molecules Article
Diarylheptanoid Glycosides of Morella salicifolia Bark Edna Makule 1,2 , Thomas J. Schmidt 3 , Jörg Heilmann 1, * and Birgit Kraus 1, * 1 2 3
*
Department of Pharmaceutical Biology, Faculty of Chemistry and Pharmacy, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany;
[email protected] Department of Food Biotechnology and Nutritional Sciences, Nelson Mandela African Institution of Science and Technology, P.O. Box 447 Arusha, Tanzania Institute of Pharmaceutical Biology and Phytochemistry (IPBP), Westfälische Wilhelms-Universität Münster, PharmaCampus—Corrensstraße 48, D-48149 Münster, Germany;
[email protected] Correspondence:
[email protected] (J.H.);
[email protected] (B.K.); Tel.: +49-941-943-4759 (J.H.); +49-941-943-4494 (B.K.); Fax: +49-941-943-4990 (J.H.); +49-941-943-4990 (B.K.)
Received: 26 October 2017; Accepted: 4 December 2017; Published: 19 December 2017
Abstract: A methanolic extract of Morella salicifolia bark was fractionated by various chromatographic techniques yielding six previously unknown cyclic diarylheptanoids, namely, 7-hydroxymyricanol 5-O-β-D-glucopyranoside (1), juglanin B 3-O-β-D-glucopyranoside (2), 16-hydroxyjuglanin B 17-O-β-D-glucopyranoside (3), myricanone 5-O-β-D-gluco-pranosyl-(1→6)-β-D-glucopyranoside (4), neomyricanone 5-O-β-D-glucopranosyl-(1→6)-β-D-glucopyranoside (5), and myricanone 17-O-α-L-arabino-furanosyl-(1→6)-β-D-glucopyranoside (6), respectively, together with 10 known cyclic diarylheptanoids. The structural diversity of the diarylheptanoid pattern in M. salicifolia resulted from varying glycosidation at C-3, C-5, and C-17 as well as from substitution at C-11 with hydroxy, carbonyl or sulfate groups, respectively. Structure elucidation of the isolated compounds was achieved on the basis of one- and two-dimensional nuclear magnetic resonance (NMR) as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) analyses. The absolute configuration of the glycosides was confirmed after hydrolysis and synthesis of O-(S)-methyl butyrated (SMB) sugar derivatives by comparison of their 1 H-NMR data with those of reference sugars. Additionally, absolute configuration of diarylheptanoid aglycones at C-11 was determined by electronic circular dichroism (ECD) spectra simulation and comparison with experimental CD spectra after hydrolysis. Keywords: Morella salicifolia; Myricaceae; diarylheptanoid glycosides; myricanol; juglanin
1. Introduction Morella salicifolia (H OCHST. EX A. R ICH.) VERDC. & POLHILL belongs to the family Myricaceae in the order Fagales. It was formerly named Myrica salicifolia HOCHST. ex A. RICH. until the genus Myrica was divided into two genera: Myrica and Morella. M. salicifolia now belongs to the genus Morella, with the new accepted name Morella salicifolia (HOCHST. ex A. RICH.) VERDC. & POLHILL. (syn. Myrica salicifolia HOCHST. ex A. RICH.) [1]. M. salicifolia is reported to be spread in many mountainous ranges in Tanzania above 1200 m and prefers shallow soil, heath, and rocky areas [2]. The species is distributed mainly in Tanzania, Kenya, Uganda, Rwanda, Burundi, Ethiopia, Democratic Republic of the Congo, Yemen, and Saudi Arabia [2,3]. Traditional medicinal use of M. salicifolia has been previously reported in Tanzania where it is used for the treatment of cough, toothache, decoction, tonic, stomach troubles, skin diseases [4], headaches [5], and opportunistic diseases of human immunodeficiency virus/acquired immune deficiency syndrome such as tuberculosis, chronic diarrhoea, cryptococcal meningitis, and herpes simplex [6]. Further, traditional medicinal uses of
Molecules 2017, 22, 2266; doi:10.3390/molecules22122266
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M. salicifolia have been reported in other countries such as Ethiopia where it is used for the treatment of skin diseases [7], pain, inflammation, and respiratory disorders [8,9]. In Uganda, M. salicifolia has been used for the treatment of male sexual impotence and erectile dysfunction [10]. Despite the many traditional uses of M. salicifolia, literature describing its phytochemical and pharmacological investigation is scarce. A preliminary phytochemical screening of the methanolic extracts of the stem bark and leaves showed the presence of polyphenols, unsaturated sterols/triterpenes, saponins, glycosides, and carbohydrates [11]. Moreover, results from previously conducted biological activity studies on M. salicifolia showed that a methanolic extract of M. salicifolia stem bark was effective against Bacillus cereus, Neisseria gonorrhoeae, Shigella dysenteriae, and Staphylococcus aureus [11,12]. In vivo testing of a methanolic extract of M. salicifolia in mice showed potent analgesic and antipyretic activity at a concentration of 100 mg/kg [13]. To date, no study has reported the isolation of individual compounds from M. salicifolia. Therefore, the aim of this work was the isolation and structure elucidation of secondary metabolites from crude methanolic extract of M. salicifolia bark to explore the documented activity and traditional usage of the drug in future studies. 2. Results Fractionation of a methanolic extract of M. salicifolia bark resulted in the isolation of 6 unknown (1–6) and 10 known (7–16) cyclic diarylheptanoids of the sub-group meta-meta cyclophane (Figure 1). The known diarylheptanoids were juglanin B-sulfate (7) [14,15], myricanol (8) [16–19], myricanone (9) [16,19], myricanol 5-O-β-D-glucopyranoside (10) [20], myricanone 5-O-β-D-glucopyranoside (11) [21], myricanol 11-O-β-D-xylopyranoside (12) [22], myricanol 5-O-β-D-(6’-O-galloyl)-glucopyranoside (13) [23], myricanone 5-O-β-D-(6’-O-galloyl)-glucopyranoside (14) [15], myricanol 5-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside (15) [24], and myricanol gentiobiosidse: myricanol 5-O-β-D-glucopranosyl-(1→6)-β-D-glucopyranoside (16) [23], respectively. Structure elucidation was done by comprehensive one- and two-dimensional nuclear magnetic resonance (NMR), as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS), sugar hydrolyses, circular dichroism (CD)-analyses, and comparison with published data. The HR-ESI-MS of compound 1 exhibited ions at m/z 535.2185 [M − H]− and 1071.4445 [2M − H]− which are consistent with the molecular formula C27 H36 O11 . The ultraviolet (UV) spectrum (MeOH) of 1 showed absorption maxima at 213, 250, and 295 nm, which are typical for cyclic biphenyl-type diarylheptanoids [22,25]. The 1 H-NMR spectrum of 1 was similar to published data on compound 10 [20] showing four aromatic protons resonating at δH 7.02 (1H, dd, J = 2.2, 8.2, H-15), δH 6.77 (1H, d, J = 8.2, H-16), and δH 6.87 (2H, s, H-18, H-19). Two aromatic methoxy groups were observed as one singlet at δH 3.95 (6H, H3 -20 and H3 -21) and were placed at C-3 and C-4 due to the [1 H-13 C]-heteronuclear multiple bond correlations (HMBC). Five high-field shifted methylene groups were observed as multiplets between δH 1.12 and 2.88 and assigned with the help of [1 H-1 H]-correlated spectroscopy (COSY) and [1 H-13 C]-HMBC experiments to H-8 (δH 2.13, m and δH 1.97, m), H-9 (δH 1.52, m and δH 1.15, m), H-10 (δH 1.79, m and δH 1.52, m), H-12 (δH 2.13, m and δH 1.63, m), and H-13 (δH 2.84, m). Two methane groups bearing a hydroxy group resonated at δH 3.84 (m) and 4.92 (dd, J = 3.6, 11.4) and were assigned to H-11 and H-7 following a proton coupling network from the COSY and HMBC long-range correlations. Sugar protons typical of a glucose resonating between δH 3.32 and 3.80 were assigned to H-20 –H-60 based on the COSY experiment. The anomeric proton of the glucose moiety was observed at δH 5.05 (H-10 , J = 7.4 Hz), suggesting β-configuration. The position of the glycosidic linkage was elucidated from the HMBC long-range correlation between H-10 and C-5 (δC 150.0). From obtained the UV, NMR (Tables 1 and 2), and HR-MS data, compound 1 was concluded to be a hitherto undescribed diarylheptanoid, 7-hydroxymyricanol 5-O-β-D-glucopyranoside, and was named salicimeckol.
