroside prolonged the reaction time by 68.0% and 100%, which suggests that it exerts centrally mediated analgesic action. In the writhing test, 100 mg/kg aspirin, used as the positive control, decreased writhing number by 75.6%, which is indicative of peripherally mediated analgesic action, while administration of 10 and 20 mg/kg salidroside inhibited the writhing number by 38.6% and 47.9%, respectively. These results indicate that salidroside has both centrally and peripherally mediated analgesic activity.

Fig. (2) Anti-nociceptive effect of salidroside from A. tegmentosum heartwood in hot-plate test (A) and acetic acid-induced writhing syndrome (B) in mice. Values followed by superscripted letters are significantly different (p < 0.05) by Duncan’s multiple range test.

As shown in Fig. (3), cimetidine (100 mg/kg, p.o.), a positive control, inhibited the gastric lesion caused by HCl/EtOH by 91.4%, while administration of salidroside inhibited the gastric lesion diameter by 51.5% and 65.8% at the 10 and 20 mg/kg dosages, respectively. In the indomethacin/bethanechol-induced gastric lesion, administration of 10 and 20 mg/kg of salidroside reduced the ulcerative index by 31.8% and 38.8%, whereas omeprazol, a positive control, decreased the ulcerative index by 61.8%. These results suggest that salidroside has anti-gastropathic activity. This compound also increased the pH of gastric juice and further decreased the volume of gastric juice and the total acid output, although its activities were weaker than those of cimetidine (Fig. 4).

Fig. (3) Effect of salidroside from A. tegmentosum heartwood on HCl/ethanol- (A) and indomethacin/bethanechol- (B) induced gastric ulcers in mice. Values followed by superscripted letters are significantly different (p < 0.05) by Duncan’s multiple range test.

Fig. (4) Effect of salidroside from A. tegmentosum heartwood on the biochemical parameters of gastric juice obtained from pylorus-ligated mice. Compared among the bars with the same figure, values followed by the same superscripted letters are not significantly different (p < 0.05) by Duncan's multiple range test.