Compound 1 was isolated as an optically active yellowish amorphous powder, [α]_D²⁵−77.0 (c 0.03, MeOH). Its molecular formula was established as C₃₀H₂₂O₁₀ by ESI-MS and HR-ESI-MS, with 20 degrees of unsaturation and a dimer composed of a flavone moiety and a flavanol moiety was proposed. The observed UV absorption peaks at 210, 217 sh, 226, 315 sh, and 335 sh nm in conjunction with a bathochromic shift observed after the addition of aq. KOH suggests the presence of a phenolic flavonoid moiety [7]. The IR spectrum showed absorption bands at 3450 cm⁻¹ for a hydroxy group and at 1647 cm⁻¹ for a carbonyl group. The ¹H- and ¹³C-NMR spectra (Table 1) of 1 showed a trioxygenated flavone moiety [δ_C 160.7 (C-4′), 183.6 (C-4), 163.3 (C-5), and 166.0 (C-7)] with two meta-coupled protons [δ_H 6.18 (1H, d, J = 2.4 Hz, H-6), 6.32 (1H, d, J = 2.4 Hz, H-8)] in ring A and with a para-oxygenated phenyl group [δ_H 6.67 (2H, d, J = 9.0 Hz, H-3′, 5′), 7.26 (2H, d, J = 9.0 Hz, H-2′, 6′)] in ring C; and another 4‴, 3″, 5″, 7″-tetraoxygenated flavan moiety (δ_C 158.1, 68.9, 154.8 and 156.0) with a para-oxygenated phenyl group [δ_H 6.70 (2H, d, J = 9.0 Hz, H-3‴, 5‴), 7.10 (2H, d, J = 9.0 Hz, H-2‴, 6‴)] in ring C′ and an aromatic proton [δ_H 6.04 (1H, s), H-8″] in ring A′. The HMBC spectrum (Figure 2) showed correlations between the singlet H-8″ (δ_H 6.04) and C-6″ (δ_C 100.7) and the H-3 in ring C was lack in flavone moiety. These supported the connection between the flavone moiety and the flavan-3-ol moiety is via C-3 and C-6″ linkage. The molecular formula C₃₀H₂₂O₁₀ supported the presence of seven hydroxy groups, which were confirmed by ¹³C-NMR, HMQC, NOESY and HMBC (Figure 2) experiments. The relative configurations of H-2″ and H-3″ were determined by NOESY spectrum, where H-2″ showed the correlation with H-3″. Comparing with the aglycone levorotatory optical activity of epiafzelechin 5-glucoside [α]_D²⁵ −63 (MeOH) [8], and the absolute configuration of 1 with [α]_D²⁵ −77.0 (c 0.03, MeOH) was suggested to be (2″R:3″R). It was found that when dissolved in different solvents, other compounds with (2″R:3″R) structure, e.g., (−)-epicatechin [9] and (−)-epigallocatechin [10] also displayed levorotatory optical activity [9,10]. Based on the evidence above, the structure of 1 was elucidated to be 5,7-dihydroxy-2-(4′-hydroxyphenyl)-3-(3″R,5″,7″-trihydroxy-2″R-(4‴-hydroxyphenyl)-3″,4″-dihydro-2H-chromen-6″-yl) -4H-chromen-4-one, and named wikstaiwanone A.

Compound 2 was obtained as pale yellowish amorphous powder with [α]_D²⁵ −23.3 (c 0.03, MeOH). Its molecular formula, C₃₀H₂₂O₁₀, was same as 1 and established by ESI-MS and HR-ESI-MS. The IR, UV, and NMR (Table 1) spectra of 2 were similar to those of 1, except that the different resonance of H-2″ (δ_H 4.53 (1H, d, J = 8.2 Hz), H-3″ (δ_H 3.68 (1H, br d, J = 8.2 Hz)) in 2 and H-2″ (δ_H 4.13 (1H, d, J = 7.8 Hz), H-3″ (δ_H 3.90 (1H, ddd, J = 8.4, 7.8, 6.0 Hz)) in 1. This proposed 2 is a stereoisomer of 1. Referring to the coupling constant J = 8.1 Hz of (−)-catechin with [α]_D²⁵ −14.9 (c 1.0, 50% aqueous acetone) [9], 2 was suggested to be the 3-epimer of 1 and H-2″/H-3″ were in trans-orientation. Thus the absolute configuration of 2 was determined as (2″S:3″R). Based on the above data, compound 2 was determined to be 5,7-dihydroxy-2-(4′-hydroxyphenyl)-3-(3″R,5″,7″-trihydroxy-2″S-(4‴-hydroxyphenyl)-3″,4″-dihydro-2H-chromen-6″-yl)-4H-chromen-4-one, namely wikstaiwanone B, which was further confirmed by HMQC, DEPT, NOESY, and HMBC (Figure 2) experiments.