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Figure 1. Structures of the isolated diarylheptanoids from methanolic extract of Morella salicifolia bark. Figure 1. Structures of the isolated diarylheptanoids from methanolic extract of Morella salicifolia bark. Compounds 1–6 (new) and 7–16 (known). Compounds 1–6 (new) and 7–16 (known).
Compound 2, as a second previously unknown cyclic diarylheptanoid monoglycoside, was Compound 2, asB 3-O-βa second previously unknown diarylheptanoid D-glucopyranoside and namedcyclic salicireneol A. HR-ESI-MSmonoglycoside, showed ions identified as juglanin was as [M juglanin B 3-O-βD -glucopyranoside and namedtosalicireneol HR-ESI-MS showed at identified m/z 489.2137 − H]− and m/z 979.4324 [2M − H]− corresponding a molecularA. formula of C26H 34O9. Structure was in analogy to compound by − extensive 1D and 2D measurements. ions at m/z elucidation 489.2137 [M − done H]− and m/z 979.4324 [2M −1 H] corresponding to aNMR molecular formula of C26 H34 O9. Structure elucidation was done in analogy to compound 1 by extensive 1D and 2D NMR
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measurements. Accordingly, the 1 H- and 13 C-NMR data for 2 are in agreement with published data of juglanin B [26,27] complemented by additional sugar signals (Tables 1 and 2). The molecular mass of compound 3 was deduced from HR-ESI-MS ions at m/z 505.2078 [M − H]− and 551.2137 [M + HCOO]− calculated for the molecular formula of C26 H34 O10 . The 1 H-NMR of 3 showed four aromatic protons, at δH 7.04 (H-5, brs), δH 6.87 (H-19, brs), δH 6.64 (H-18, brs), and δH 5.66 (H-15, brs). Furthermore, signals attributed to six aliphatic methylene groups were detected between δH 0.90 and 2.78 and were assigned to positions H-7–H-10, H-12, and H-13 (Table 1). One methoxy group resonated at δH 3.80 (s, 3H) and was assigned to H3 -20 by [1 H-1 H]-rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY) and HMBC correlations. The anomeric proton of a β-glucopyranosyl moiety was observed at δH 5.00 (H-1’, d, J = 7.6 Hz) and the glycosidic linkage of 3 was elucidated by an HMBC experiment showing a long-range correlation to C-17 (δC 144.7 ppm, Table 2). The position of the hydroxy group at C-11 was deduced from COSY due to lacking long-range correlation signals of H-11 (δH 3.05, m) in the HMBC experiment. Therefore, compound 3 is 16-hydroxyjuglanin B 17-O-β-D-glucopyranoside, a hitherto unknown compound and named salicireneol B. HR-ESI-MS of 4 and 5 showed ions at m/z 679.2682 [M − H]− and m/z 679.2622 [M − H]− with the common molecular formula of C33 H44 O15 and calculated mass of 680 Da. The 1 H-NMR of 4 showed signals at δH 3.94 and 3.80 (both s, 3H), which were assigned to the two methoxy groups H3 -20 and H3 -21. Four aromatic protons were assigned to H-15 (7.05, dd, J = 2.1, 8.2), H-16 (6.80, d, J = 8.2), H-18 (6.64, brs), and H-19 (6.57, s) of the aglycone (Table 1). The carbonyl groups at δC 217.2 ppm were assigned to position C-11 as deduced from the HMBC experiment due to long-range correlations between H-9 (δH 1.70, m, 2H), H-10 (δH 2.78, m, 1H and δH 2.65, m, 1H), H-12 (δH 2.91, m, 2H), H-13 (δH 2.78, m, 1H), and the carbonyl carbon. Further confirmation was achieved from the COSY experiment due to observed cross peaks between H-7 (δH 2.91, m, 1H and δH 2.78, m, 1H) and H-8 (1.89, m, 2H), H-8 and H-9 (1.70, m, 2H), H-9 and H-10 (δH 2.78, m, 1H and δH 2.65, m, 1H), and H-12 (δH 2.91, m, 2H) and H-13 (δH 2.78, m, 1H). The anomeric protons of two glucosyl moieties resonated at δH 4.99 (d, J = 7.2 Hz, H-1’) and δH 4.28 (d, J = 7.8 Hz, H-1”). Long-range correlations were observed between the anomeric proton H-1’ and C-5 (δC 149.8 ppm) of the aglycone and the anomeric proton H-1” to C-6’ of the glucose. Therefore, two sugar groups were attached to C-5 of the aglycone and they were confirmed to be two D-glucose molecules. Hence, the structure of 4 was confirmed to be the hitherto unknown myricanone 5-O-β-D-glucopranosyl-(1→6)-β-D-glucopyranoside and was given the name saliciclaireone A. The carbon data of 5 was found to be similar to the published data of neomyricanone 5-O-β-D-glucopyranoside [22], except that 5 was found to have two 1→6 connected glucose moieties attached to C-5. Extensive one- and two-dimensional NMR revealed that compound 5 is neomyricanone 5-O-β-D-glucopranosyl-(1→6)-β-D-glucopyranoside, a hitherto undescribed compound which was thus named saliciclaireone B. The summarized 1D-NMR data for 4 and 5 were depicted in Tables 1 and 2, respectively. The HR-ESI-MS of 6 showed a pseudomolecular ion at m/z 649.2509 [M − H]− in the negative mode, consistent with the molecular formula of C32 H41 O14 . The 1 H-NMR spectrum shows 2 methoxy groups at δH 3.81 and 3.93 ppm (both s, H3 -21 and H3 -20), 4 aromatic protons, and 6 methylene groups. A carbonyl group resonating at δC 216.1 was assigned to C-11 based on the HMBC experiment showing long-range correlations to δH 2.80/2.61 (H-10), δH 2.93/2.79 (H-12), and δH 2.95 ppm (H-13, 2H). Two anomeric protons observed at δH 4.97 (H-1’, d, J = 7.2) and δH (4.83 H-1” brs), indicated the presence of two sugar moieties with a β-D and α-L-configuration. The positions of glycosidic linkages were elucidated from the HMBC experiment. Long-range correlation was observed between H-1’ (δH 4.97) and C-17 (δC 152.7) of the aglycone as well as H-1” (δH 4.83) and C-6’ (δC 68.0) of the glucose. Therefore, it was concluded that the two sugar groups are attached to C-17 of the aglycone. The NMR signals of the aglycone were similar to those of myricanone (compound 12).
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Table 1. 1 H-Nuclear magnetic resonance (NMR) data of 1–6 (600 MHz, methanol-d4 , 298 K, δ ppm, mult, J, Hz). Number
1
5
2
3
6.85 brs
7.04 brs
4
5
6
7
4.92 dd (3.6, 11.4)
2.98 m 2.54 m
2.78 m 2.64 m
2.91 m * 2.78 m *
3.19 m 2.97 m
2.73 m 2.96 m
8
2.13 m 1.97 m
1.97 m 1.83 m *
1.74 m 1.59 m
1.89 m * 1.89 m *
2.87 m * 2.87 m *
1.90 m * 1.90 m *
9
1.52 m * 1.15 m
1.61 m 1.43 m
1.27 m * 1.03 m
1.70 m * 1.70 m *
10
1.79 m 1.53 m *
1.83 m * 1.52 m
1.27 m * 0.90 m
2.78 m * 2.65 m *
11
3.84 m
3.91 m
3.05 m
12
2.13 m 1.63 m
2.23 m 1.63 m
1.43 m (2H)
2.91 m * (2H)
1.97 m * (2H)
2.79 m * 2.93 m *
13
2.84 m * 2.84 m *
2.85 m 2.01 m
2.64 m * 2.60 m *
2.78 m *
2.75 m * 2.64 m *
2.95 m * 2.95 m *
15
7.02 dd (2.2, 8.2)
7.04 dd (2.3, 8.2)
5.66 brs
7.05 dd (2.1, 8.2)
7.00 dd (2.2, 8.2)
7.03 dd (2.9, 8.7)
16
6.77 d (8.2)
6.78 d (8.2)
6.80 d (8.2)
6.82 d (8.2)
6.78 d (8.2)
18
6.87 brs
7.07 brs
6.64 brs
6.64 brs
6.87 d (1.9)
6.67 d (2.3)
19
6.87 s
6.76 brs
6.87 brs
6.57 s
6.42 s
6.58 s
20
3.95 s
3.88 s
3.80 s
3.94 s
3.84 s
3.93 s
21
3.95 s
3.80 s
3.95 s
3.81 s
1’
5.05 d (7.4)
4.99 d (7.3)
5.00 d (7.6)
4.99 d (7.2)
4.88 d (7.6)
4.97 d (7.2)
2’
3.50 m *
3.38 m *
3.54 m *
3.50 m *
3.52 m *
3.44 m *
3’
3.39 m *
3.20 m *
3.49 m *
3.50 m *
3.39 m *
3.43 m *
4’
3.47 m *
3.07 m
3.42 m *
3.50 m *
3.53 m *
3.44 m *
5’
3.32 m *
3.29 m *
3.42 m *
3.41 m
3.46 m
3.38 m
6’
3.80 dd (2.4, 11.8) 3.64 dd (5.7, 11.8)
3.45 m * 3.45 m *
3.89 brd (12.3) 3.71 (dd, 4.0, 1.8)
4.08 dd (1.8, 11.5) 3.78 m
4.11 dd (2.2, 11.2) 3.79 m
3.96 dd (2.1, 11.0) 3.56 m * 4.83 brs
1.74 m * 1.74 m * 2.75 m * 2.64 m *
2.80 m * 2.61 m
1.74 m * (2H)
1”
4.28 d (7.8)
4.25 m
2”
3.18 m
3.24 m *
3.91 m
3”
3.30 m *
3.22 m *
3.78 m *
4”
3.25 m *
3.27 m *
3.83 m * 3.67 dd (3.2, 11.7) 3.58 m *
5”
3.25 m *
3.27 m *
6”
3.84 m 3.63 dd (5.2, 11.9)
3.81 dd (2.5, 11.9) dd (5.4, 11.9)
* Overlapping signals.
