Dendrobium, one of the largest genera in the Orchidaceae family with more than 1100 species identified, is widely distributed throughout Asia, Europe, and Australia [1]. There are 75 species and two varieties of Dendrobium in China [2], and the stems of many species are used as “Shi-Hu” in traditional Chinese medicines to nourish the stomach, promote the production of body fluid, and reduce fever. Dendrobium primulinum Lindl. is distributed in southwest of China, India, Nepal, Burma, Laos, Vietnam, and Thailand and is widely used as a medicinal Dendrobium plant in “Dai Medicine.” Previous phytochemical studies on D. primulinum have revealed the presence of a series of compounds such as alkaloids, nucleosides, and flavonoids [3, 4]. Our further investigation on this plant species led to the isolation of a novel 9,10-dihydrophenanthrene glycoside (1) and 11 known compounds 2–12.

Compound 1 was obtained as a white amorphous powder from MeOH with [α] 25D –27° (c 0.3; MeOH). The molecular formula of C20H22O9 was deduced from the positive HR-ESI-MS m/z 429.1160 [M + Na]+ (calcd 429.1162) and its NMR data (Table 1), indicating 10 degrees of unsaturation. The negative-ion ESI-MSn data showed m/z 243 [M – H – 162]–, suggesting the presence of one hexose. Its UV maxima absorptions at 211.2, 270.2, and 296.4 nm were similar to those of 9,10-dihydrophenanthrene derivatives [5]. The IR spectrum showed absorption bands for hydroxyl (3396 cm–1) and aromatic (1618, 1451 cm–1) groups. The aromatic region of the 1H NMR spectrum showed three adjacent aromatic protons at δ 7.14 (1H, t, J = 7.8 Hz), 7.05 (1H, d, J = 7.8 Hz), and 6.84 (1H, d, J = 7.8 Hz) and a pair of meta-coupled protons at δ 6.62 (1H, d, J = 1.8 Hz) and 6.50 (1H, d, J = 1.8 Hz), indicating a 1,2,3-trisubstituted benzene ring and a 1,3,4,5-tetrasubstituted benzene ring, respectively. In the aliphatic region, two methylene protons at δ 2.72 (1H, dd, J = 14.4, 3.6 Hz) and 2.62 (1H, dd, J = 14.4, 9.6 Hz) and one oxygenated methine proton at δ 4.48 (1H, dd, J = 9.6, 3.6 Hz) were attributed to a -OCHCH2 group by 1H–1H COSY correlations, and one anomeric signal at δ 5.13 (1H, d, J = 7.8 Hz) and six protons in the range 3.23–3.72 were due to a sugar residue. The 13C NMR and DEPT spectrum confirmed the presence of 12 aromatic carbons (three bearing oxygen atoms), one oxygenated methine carbon at δ 67.7, one methylene carbon at δ 39.7, together with six hexose carbons. In addition, based on the IR absorption at 3396 cm–1 and its molecular formula and oxygenated C-atoms (δ 157.7, 153.1, 153.0), three hydroxyl groups were assumed. Acid hydrolysis of 1 afforded D-glucose, which was identified by GC analysis, and the J value of the anomeric proton δ 5.13 (1H, d, J = 7.8 Hz) indicated a β-configuration. In the HMBC spectrum, the anomeric proton (H-1′) showed correlation with C-4, indicating the attachment of β-D-glucose to C-4. The NOESY correlations between H-1′ to H-3 and H-5′ to H-3 confirmed the location of glucose (Fig. 1).

Table 1 1H (600 MHz), 13C NMR (150 MHz) Data, and HMBC Correlations of Compound 1 (DMSO-d6+ CD3OD, δ, ppm, J/Hz)
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Fig. 1
figure 1

Structure of compound 1 and its HMBC and NOESY correlations.

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The CD spectrum of 1 showed a negative Cotton effect at 233 nm and a positive Cotton effects at 268 nm, which were in agreement with that of (+)-(9S)-hydroxy-9,10-dihydrophenanthrene [6]. Therefore, the structure of compound 1 was deduced as 2,4,5,9S-tetrahydroxy-9,10-dihydrophenanthrene 4-O-β-D-glucopyranoside.