Compound 3 was isolated as pale yellowish amorphous powder with [α]_D²⁵ +53.1 (c 0.058, MeOH). Its molecular formula was determined as C₃₁H₂₄O₁₀ by ESI-MS and HR-ESI-MS with 15 unsaturated degrees. The ¹H- and ¹³C-NMR spectra (Table 2) of 3 were similar to those of chamaejasmine [5] except a methoxy signal [δ_H 3.79 (3H, s, OCH₃-4′), δ_C 159.6 (C-4′)] in 3 was in place of a hydroxy group at C-4′ in chamaejasmine. Thus the planar structure of 3 was determined as 3-(5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochroman-3-yl)-5,7-dihydroxy-2-(4-methoxyphenyl)-2,3-dihydrochromen-4-one. The relation configurations at C-2/C-3 and C-3″/C-2″ positions were decided as trans-trans by comparison of J values of H-2/H-3 with 12.0 Hz and H-3″/H-2″ with 12.0 Hz, and no correlations between H-3/H-2, H-3″/H-2″ were observed. Additionally, H-3 and H-3′ (δ_H 2.60 (2H, dd, J = 18.0, 12.0 Hz)) with same chemical shift supported the H-3 and H-3′ being at same side. Referring to chamaejasmine [5] with [α]_D −61.2 (c 0.49, MeOH), compound 3 with dextrorotatory optical activity, [α]_D +53.1 (c 0.058, MeOH), indicated 3 is an enantiomer of chamaejasmine. According to the above evidence, the structure of 3 was elucidated as to be (2R,3S)-3-((2″R,3″S)-5″,7″-dihydroxy-2″-(4‴-hydroxyl-phenyl)-4-oxochroman-3-yl)-5,7-dihydroxy-2-(4-methoxyphenyl)-2,3-dihydrochromen-4-one, named wikstaiwanone C, which was further confirmed by HMQC, DEPT, NOESY and HMBC (Figure 2) techniques.

The amount of sample isolated for 1–7 ranged from about 3 to 42 mg, although all compounds underwent structural characterizations, only Shikokianins B (4) and C (5) (42.3 mg and 35.7 mg respectively) provided sufficient quantity for evaluating their in vitro antitubercular activity against M. tuberculosis H₃₇Rv with the clinical used drug ethambutol (MIC 6.25 μg/mL) as a positive control. Results showed that both 4 and 5 have MIC values of about 15 μg/mL.

Optical rotations were measured on a polarimeter (JASCO, Japan), UV spectra were obtained with a Jasco V-530 UV/VIS spectrophotometer. IR spectroscopic data were recorded on a Genesis II FTIR spectrophotometer with KBr pellets. NMR spectra were obtained on a Varian Unity Plus 400 spectrometer (400 MHz for ¹H-NMR, 100 MHz for ¹³C-NMR) and Varian Unity Inova 600 spectrometer (600 MHz for ¹H-NMR, 150 MHz for ¹³C-NMR). Chemical shifts were reported with respect to acetone-d₆, methanol-d₄ or DMSO solvents. Low-resolution MS spectra were obtained with Micromass Trio-2000 GC/MS, VG Biotech Quattro 5022, and JEOL-JMS-HX 100 mass spectrometers. The HRMS spectra were recorded on JEOL JMS-SX102A GC/LC/MS and Finnigan MAT-95XL high-resolution mass spectrometers. Silica gel (70–230 and 230–400 mesh; Merck) and Spherical C18 100 Å reversed phase silica gel (RP-18; particle size 20–40 μm; Silicycle) were used for column chromatography, and silica gel 60 F254 (Merck) and RP-18 F254S (Merck) were used for TLC and preparative TLC. Further purification was performed by HPLC (Hitachi; pump: L-2130; UV detector: L-2400).

Stems of W. taiwanensis were collected in May, 2008 at Mountain Goshifer, Pingtung County, and identified by Professor Ih-Sheng Chen. A voucher specimen (Chen 6163) has been deposited in the Herbarium of the College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.