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Table 2.
13 C-NMR
data of 1–6 (150 MHz, methanol-d4 , 298 K, δ ppm).
Number
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3” 4” 5” 6”
126.8 120.2 147.0 152.2 150.0 131.0 66.7 35.6 21.8 40.0 68.9 35.5 27.7 131.3 131.0 117.1 153.1 135.8 130.8 61.1 61.9 104.8 75.5 71.4 77.7 78.5 62.1
128.0 128.2 140.9 152.7 113.6 131.8 31.6 27.5 23.8 40.2 69.1 35.5 27.9 132.0 130.6 117.2 152.1 135.0 127.6 56.6
126.2 131.5 144.8 154.0 115.3 137.9 36.2 31.2 23.4 39.5 72.2 37.4 29.3 136.3 115.2 151.6 144.7 122.7 124.2 56.4
104.8 75.3 71.1 77.5 78.6 62.6
102.9 74.7 77.8 71.1 78.0 62.3
126.7 129.4 146.6 149.2 149.8 131.8 28.4 25.7 22.9 46.2 217.2 43.1 29.1 133.2 130.2 117.5 152.8 133.9 130.0 62.3 61.8 105.0 75.4 77.0 71.0 77.5 69.5 104.4 75.0 77.7 71.4 77.7 62.6
125.4 129.6 148.3 146.3 148.7 131.9 23.6 41.8 215.8 44.6 22.7 26.2 31.8 132.1 130.9 117.4 152.2 133.7 129.5 62.1 62.2 105.2 74.8 76.3 70.5 77.0 69.3 103.8 74.3 77.0 70.8 77.2 62.2
126.5 129.6 146.5 148.9 149.9 127.6 28.2 25.6 22.9 45.8 216.1 42.7 28.7 132.8 129.8 117.1 152.7 133.8 129.7 61.8 61.6 104.9 75.3 77.5 75.3 76.6 68.0 109.6 82.8 78.6 85.6 62.7
The attached glycosides were confirmed for β-D-glucopyranoside and α-L-arabinofuranoside as described in section of absolute configuration of isolated diarylheptanoids. The furan ring of the α-L-arabinose was concluded based on its carbon chemical shifts as described by Beier and Mundy [28]. Based on the obtained data, compound 6 was identified to be myricanone 17-O-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside and was named saliciclaireone C. The 1D-NMR data of compound 6 are summarized in Tables 1 and 2. 1 H-NMR of compounds 1–6 can be found in the Supplementary Materials. The absolute configuration at C-11 was determined as 11R for all isolated diarylheptanoids by measurement of the CD spectra of the aglycone and comparison with electronic CD spectra simulation. Recorded experimental CD spectra of the isolated diarylheptanoids and the aglycone obtained from enzyme hydrolysis were found to be very similar (Figure 2) and it was hence concluded that the attached glycosides and sulfate moieties had no influence on conformation of the aglycone and its chromophore. Therefore, CD spectra simulation was performed using myricanol as a model compound. The myricanol structure contains one chiral center at C-11, which is axially dissymmetric due to the twisted biphenyl. Thus, the structure of myricanol can occur as two pairs of enantiomers i.e., (R,Ra), (S,Sa), and (R,Sa), (S,Ra), where “a” stands for the chirality of the axially dissymmetric biphenylic system [18,29].
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Figure 2. 2. Recorded experimental circular circular dichroism dichroism (CD) (CD) spectra spectra of of isolated isolated diarylheptanoids diarylheptanoids (molar (molar ellipticity ellipticity vs. vs. wavelength. wavelength. (nm)). (nm)). Figure Recorded experimental
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Molecular models generated for the R-enantiomer of myricanol resulted in three conformations Molecular models generated for the R-enantiomer of myricanol resulted in three conformations corresponding to the 11R,Ra, 11R,Sa (a), and 11R,Sa (b) forms (Figure 3A–C). corresponding to the 11R,Ra, 11R,Sa (a), and 11R,Sa (b) forms (Figure 3A–C).
Figure 3. Three-dimensional structures of the conformations of R-myricanol. (A) 11R,Ra myricanol. Figure 3. Three-dimensional structures of the conformations of R-myricanol. (A) 11R,Ra myricanol. RB3LYP/6-31G(d,p) energy: –1192.01521911 a.u. Energy difference from lowest conformer: 2.01 RB3LYP/6-31G(d,p) energy: –1192.01521911 a.u. Energy difference from lowest conformer: 2.01 kcal/mol; kcal/mol; (B) 11R,Sa myricanol (a). RB3LYP/6-31G(d,p) energy: −1192.01342066 a.u. Energy difference (B) 11R,Sa myricanol (a). RB3LYP/6-31G(d,p) energy: −1192.01342066 a.u. Energy difference from the from the lowest conformer: 3.31 kcal/mol; (C) 11R,Sa myricanol (b). Conformation corresponds to the lowest conformer: 3.31 kcal/mol; (C) 11R,Sa myricanol (b). Conformation corresponds to the X-ray X-ray crystal structure [19]. RB3LYP/6-31G(d,p) energy: –1192.01842217 a.u. Energy difference from crystal structure [19]. RB3LYP/6-31G(d,p) energy: –1192.01842217 a.u. Energy difference from the the lowest conformer: 0.000 kcal/mol. lowest conformer: 0.000 kcal/mol.