The known compounds were identified as 2,4,7-trihydroxy-9,10-dihydrophenanthrene (2) [7], denthyrsinol (3) [8], moscatin (4) [9], moscatilin (5) [10], gigantol (6) [11], batatasin III (7) [12], tristin (8) [13], 3,4′,5-trihydroxybibenzyl (9) [14], 3,6,9-trihydroxy-3,4-dihydroanthracen-1(2H)-one (10) [15], β-sitosterol (11) [16], and β-daucosterol (12) [17] by comparing their spectroscopic data with literature data.

All the above compounds were isolated from this plant for the first time.

Experimental

General Procedures. Optical rotations were taken on a PerkinElmer 341 polarimeter. IR spectra were carried out on a Nicolet-Nexus 670 infrared spectrophotometer with KBr pellet. NMR spectra were run on a Bruker AV-600 NMR spectrometer using TMS as an internal standard. ESI-MS and HR-ESI-MS were recorded on a Waters Micromass Q-TOF micro mass spectrometer. The CD spectra were obtained on a Chirascan spectrometer. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 silicagel (ODS, 50 μm, YMC), and Sephadex LH-20 (Pharmacia).

Plant Material. The whole plant of D. primulinum was collected in Longling, Yunnan Province, China, in November 2012, and identified by Prof. Xinjia Ming, Chongqing Academy of Chinese Materia Medica. A voucher specimen (No. 20121101) was deposited at the Shanghai Institute of Pharmaceutical Industry, Shanghai, China.

Extraction and Isolation. The air-dried and powdered stems of D. primulinum (4 kg) were extracted with 95% ethanol (four times, each 40 L) at room temperature. Evaporation of the solvent under reduced pressure afforded a brown crude extract (300 g). The extract was suspended in water and then partitioned with petroleum ether (60–90°C), EtOAc, and n-BuOH successively to obtain four fractions. The EtOAc fraction (40 g) was subjected to silica gel column chromatography and eluted with CH2Cl2–MeOH (100:1, 50:1, 30:1, 15:1, 10:1, 0:1) to yield six fractions (Fr. 1–Fr. 6). Fraction 2 (11.8 g) was isolated by silica gel column chromatography with CH2Cl2–acetone (40:1–10:1) to give six subfractions (Fr. 2.1–Fr. 2.6). Fraction 2.2 (3.0 g) was rechromatographed on silica gel column with petroleum ether–acetone (5:1–3:1) and then purified over a Sephadex LH-20 column with CH2Cl2–MeOH (1:1) and recrystallized (petroleum ether–acetone) to afford compounds 4 (112 mg), 5 (210 mg), 6 (738 mg), and 11 (80 mg). Fraction 2.4 (4.2 g) was further subjected to silica gel column chromatography and eluted with petroleum ether–EtOAc (3:1–1:1) and then purified over a Sephadex LH-20 column with CH2Cl2–MeOH (1:1) and preparative TLC to obtain compounds 3 (9 mg) and 8 (170 mg). By the same method, compound 7 (103 mg) was obtained from Fr. 3 (4.2 g), compounds 2 (30 mg) and 9 (20 mg) were obtained from Fr. 4 (4.5 g), and compound 10 (6 mg) was obtained from Fr. 5 (3.3 g). The n-BuOH fraction (42 g) was subjected to silica gel column chromatography and eluted with CH2Cl2–MeOH–H2O (30:1:0, 15:1:0, 10:1:0, 4:1:0.1, 7:3:0.5, 0:1:0) to yield six fractions (Fr. I–VI). Fraction III (7.4 g) was subjected to Sephadex LH-20 column chromatography and eluted with EtOH and recrystallized (MeOH) to give compound 12 (19 mg). Fraction IV (10.5 g) was subjected to ODS column chromatography and eluted with a step gradient of MeOH–H2O (1:9–6:4) to yield nine subfractions (Fr. IV.1–Fr. IV.9). Fraction IV. 5 was purified over a Sephadex LH-20 column with EtOH and silica gel column chromatography with CH2Cl2–MeOH–H2O (6:1:0.1) to obtain compound 1 (43 mg).