Dried stems (9.8 kg) of W. taiwanensis were sliced to about 0.5cm in length and extracted three times, each time for 8 hours, with cold MeOH at room temperature to yield a MeOH extract, which was partitioned between EtOAc:H₂O (1:1) to provide an EtOAc-soluble fraction (390 g). Both the aqueous and the EtOAc-soluble fractions were studies for their biological activities. The active EtOAc-soluble fraction (70 g) was subjected to Sephadex LH-20 column with CH₂Cl₂-MeOH (1:1) to obtained 4 fractions (1–4). The bioactive fraction 3 (24 g) was chromatographed on Silica gel CC and eluted with a CH₂Cl₂-MeOH-H₂O gradient solvent system (50:3:1 to 6:4:1) to give fractions 3a-q. Fraction 3b (1.17 g) was subjected to a silica gel CC with CH₂Cl₂-EtOAc (15:1) to give 10 fractions (3b-1 to 3b-10). Fraction 3b-7 was subjected to MPLC and eluted with CH₂Cl₂-acetone (15:1) to give 5 (35.7 mg); Fraction 3b-8 was applied to MPLC eluting with CH₂Cl₂-MeOH (20:1) to give 6 (8.3 mg); and Fraction 3b-9 was purified with MPLC (RP₁₈) eluting with MeOH-H₂O (2:1) to give 4 (42.3 mg). Fraction 3e (2.56 g) was subjected to Sephadex LH-20 column and RP18 column successively to give 1 (3.8 mg), 2 (6 mg) and 7 (3 mg). Fraction 3f (708 mg) was eluted with MeOH–H₂O (1:2) over RP₁₈ column chromatography, purified with Sephadex LH-20 (MeOH) and ODS HPLC (254 nm, 250 × 10 mm, 70% CH₃CN(aq.), 2.0 mL/min) to give 3 (12 mg).

Wikstaiwanone A (1): C₃₀H₂₂O₁₀; Pale yellowish amorphous powder; [α]_D²⁵ = −77.0° (c = 0.03, MeOH); UV (MeOH): 210 (4.69), 217 sh (4.68), 266 (4.29), 315 sh (3.98), 355 sh (4.00); IR ν_max (KBr): 3450 (OH), 1647 (C=O) cm⁻¹; ESI-MS, m/z 565 ([M+Na]⁺), HR-ESI-MS at m/z 565.1107 ([M+Na]⁺, C₃₀H₂₂O₁₀Na, calc. 565.1111); ¹H- and ¹³C-NMR data, see Table 1.

Wikstaiwanone B (2): C₃₀H₂₂O₁₀; Pale yellowish amorphous powder; [α]_D²⁴ = −23.3° (c = 0.03, MeOH); UV (MeOH): 202 (4.72), 207 (4.72), 225 sh (4.51), 264 (4.36), 313 sh (4.04), 348 sh (3.94); IR ν_max (KBr): 3395 (OH), 1638 (C=O) cm⁻¹; ESI-MS m/z 565 ([M+Na]⁺); HR-ESI-MS at m/z 565.1114 ([M+Na]⁺, C₃₀H₂₂O₁₀Na, calc. 565.1111); ¹H- and ¹³C-NMR data, see Table 1.

Wikstaiwanone C (3): C₃₁H₂₄O₁₀; Pale yellowish amorphous powder; [α]_D²⁵ = +53.1° (c = 0.058, MeOH); UV (MeOH): 212 (4.61), 230 sh (4.47), 294 (4.55), 330 sh (3.77); IR ν_max (KBr): 3395 (OH), 1631, 1617 (C=O) cm⁻¹; ESI-MS m/z 579 ([M+Na]⁺); HR-ESI-MS at m/z 579.1269 ([M+Na]⁺, C₃₁H₂₄O₁₀Na, calc. 579.1267); ¹H- and ¹³C-NMR data, see Table 2.

The in vitro antitubercular activity of each tested compound was evaluated using the Mycobacterium tuberculosis H₃₇Rv. Middlebrook 7H10 agar was used to determine the minimum inhibitory concentration (MIC) values as recommended by the proportion method [22]. Briefly, each test compound was added to Middlebrook 7H10 agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) at 50–56° by serial dilution to yield a final concentrations of 100 to 0.8 μg/mL. Ten milliliter samples of each concentration of test compound-containing medium, was dispensed into plastic quadrant Petri dishes. Several colonies of the test isolate of M. tuberculosis were selected to make a suspension with Middlebrook 7H10 broth, and used as the initial inoculum. The inoculum of test isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook 7H10 broth until turbidity was reduced to that equivalent to that of the McFarland No. 1 standard. Final suspensions were prepared by adding Middlebrook 7H10 broth, and preparing 10⁻² dilutions of the standardized bacterial suspensions. After solidification of the Middlebrook 7H10 medium, 33 μL of the 10⁻² dilution of the standardized bacterial suspensions was placed on each quadrant of the agar plates. The agar plates were then incubated at 35° with 10% CO₂ for 2 weeks. The MIC is the lowest concentration of test compound that completely inhibited the growth of the test isolate of M. tuberculosis, as detected by the unaided eye.