The 11R,Sa (b) conformation corresponds to the X-ray structure of myricanol determined by Theet11R,Sa (b)The conformation corresponds structure of myricanol determined by Begley al. [19]. simulated CD spectrum to of the the X-ray energetically most favorable 11R,Sa-myricanol Begley et al. C, [19]. The 3C) simulated CDto spectrum most favorable 11R,Sa-myricanol (conformer Figure was found be similaroftothe theenergetically experimentally determined spectrum (Figure 4C). (conformer C, Figure 3C) was found to be similar to the experimentally determined spectrum The major cotton effects are of equal sign, indicating that the compound indeed has the 11R,Sa(Figure 4C). The major cotton effects are of equal sign, indicating that the compound indeed has the configuration, as would also be expected based on its reported crystal structure. The spectrum would 11R,Sa-configuration, as wouldifalso expected based onwas its reported crystal structure. appear exactly the opposite the be 11S,Ra-enantiomer present. The spectrum forThe the spectrum much less would appear exactly the opposite if the 11S,Ra-enantiomer was present. The spectrum for the much favorable 11R,Ra-atropisomer (conformer 1, Figure 3A) shows almost entirely the opposite sign less favorable 11R,Ra-atropisomer (conformer 1, Figure 3A) shows almost entirely the opposite sign (Figure 4A). Since its internal energy is predicted to be more than 2 kcal/mol higher than that of (Figure 4A).C, Since its internal energy is to be more in than kcal/mol higher than that The of conformer it would only represent anpredicted insignificant fraction the2conformational equilibrium. conformer C, it would only represent an insignificant fraction in the conformational equilibrium. two different conformers of 11R,Sa myricanol (2 and 3, Figure 3B,C) show similar signs with the The two different conformers of 4B,C) 11R,Sa myricanol (2 and Figure of 3B,C) similar signs with experimental spectrum (Figure which supports the 3, presence the show 11R,Sa-configuration. The the experimental spectrum (Figure 4B,C) which supports the presence of the 11R,Sa-configuration. Boltzmann distribution calculated on the basis of the B3LYP/6-31D(d,p) energies of the three The Boltzmann distribution calculated onlower the basis of the B3LYP/6-31D(d,p) energies ofanthe three conformers would correspond to an even amount of conformer 2 (0.3%). Therefore, averaged conformers would correspond to an even lower amount of mixture conformer 2 (0.3%). Therefore, averaged spectrum was generated for the theoretical equilibrium corresponding to 97%an R,Sa and 3% spectrum was generated for the theoretical equilibrium mixture corresponding to 97% R,Sa R,Ra-myricanol and compared with the experimental CD spectrum of myricanol (Figure and 4D), 3% R,Ra-myricanol and compared with the experimental CD spectrum of myricanol (Figure 4D), presenting a very good match with the experimental spectrum. A slight inconsistency observed in presenting very good match with the A slight observedvery in the 230 nma range (Figure 4B–D) can beexperimental explained byspectrum. an exchange of twoinconsistency electronic transitions the 230 nm range (Figure 4B–D) can be explained by an exchange of two electronic transitions very close to each other, at 217 and 223 nm. However, this does not change the general picture of the main close to at each other, 217 wavelength and 223 nm. being However, this does not According change theto general picture of thenatural main bands long andat short of the same sign. these calculations, myricanol was concluded to be 11R and very predominantly Sa-configured. This corresponds to the X-ray structure and matches the CD data of myricanol reported in literature [19,30]. It should hence
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bands at long and short wavelength being of the same sign. According to these calculations, natural myricanol was concluded to be 11R and very predominantly Sa-configured. This corresponds to9 of the Molecules 2017, 22, 2266 15 X-ray structure and matches the CD data of myricanol reported in literature [19,30]. It should hence be clear thatthat in the case of of thethe 11S-configured compound, be clear in the case 11S-configured compound,the theRa-atropisomer Ra-atropisomer(i.e., (i.e.,11S,Ra, 11S,Ra,representing representing the enantiomer of the depicted structure) would be the energetically most favorable the enantiomer of the depicted structure) would be the energetically most favorable form form and and the the cotton effects in the CD spectrum would show exactly opposite sign. It is possible that a very small cotton effects in the CD spectrum would show exactly opposite sign. It is possible that a very small amount amount of of the the Ra-atropisomer Ra-atropisomer is is present present in in the the conformational conformational equilibrium. equilibrium. Finally, Finally, aa confirmation confirmation of of 11R-configuration 11R-configurationof ofdiarylheptanoids diarylheptanoids(1–3, (1–3,7,7,8, 8,10, 10, 12, 12, 13, 13, 15, 15, and and 16) 16) was was demonstrated demonstrated by by their their very very similar CDspectra spectra(Figure (Figure2)2)showing showingcongruency congruency that myricanol. Experimental similar experimental experimental CD toto that of of myricanol. Experimental CD CD spectrum of myricanol in comparison to averaged CD spectrum for the S,Sa (87%) and spectrum of myricanol in comparison to averaged CD spectrum for the S,Sa (87%) and S,Ra S,Ra form form (13%) (13%) can canbe befind findin inthe theSupplementary SupplementaryMaterials. Materials.
Figure 4.4. (A) (A) Conformer Conformer 1:1: 11R,Ra 11R,Ra myricanol. myricanol. Blue: Blue: Experimental Experimental CD CD spectrum spectrum of of myricanol. myricanol. Red: Red: Figure CD spectrum simulated for conformer 1 by time-dependent density functional theory (TDDFT): CD spectrum simulated for conformer 1 by time-dependent density functional theory (TDDFT): RB3LYP/6-31G(d,p), nstates NoNo shift, no scaling of calculated spectrum; (B) Conformer 2: 11R,SaRB3LYP/6-31G(d,p), nstates= =30.30. shift, no scaling of calculated spectrum; (B) Conformer 2: myricanol (a). Blue: Experimental CD spectrum of myricanol. Red: CD spectrum simulated for 11R,Sa-myricanol (a). Blue: Experimental CD spectrum of myricanol. Red: CD spectrum simulated conformer 2 by2TDDFT: RB3LYP/6-31G(d,p), nstatesnstates = 30. No shift,No no shift, scaling calculated spectrum; for conformer by TDDFT: RB3LYP/6-31G(d,p), = 30. noofscaling of calculated (C) Conformer 3: 11R,Sa-myricanol (b), conformation corresponds to the X-ray structure. Blue: spectrum; (C) Conformer 3: 11R,Sa-myricanol (b), conformation corresponds to the X-ray structure. Experimental CD spectrum of myricanol. Red: CD spectrum simulated for conformer 3 by TDDFT: Blue: Experimental CD spectrum of myricanol. Red: CD spectrum simulated for conformer 3 by RB3LYP/6-31G(d,p), nstates = 30. No shift, scaling (D) Blue: Experimental TDDFT: RB3LYP/6-31G(d,p), nstates = 30.noNo shift,ofnocalculated scaling ofspectrum. calculated spectrum. (D) Blue: CD spectrum CD of myricanol. Averaged CDAveraged spectrumCD forspectrum the R,Sa for (97%) Experimental spectrum ofRed: myricanol. Red: the and R,Sa R,Ra (97%)form and (3%). R,Ra TDDFT: RB3LYP/6-31G(d,p), nstates = 30. Calculated spectrum was red-shifted by −0.15 eV and scaled form (3%). TDDFT: RB3LYP/6-31G(d,p), nstates = 30. Calculated spectrum was red-shifted by −0.15 eV by factor and scaled0.5. by factor 0.5.
3. Discussion A systematic investigation of the diarylheptanoid pattern of Morella salicifolia revealed the presence of 16 cyclic diarylheptanoids (meta, meta-bridged biphenyls), among them six previously unknown compounds. The secondary metabolite pattern of M. salicifolia demonstrated the close taxonomic relationship of the new genera Morella and Myrica resulting from the taxonomic reorganization of the former postulated genus Myrica [1]. The taxonomic relationship was deduced from comparison of isolated known compounds (7–16) to the same compounds also reported from
