Structural Elucidation and Bioactivity of Biflavonoids from the Stems of Wikstroemia taiwanensis

Three new biflavonoids, wikstaiwanones A–C (1–3), along with four known compounds (4–7) were isolated from the stems of Wikstroemia taiwanensis (Thymelaeaceae). Their structures were elucidated by spectroscopic analysis. Compounds 4 and 5 showed antitubercular activity against Mycobacterium tuberculosis with MIC values of 15 μg/mL, respectively.


Introduction
Tuberculosis (TB), including multidrug resistant tuberculosis and extensively drug resistant tuberculosis, remains an epidemic in many regions of the world, with an estimated 8.8 million new cases and caused 1.1 million deaths globally in 2010 [1]. In Taiwan, it is also one of the most important communicable infectious diseases subject to official notification, with the highest number of confirmed cases and deaths [2]. Searching new sources of antitubercular agents from nature is crucial to curb the rise of drug-resistant strains. Examining an antitubercular activity from crude extracts of several Taiwan endemic plants, we found the methanolic extract of the stems of

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Wikstroemia taiwanensis showed potent activity against Mycobacterium tuberculosis H 37 Rv in vitro W. taiwanensis (Thymelaeaceae) is an endemic deciduous shrub growing in the south of Taiwan. Analysis of the chemical constituents and bioactivity of this plant has not been conducted previously. Bioactivity-directed fractionation of the active EtOAc layer of the stems of this species led to the isolation and characterization of three biflavonoids, wikstaiwanones A-C (1-3), along with three known biflavonoids, sikokianins B (4) [3], C (5) [4], isochamaejasmin (6) [5], and, methyl 4-hydroxybenzoate (7) [6] (Figure 1). This paper reports the structure elucidation of 1-3 and their antitubercular activities.

Results and Discussion
The present study focused on the fraction of the W. taiwanensis stem extract that displayed biological activity, which was confined to the EtOAc-soluble fraction. When this fraction was further separated by gel filtration into four parts, only one of them showed activity against Mycobacterium tuberculosis H 37 Rv. On separating this active part, compounds 1-7 were isolated, characterization of these compounds indicated 1-3 were new compounds.

Structure Elucidation of the New Isolates
Compound 1 was isolated as an optically active yellowish amorphous powder, [α] 25 D −77.0 (c 0.03, MeOH). Its molecular formula was established as C 30 H 22 O 10 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 1 H-and 13 C-NMR spectra ( Table 1)  25 D −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-

Antitubercular Activity
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 H 37 Rv 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.

Plant Material
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.

Antitubercular Activity Assay
The in vitro antitubercular activity of each tested compound was evaluated using the Mycobacterium tuberculosis H 37 Rv. 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% CO 2 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.

Conclusions
Three new biflavonoids (1-3) together with four known compounds (4-7) were isolated from the stem of W. taiwanensis in this study. Several biflavonoids from Thymelaeaceae have been reported in a number of studies [11][12][13][14][15][16][17][18] and their biological activities evaluated [19][20][21]. Most of them contain C-3/C-3'' linkage with a symmetrical structure [11][12][13][14], except for wikstrols [4], genkwanols and stelleranol [14][15][16][17]. Compounds 1 and 2 were constituted by a flavone and a flavan-3-ol moieties and connected by an unusual C-3/C-6'' linkage, which was found for the first time in nature. The solubility in acetone of 1 and 2 is remarkably different, where 1 dissolved in acetone-d 6 slightly, 2 was readily soluble. Incidentally, their antitubercular properties have not previously been reported. In the study, compounds 4 and 5 were evaluated in this respect, and it was revealed that both have MIC values of 15 μg/mL, which is of the same order as the MIC value for ethambutol, a clinically used drug for