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3. Discussion A systematic investigation of the diarylheptanoid pattern of Morella salicifolia revealed the presence of 16 cyclic diarylheptanoids (meta, meta-bridged biphenyls), among them six previously unknown compounds. The secondary metabolite pattern of M. salicifolia demonstrated the close taxonomic relationship of the new genera Morella and Myrica resulting from the taxonomic reorganization of the former postulated genus Myrica [1]. The taxonomic relationship was deduced from comparison of isolated known compounds (7–16) to the same compounds also reported from Myrica species [31–33]. This makes it probable that cyclic diarylheptanoids are characteristic constituents of both Morella and Myrica species. Additionally, a comparison of experimental data with simulation of electronic circular dichroism (ECD) data revealed that there is no effect of an attached glycoside or sulfate group on the natural aglycone absolute configuration. The obtained absolute configuration of natural myricanol in this study matches very well to that achieved by X-ray in the group of Begley [19]. However, in this study the established CD calculation of myricanol was further used as a reference and a non-destructive method to determine the absolute configuration of isolated diarylheptanoids containing glycosides or sulfate groups. The method used is reliable as the obtained results are in agreement with the published crystal structure. It should also be noted that this method was used to determine the absolute configuration of the meta,meta-bridged biphenyls with chiral center at position 11. There was no sample to prove if the same method can also be applied for the cyclic diarylheptanoids of the meta,para- diphenyl ether type as well as for the ones without chiral center at position 11. 4. Materials and Methods 4.1. General Experimental Procedures Optical rotations were measured in methanol (MeOH for spectroscopy, Merck, Darmstadt, Germany) at 25 ◦ C on a Unipol L 1000 polarimeter (Schmidt and Haensch, Berlin, Germany). CD spectra were recorded on a Jasco J-710 spectrometer (Jasco, Groß-Umstadt, Germany) at a wavelength range of 195–350 nm. UV spectra (MeOH for spectroscopy, Merck, Darmstadt, Germany) were measured using a Cary 50 Scan UV spectrophotometer (Varian, Darmstadt, Germany) equipped with Cary WinUV 3.00 software (Varian, Darmstadt, Germany). The 1D-1 H, 1D-13 C, [1 H-13 C]-heteronuclear single quantum coherence (HSQC), [1 H-13 C]-HMBC, [1 H-1 H]-COSY and [1 H-1 H]-ROESY NMR experiments were recorded on a Bruker AVANCE 600 spectrometer (600.25 MHz for 1 H- and 150.93 MHz for 13 C-NMR (Bruker, Ettlingen, Germany), and referenced to tetramethylsilane (TMS). Samples were dissolved in methanol-d4 (99.8%, Sigma-Aldrich, Taufkirchen, Germany), acetone-d6 (99.8%), or pyridine-d5 (99.5%, both Deutero, Kastellaun, Germany). Low-resolution (LR)-ESI-MS was measured using the TSQ 7000 spectrometer (Thermo Quest, Finnigan, Egelsbach, Germany) and high-resolution (HR)-ESI-MS was measured using the Q-TOF 6540 UHD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). MeOH and acetonitril of HPLC grade (Merck, Darmstadt, Germany) were used for chromatography. For extraction and analysis of MeOH, dichloromethane (DCM) and EtOAc of analytical grade (Acros Organics, Morris Plains, NJ, USA) were used. Open column chromatography was performed using Sephadex® LH-20 (25–100 µm, 265 g, 90 × 4.76 cm, GE Healthcare GmbH, München, Germany) and fraction control was done by analytical thin layer chromatography (TLC, silica gel 60 F254 aluminium sheets, 20 × 20 cm). The standard mobile phase used for all TLC analyses was ethylacetate/water/acetic acid/formic acid (100 + 26 + 11 + 11). Detection was done at visible light (VIS), and UV 254 and 366 nm before and after spraying a plate with anisaldehyde-sulfuric acid and heating a TLC sheet at 105 ◦ C for 3–10 min. Centrifugal partition chromatography (CPC) was performed using SPOT centrifugal partition chromatography (Armen Instrument, Saint-Avé, France) with EtOAc (for analysis, Acros Organics) and water as solvent system. Two modes were used; ascending mode, whereby EtOAc was a
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mobile phase, and descending mode, whereby water was as a mobile phase: rotation = 800 rpm, flow rate 5 mL/min, volume collected in a test tube = 10 mL. Changing from ascending to descending mode was done when no more spots were detected on a TLC sheet when visualized under 254 nm. Final purification of isolated compounds was achieved on semi preparative HPLC using the ProStar HPLC (Varian, Darmstadt, Germany) coupled with the Purospher STAR RP18 column (Eclipse XDB-C18, 250 × 1.4 mm, 5 µm). 4.2. Plant Material The bark of Morella salicifolia (HOCHST. ex A. RICH.) VERDC. & POLHILL was collected in February 2013 at the Monduli mountain ranges in the Arusha region, Tanzania. Identification of the plant was done by Mr. Daniel Sitoni (a senior botanist from the Tanzania National Herbarium (TNH)). Specimen was stored at the TNH with voucher number CK 7792. The collected bark material was spread on a clean cotton cloth under direct sunlight with a temperature between 30 and 35 ◦ C until it was completely dried. 4.3. Extraction and Isolation Procedure Dried pulverized M. salicifolia bark (390.1 g) was mixed with 400 g of sea sand, packed in a column, and macerated overnight with 1 L of dichloromethane (DCM). After maceration, bulk extraction of M. salicifolia bark was performed using four different solvents with increasing polarity, approximately 4 L each. The solvents used for the extraction were DCM, EtOAc, MeOH 100%, and MeOH 50% (v/v). The extraction resulted in respective DCM (7.7 g), EtOAc (1.1 g), MeOH (162.1 g) and MeOH 50% (25.9 g) extracts after evaporation using a rotary vacuum evaporator at 40 ◦ C for complete dryness, mixing with water, and complete freezing at −20 ◦ C followed by lyophilization has been done. The dried powder crude extracts were stored at 4 ◦ C in the refrigerator. MeOH extract (162.1 g) was subjected to Sephadex® LH-20 column chromatography with a 12-g crude portion of extract in each run, aimed at separating tannins from non-tannin compounds (0–1240 mL). Two eluents were used, ethanol 70% (v/v) and acetone 70% (v/v). Seven fractions (S1–S7) were obtained. Fractions S1–S6 were eluted with ethanol 70%. Fraction S7, which comprised proanthocyanidins polymers, was retained in the column and could not be eluted with ethanol 70%. Its elution was achieved by acetone 70%. For isolation of diarylheptanoids, fraction S2 (15.7 g, 450–560 mL) was fractionated by means of flash chromatography with the RP-18 pre-packed column eluted with water (A) and MeOH (B), flow rate of 40 mL/min, and a gradient of 20–40% B (10 min) → 40–100% B (20 min) → 100% B (30 min). Seven fractions (F1–F7) were obtained. The gradient starting at polar conditions resulted in elution of most of the sugars at the beginning of the chromatography (fraction F1), and hence their separation from other compounds. F5 (1.44 g, 851–920 mL) was fractionated by CPC (EtOAC/water system, flow = 5 mL/min, 800 rpm) and resulted in nine fractions (F5.C1–F5.C9). F5.C1 (79.5 mg, 101–160 mL) was subjected to semi-preparative HPLC eluted with water (A) and MeOH (B), flow rate 3 mL/min, gradient 50–70% B (20 min) → 70–100% B (0.1 min) → 100% B. Three peaks were eluted, whereby two peaks resulted from the isolation of compounds 14 (3.1 mg, tR = ~12 min), and 10 (6.5 mg, tR = ~13.3 min). The third peak at tR = ~14 min (42.9 mg) was not pure, and was hence subjected to a subsequent purification step with flash chromatography (column: silica gel Reveleris Flash Cartridges, 20 µm, 12 g) eluted with CHCl3 (A) and MeOH (B), flow rate: 15 mL/ min, gradient: 5% B (40 min) → 5–100% B (25 min) → 100% B (15 min), and resulted in isolation of compounds 11 (10.2 mg, tR = 6 min) and 13 (13.3 mg, tR =9 min). Similarly, separation and purification of F5.C3 (21.8 mg, 281–480 mL) using preparative HPLC with the same procedure as for purification of F5.C1 led to the isolation of compounds 3 (0.7 mg, tR = ~7.5 min), 2 (0.6 mg, tR = ~5.8 min), and 10 (1.81 mg). Further separation and purification of F5.C4 (22.7 mg, 481–810 mL) by preparative HPLC eluted with water + 0.02% trifluoroacetic acid (TFA) (A), and MeCH + 0.02% TFA (B), flow rate: 2 mL/min, gradient: 25–35% B (30 min) → 35–65% B (1 min) → 65% B (4 min), resulted in compound 6 (0.9 mg, tR = ~24.2 min).
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(30 min) → 12–60% B (1 min) → 60–100% B (9 min) → 100% B (10 min), and resulted in the 10 subfractions F6.1–F6.10. Subfraction (43.4 mg, 21–40 mL) mL) was by subjected to preparative RP-18 Furthermore, purification of F6.3 F5.C5, (15.5 mg, 811–1330 RP-18 (semi preparative), Molecules 2017, 22, 2266 12 ofeluted 15 (semi-preparative) HPLC, eluted with H 2O (A) and MeOH (B), flow rate 2 mL/min, gradient: 75–80% with H2 O (A) and MeOH (B), flow rate: 3 mL/min, gradient: 50–60% B (15 min) → 60–100% B (1 min) B (15→min) → 80–100% B (1tomin) → 100% B (3 min), 1and tot the isolation of compounds 8 t(30.5 mg,min). 100% (4 min),Bled isolation of compound (2.5led mg, ~10.5 (2.2 mg, ~14 (30 min) → B12–60% (1 min) → 60–100% B (9 min) → 100% (10 min) min),and and15resulted in 10 R =B R =the tR = F5.C7 ~10.5 min) and 9 (6.6 mg, t R = ~11.8 min). F6.5 (1.8 mg, 96–140 mL) was further purified using (39.5 mg, 1140–1160 mL) and F5.C8 (11.0 mg, 1161–1200 mL) were separated using the same subfractions F6.1–F6.10. Subfraction F6.3 (43.4 mg, 21–40 mL) was subjected to preparative RP-18 RP-18 semi-preparative HPLC eluted with water (A) and MeOH (B), flowmin), 2 mL/min, gradient: 70% Bmin) Molecules 2017, 22,as 2266 12 of 15 procedure for F5.C5 and resulted in compounds 7 (1.0 mg, t = ~6.3 16 (2.6 mg, t = ~11.9 (semi-preparative) HPLC, eluted with H2O (A) and MeOH (B), flow R rate 2 mL/min, gradient: R 75–80% (15 min) 70–100% (1 min) → 100% B (4 min),resulted and resulted in the isolation of compound 12 4→ (1.0 mg, tR =BB~10.5 min) F5.C7. theisolation reisolation of 3 (1.8 mg,8 t(30.5 B (15and min) → 80–100% (1 min) →for 100% B (3 F5.C8 min), and led tointhe of compounds mg,min) R = ~12 (30 min) 12–60% B (1 min) → 60–100% B (9 min) → 100% B (10 min), and resulted in the 10 (0.7 mg, t R→ = ~11 min). 4 (4.2 ~10.5 min) compound 5 (0.9 min). tR = and ~10.5 min)mg, andtR9=(6.6 mg, tR =and ~11.8 min). F6.5 (1.8 mg, mg,t96–140 mL) was further purified using R = ~17.1 subfractions F6.1–F6.10. Subfraction F6.3 (43.4 mg, 21–40 mL) was subjected to preparative RP-18 F6 (from was fractionated using flash chromatography (column: gel Reveleris Flash Salicimeckol (1). amorphous powder; [α] −56 (c MeOH 0.1, MeOH); UV2 λmL/min, max silica (MeOH) (log Ɛ) 213 RP-18 semi-preparative HPLC eluted with water (A) and (B), flow gradient: 70% B of 15 Molecules 2017,White, 22,S2) 2266 12 (semi-preparative) HPLC, eluted with H21O (A) and MeOH (B), flow rate 2 mL/min, gradient: 75–80% 13C-NMR 20and µm,295 eluted with CHCl and (B), flow: 15 mL/min, gradient:m/z 5–12% (15 Cartridges, min) 70–100% B12(1g), min) → 100% B (4 min), and MeOH resulted inTables the isolation compound 12 and data see 1 and 2;ofHR-ESI-MS (3.90), 250→(3.88), (3.67) nm; for H3 (A) B (15 min) → 80–100% B (1 min) → 100% B (3 min), and led to the isolation of compounds 8 (30.5 mg, −12–60% (30min) 12–60% (1 min) B (9 min) 100% B (10 min), resulted → BB (1for min) →35→ 60–100% B (9 min) →→ 100% B (10 min), andand resulted in in thethe 10 535.2185 H]→ (calculated C27H O 1160–100% 535.2185). (0.7 B(30 mg, t[M Rmin) = −~11 min). tR = ~10.5 min) and 9 (6.6 mg, tR = ~11.8 min). F6.5 (1.8 mg, 96–140 mL) was further purified using 10 subfractions F6.1–F6.10. Subfraction F6.3(43.4 (43.4mg, mg,21–40 21–40mL) mL)was was subjected subjected to preparative RP-18 subfractions F6.1–F6.10. Subfraction F6.3 RP-18 Salicireneol A2017, (2).White, White, amorphous powder; [α] (A) −49.0 0.1,MeOH); MeOH); UV λmax max(MeOH) (MeOH) (log Ɛ) 214 Salicimeckol (1). powder; [α] −56 (c (c 0.1, UV (log 213 RP-18 semi-preparative HPLC eluted with water and MeOH (B), flow 2λ mL/min, gradient: 70% B of 15 Molecules 22, 2266 amorphous 12 (semi-preparative) HH22O (A) and MeOH (B), flow rate 22mL/min, gradient: 75–80% (semi-preparative)HPLC, HPLC,eluted elutedwith with O (A) and MeOH (B), flow rate mL/min, 1313 1HC-NMR data 11 and (4.3), 254 (4.02), and for (15 min) 70–100% B (1(3.91) min)nm; → 100% B and (4 min), and resulted the isolation compoundm/z 12 and C-NMR data see seeinTables Tables and 2; 2;ofHR-ESI-MS HR-ESI-MS (3.90), 250→ (3.88), and285 295 (3.67) nm; for1HBB (15 (15 min) min) → →−−80–100% B (1 min) → 100% B (3 min), and led to the isolation of compounds 8 (30.5 mg, (30 min) → 12–60% B (1for min) →33 60–100% B (9 min) → 100% B (10 min), and resulted in the 10 489.2137 H]min). (calculated C26 911489.2130). 535.2185 27H 35O 535.2185). (0.7 mg, t[M R =− ~11 ttRR = ~10.5 min) and 9 (6.6 mg, F6.5 (1.8 mg, 96–140 mL) was ~10.5 min) mg, ttRR = ~11.8 ~11.8 min). F6.5 was further further purified purified using using subfractions F6.1–F6.10. Subfraction F6.3 (43.4 mg, 21–40 mL) was subjected to preparative RP-18 Salicireneol B (3). White, amorphous powder; −33.6 (c 0.1, MeOH); UV (MeOH)gradient: (log Ɛ) A2017, (2).White, White, amorphous powder; [α] −49.0 (c 0.1, MeOH); UV max Ɛ) 216 214 RP-18 semi-preparative HPLC with water (A) MeOH (B), flow 2max 70% BB Salicimeckol (1). powder; [α] −56 (cand 0.1, MeOH); UV λλλ max (MeOH) (log 213 RP-18 semi-preparative HPLCeluted eluted with[α] water (A) and MeOH (B), flow 2mL/min, mL/min, gradient: 70% Molecules 22, 2266 amorphous 12 of 15 (semi-preparative) HPLC, eluted with H2O (A) and MeOH (B), flow rate 2 mL/min, gradient: 75–80% 1 13 1 13 1 13 (15 min) →→70–100% B(4.04) (1 min) → for 100% B and (4 and resulted in the isolation of2; 12 m/z (0.7 mg, Hdata see Tables 11 and HR-ESI-MS (4.11), 253 (4.0), and nm; (4.3), 254 (4.02), 285 (3.91) (15 min) 70–100% B (1 min) → Bmin), (4C-NMR min), and resulted in the isolation of compound 12 Hand C-NMR data see Tables and 2;compound HR-ESI-MS (3.90), 250 (3.88), and298 295 (3.67) nm; for100% B (15 min) →−− 80–100% B (1 min) → 100% B (3 min), and led to the isolation of compounds 8 (30.5 mg, (30 12–60% min) →33 60–100% B (9 min) → 100% B (10 min), and resulted in the 10 505.2078 [M −min). H] (calculated C26 10 505.2079). t(0.7 ~11 489.2137 26H 33O 9 489.2130). 535.2185 27 35 11 535.2185). mg, tR→ = ~11 min).B (1for R = min) tR = ~10.5 min) and 9 (6.6 mg, tR = ~11.8 min). F6.5 (1.8 mg, 96–140 mL) was further purified using subfractions F6.1–F6.10. Subfraction F6.3 (43.4 mg, 21–40 mL) was subjected to preparative RP-18 25 Saliciclaireone A (1). (4). White, amorphous powder; [α] −51.6 (cMeOH 0.1, MeOH); UV λmax max (MeOH) Ɛ) B (3). White, amorphous powder; (c (c 0.1, MeOH); λmax (MeOH) (log(log 216 Salicireneol A (2). −49.0 Ɛ) 214 Salicimeckol White, amorphous powder; − 56 MeOH); UV (MeOH) ) 213 Salicimeckol (1). amorphous powder; [α] −56 0.1, max 213 RP-18 semi-preparative HPLC eluted with[α] water and (B),UV flow 2λλ mL/min, gradient: 70% B [α]−33.6 D(A) (semi-preparative) HPLC, eluted with H 2O (A) and MeOH (B), flow rate 2 mL/min, gradient: 75–80% 1and 1313C-NMR 1H13 1for 13 1HC-NMR data seeTables Tables and 2; HR-ESI-MS m/zm/z 214 (4.21), 250 (4.10), and (4.04) (3.90), 250 (3.88), and 295 (3.67) nm; and data see Tables 11and 2;2;of HR-ESI-MS (4.11), 253 (4.0), 298 (4.04) HC-NMR data see 11and 2; (4.3), 254 (4.02), and 285 (3.91) nm;nm; for (15 min) → 70–100% B (1 min) → for 100% Band (4 min), and resulted in the isolation compound 12 Hand C-NMR data see Tables and HR-ESI-MS m/z (3.90), 250 (3.88), and300 295 (3.67) nm; for B (15 min) →−− 80–100% B (1 min) → 100% B (3 min), and led to the isolation of compounds 8 (30.5 mg, −−(calculated 679.2682 [M −t[M for Cfor 33 HC43 O27 15 505.2078 505.2079). O535.2185). 535.2185 H]min). (calculated for C 489.2137 26 33 910H 489.2130). 535.2185 [M − (calculated H] 27 H 35679.2607). O (0.7 mg, RH] =− ~11 3511 11 535.2185). tR = ~10.5 min) and 9 (6.6 mg, tR = ~11.8 min). F6.5 (1.8 mg, 96–140 mL) was further purified using 25 Saliciclaireone B (5). White, amorphous powder; [α] −43.1 (c 0.1, MeOH); UV λλλ max Ɛ) A (4). White, amorphous powder; [α] −51.6 (c 0.1, MeOH); UV max (MeOH) Salicireneol B (3). White, amorphous powder; [α] (c (c 0.1, MeOH); λUV max (MeOH) (log(log Ɛ) 216 Salicireneol A (2). White, amorphous powder; [α] −49.0 (c 0.1, MeOH); max(MeOH) Ɛ)) 214 214 Salicireneol A (2). White, amorphous powder; α − 49.0 (cMeOH (MeOH) (log Salicimeckol (1). White, amorphous powder; [α] −56 0.1, MeOH); UV max 213 RP-18 semi-preparative HPLC eluted with water and (B),UV flow 2 λmL/min, gradient: 70% B [ ]−33.6 max D(A) 1 13 1 13 1 13 1 13 1 13 1 13 C-NMR data seesee Tables and 2; HR-ESI-MS m/zm/z 214 (4.08), 250 (4.09), and 298 (4.03) HR-ESI-MS (4.21), (4.10), 300 (4.04) (4.3), 254 (4.02), and (3.91) for Hand C-NMR data Tables 111and 2;2;HR-ESI-MS Hand C-NMR data see Tables 11and 2; (4.11), (4.0), and 298 (4.04) nm;nm; for Hand C-NMR data see Tables and m/z (4.3), 254 (4.02), 285 (3.91) nm; for (15253 min) → 70–100% B (1 min) → for 100% Band (4 min), and resulted in the isolation ofHR-ESI-MS compound 12 Hand C-NMR data see Tables and (3.90), 250 (3.88), and285 295 (3.67) nm; forH−−(calculated 679.2682 [M −t[M for Cfor 33 HC43 O26 15 679.2607). 33 43 15H 679.2607). 505.2078 26 33 10 505.2079). 489.2137 H]min). (calculated for C 489.2137 26 33 9O 489.2130). 535.2185 [M −−− (calculated H] 27 H 35 O 535.2185). (0.7 mg, RH] =− ~11 3311 9 489.2130). 25 Saliciclaireone C (6). Off-white, amorphous powder; −36 0.1,MeOH); MeOH);UV UV max BBB (5). White, powder; [α] −43.1 0.1, MeOH); max (MeOH) A (4). −51.6 (log Salicireneol (3). White,amorphous amorphous powder; [α] −33.6 max(MeOH) (log Ɛ) Ɛ)) 216 216 A (2). −49.0 UVλλλλλ 214 Salicireneol (3). White, powder; 33.6 (c(c UV (MeOH) (log Salicimeckol (1). White, amorphous powder; [α] −56 (c(c 0.1, max 213 [α ][α] max D − 1 13 1 13 1 13 1 13 1 13 215 252 (4.03), and 295 (3.99) nm; for Hand C-NMR data see Tables 1 and 2; HR-ESI-MS m/z (4.08), (4.09), 298 (4.03) 214 (4.06), (4.21), 250 (4.10), 300 (4.04) (4.11), 253 (4.0), (4.11), 253(4.02), (4.0), and 298 (4.04) for Hand C-NMR data see Tables and HR-ESI-MSm/z m/z (4.3), 285 (3.91) H-and and C-NMR C-NMRdata datasee seeTables Tables111and and2;2;HR-ESI-MS (3.90),254 250 (3.88), and298 295(4.04) (3.67)nm; nm;for for H649.2509 [M −[M H]− for Cfor 32 HC41 O26 14 649.2502). 679.2607). 679.2682 33 43 15H 505.2078 505.2079). 505.2078 H]−−(calculated (calculated for C O489.2130). 489.2137 [M −−− (calculated H] 26 H 33 O 535.2185 27 35 11 535.2185). 33910 10 505.2079).
C (6). amorphous powder; −36 λmax maxλ (MeOH) Saliciclaireone BB (5). White, amorphous powder; [α] (c(c0.1, MeOH); UV λ (log Ɛ) Saliciclaireone AOff-white, (4). White, amorphous (c 0.1, 0.1, MeOH); UV λ(MeOH) max (MeOH) Ɛ)) Salicireneol (3). White, amorphous powder; [α][α] −33.6 0.1, MeOH); UVUV (log(log Ɛ) 216 A (2). −49.0 214 Saliciclaireone A (4). White, amorphous powder; α−43.1 −−51.6 51.6 (c MeOH); [[α] ]25 max D 4.4. Determination of Absolute Configuration of Glycosides 1 13 1 13 1 13C-NMR 1 13 215 214 (4.06), 252 (4.03), 295 (3.99) HC-NMR data seesee Tables 1 and 2; HR-ESI-MS m/zm/z 214 (4.08), 250 (4.09), 298 (4.03) nm; for Hand C-NMR data seeTables Tables and HR-ESI-MS 214 (4.21), 250 (4.10), and 300 (4.04) nm; for (4.21), 250 (4.10), (4.04) nm; Hand data see Tables 2;2;HR-ESI-MS H-and and C-NMR data 111and HR-ESI-MS m/z (4.11), 253(4.02), (4.0), andand 298 (4.04) nm; forfor (4.3), 254 285300 (3.91) Determination of−−(calculated the glycosides their absolute configuration was achieved by recording the 649.2509 32 41 14 649.2502). 679.2682 [M −[M H]− for Cfor 33 Hand O33 679.2607). 679.2682 [M −− (calculated H] C43 33 H15 43 O 679.2607). 505.2078 26 33 10 505.2079). 679.2682 H] (calculated for C H 489.2137 9O 489.2130). 4315 15 679.2607). 1H-NMR spectra of the per-O-(S)-2-methylbutyrate (SMB) derivatives and comparison with the 1HSaliciclaireone amorphous powder; −36 UVUV λmaxλλ(MeOH) (log (log Ɛ) Saliciclaireone BOff-white, (5). White, amorphous (c 0.1, 0.1, MeOH); UV max (MeOH) Ɛ)) (4). powder; −51.6 SalicireneolC B(6). (3). White, amorphous powder; [α][α] −33.6 (c 0.1, MeOH); Ɛ) 216 Saliciclaireone BA (5). White, amorphous powder; α]25 −−43.1 43.1 (c MeOH); (MeOH) [[α] max D NMR of the SMB derivatives of reference sugars [34]. The reference sugars used in this study were 4.4. Determination of Absolute Configuration of1 Glycosides 1 13 13 1 13 1 13 215 214 (4.06), 252 (4.03), 295 (3.99) nm; for HC-NMR data seesee Tables 1 and 2; HR-ESI-MS m/zm/z 214 (4.08), 250 (4.09), and 298 (4.03) nm; for Hand C-NMR data seeTables Tables and HR-ESI-MS (4.21), (4.10), 300 (4.04) (4.08), (4.09), (4.03) nm; Hand C-NMR data see Tables 2;2;HR-ESI-MS H-and and C-NMR data 111and HR-ESI-MS m/z (4.11), 253250 (4.0), andand 298298 (4.04) nm; forfor D- and L-glucose, D- and L-arabinose, and D- and L-xylose. A quantity of 1 mg glycoside was Determination of−−(calculated the glycosides absolute configuration was achieved by recording the 649.2509 [M −[M H]− for Cfor 32 Hand O33 679.2607). 679.2682 [M −− (calculated H] C41C 33 H14their 43 O 15 679.2607). 505.2078 26 33649.2502). 10 505.2079). 679.2682 H] (calculated for H O 679.2607). 43 15 hydrolyzed using 200 µL of 2 M trifluoroacetic acid (TFA) at 121 °C for 90 min in a Wheaton vials 1H-NMR spectra of the per-O-(S)-2-methylbutyrate (SMB) derivatives and comparison with the 1Hsealed with Teflon-lined screw cap. After hydrolysis, the solvent evaporated to complete dryness (6). Off-white, amorphous powder; [α] 0.1, MeOH);UV UV max (MeOH) (MeOH) Saliciclaireone B (5). White, amorphous powder; [α][α (c MeOH); max (log A(6). (4).Off-white, −51.6 Saliciclaireone CC amorphous powder; 36was (c(c0.1, 0.1, λλλmax (log Ɛ)) −−36 ]25−43.1 NMR of the SMB of derivatives of reference of sugars [34]. 13 TheDreference sugars used in this study were 4.4. Determination Absolute Configuration Glycosides 1 under stream. 100 µL of (S)-(+)-2-methylbutyric anhydride and11100 of pyridinem/z 215nitrogen (4.06), 252 (4.03), 295 (3.99) H-and and 13C-NMR C-NMR data data see see Tables Tables andµL HR-ESI-MS m/z 214 (4.08), 250 (4.09), and295 298 (4.03) nm; for 1H(4.21),252 (4.10),Then, 300 (4.04) 215 (4.06), (4.03), and (3.99) nm; for and 2;2;HR-ESI-MS D- and L-glucose, D- and L-arabinose, and D- and L-xylose. A quantity of 1 mg glycoside was − − Determination of the glycosides and their absolute configuration was achieved by recording the were649.2509 added to mixture and incubated 121 for 4 h. The mixture was dried under nitrogen 649.2509 3232 41at 14 649.2502). 679.2682 [Mthe − H] (calculated for 33 HH 43 O 679.2607). [M − H] (calculated forCC O679.2607). 649.2502). 4115 14 °C hydrolyzed using 200 µL of 2 M trifluoroacetic acid (TFA) at 121 °C for 90 min in a Wheaton vials 1H-NMR spectra of the per-O-(S)-2-methylbutyrate (SMB) derivatives and comparison with the 1Hstream for about 8 h and 300 µL of toluene was added to the residue evaporated. The residue sealed with Teflon-lined screw cap. After hydrolysis, the solvent was evaporated to complete dryness Saliciclaireone C amorphous powder; −36 0.1, MeOH);UV UV max (MeOH) (MeOH) B (6). (5). Off-white, White, amorphous powder; [α] [α] −43.1 (c(c0.1, MeOH); λλmax (log Ɛ) 4.4. Configuration of NMR ofDetermination the SMB reference sugars [34]. The reference sugars used this study 4.4. Determination of Absolute Configuration of1 Glycosides Glycosides was dissolved in 1derivatives mLof ofAbsolute DCMofand extracted three times with 2 mL of 2 M Na 2CO3in solution and were once under stream. 100(3.99) µL of (S)-(+)-2-methylbutyric anhydride and 1100 of pyridine m/z 215 (4.06), (4.03), and 295 nm; for H- and 13C-NMR data see Tables andµL 2; HR-ESI-MS 214nitrogen (4.08), 252 250 (4.09),Then, 298 (4.03) D- and LDetermination -glucose, D-DCM and L -arabinose, and D -their and L-xylose. A quantity of 1 achieved mg using glycoside was with 2 mL H2O. The phase containing the SMB derivatives was concentrated nitrogen of the the glycosides and absolute configuration was recording − (calculated Determination of glycosides and their absolute achieved by by recording the were649.2509 added to mixture and incubated 121 °C for 4 h.configuration The mixture was dried under nitrogen [Mthe − H] for C32 41 O14 649.2502). 679.2682 33H 43at 15 679.2607). hydrolyzed using 200 µLdried ofthe 2 by M trifluoroacetic (TFA) at 121 °C for min incomparison a Wheaton vials 1then stream, completely the addition of acid 300 µL of 2-propanol and90and evaporation. Preparation 1H-NMR 1Hthe H-NMR spectra of per-O-(S)-2-methylbutyrate (SMB) derivatives and with the spectra of the per-O-(S)-2-methylbutyrate (SMB) derivatives comparison with the stream for about 8 h and 300 µL of toluene was added to the residue and evaporated. The residue sealed withfor Teflon-lined screw cap.was After hydrolysis, the [α] solvent was evaporated to complete dryness C (6). Off-white, amorphous −36 (c 0.1, MeOH); UV λused max (MeOH) (log Ɛ) 1Saliciclaireone of samples NMR measurement done bypowder; dissolving each the obtained SMB derivatives in 0.6 H-NMR of SMB derivatives of reference sugars [34]. The reference sugars in this study of the SMB derivatives ofextracted reference sugars [34]. The2of reference used in this study were 4.4. Determination of Configuration of Glycosides was NMR dissolved inthe 1 mL ofAbsolute DCM and three times with mL of 2 Msugars Na2CO 3 solution and once 1 13 1 under nitrogen stream. 100 µL of (S)-(+)-2-methylbutyric anhydride and ofglycoside pyridine 215deuterated (4.06), 252 (4.03),Then, and (3.99) nm; for H- and C-NMR data seequantity Tables 1100 andµL 2; HR-ESI-MS m/z mL of acetone. The mixture was transferred to the NMR and their spectra was D -H and -glucose, D -295 and L -arabinose, - derivatives and L -xylose. A mg D2 - mL and L2-glucose, D- and L-arabinose, and D- D and L-xylose. Atube quantity ofof11H-NMR mg glycoside withwere O. LThe DCM phase containing theand SMB was concentrated using nitrogenwas − (calculated Determination of the glycosides and their absolute configuration wasmonosaccharides achieved by recording the werehydrolyzed added to the mixture and incubated at 121 °C for 4 h. The mixture dried under nitrogen 649.2509 [M − H] for C 32H 41procedure O 14 649.2502). ◦ were recorded at 300 MHz, 298 K. The same was applied to reference with using µL M (TFA) at C for 90 Wheaton hydrolyzed using 200 200 µL of of M trifluoroacetic trifluoroacetic acid (TFA) at 121 121 and °C for 90 min min in in aaPreparation Wheaton vials vials stream, then completely dried by22the addition of 300acid µL (SMB) of 2-propanol evaporation. 1H-NMR 1Hspectra of the per-O-(S)-2-methylbutyrate derivatives and comparison with the stream for about 8 h and 300 µL of toluene was added to the residue and evaporated. The residue the exception ofTeflon-lined hydrolysis reaction. Absolute configuration and typewas of glycoside was by sealed screw cap. After hydrolysis, the solvent evaporated to complete sealed with with Teflon-lined screwwas cap. Afterby hydrolysis, the solvent evaporated to confirmed completeindryness dryness of samples forthe NMR measurement done dissolving each thewas obtained SMB derivatives 0.6were NMR of SMB derivatives ofextracted reference sugars [34]. The2of reference sugars used inof this study 4.4. Determination of Absolute Configuration of Glycosides wasunder dissolved in 1 mL of DCM and three times with mL of 2 M Na 2CO 3 solution and once comparing chemical shifts and coupling constants of anomeric proton resonances the SMB nitrogen stream. Then, 100 of (S)-(+)-2-methylbutyric anhydride and 100 pyridine under nitrogen stream.The Then, 100 µL µL oftransferred (S)-(+)-2-methylbutyric anhydride and1H-NMR 100 µL µL of of pyridine mL of deuterated acetone. mixture was toLthe NMRAtube and their spectra D2- mL andto D- and L -arabinose, and D- and -xylose. quantity of 1with mg two glycoside was withwere HL2-glucose, O. to The DCM phase containing the SMB derivatives was concentrated using nitrogen ◦C derivative that of reference monosaccharides derivative. For compounds sugar added the mixture and incubated at 121 for 4 h. The mixture was dried under nitrogen Determination of the glycosides and their absolute configuration was achieved by recording the were added to mixture incubated at 121 was °C for 4 h. The mixture monosaccharides was dried under with nitrogen werehydrolyzed recorded atusing 300the MHz, 298 K.and The same procedure applied to reference 200 µL of 2 M trifluoroacetic acid (TFA) at 121 °C for 90 min in a Wheaton vials 1 1 stream, then completely dried by the addition of 300 µL of 2-propanol and evaporation. Preparation substitutions, H-NMR of SMB derivatives were compared to a H-NMR of the SMB derivative of a 1H-NMR 1 stream for about 8 h and 300 µL of toluene was added to the residue and evaporated. The residue was of per-O-(S)-2-methylbutyrate (SMB) and with the stream forspectra 8 hthe and 300 µLAbsolute of toluene was added to derivatives the residue and comparison evaporated. The residue the exception ofabout hydrolysis reaction. configuration and type of glycoside was by Hsealed with Teflon-lined screw cap.done After hydrolysis, the solvent evaporated to confirmed completeindryness of samples forthe NMR measurement was by dissolving each of thewas obtained SMB derivatives 0.6once mixture of the two concerning reference sugars. dissolved in 1 mL of DCM and extracted three times with 2 mL of 2 M Na CO solution and NMR of SMB derivatives of reference sugars [34]. The reference sugars used in this study were 2 3 was dissolved in 1 mL of DCM and extracted three times with 2 mL of 2 M Na 2 CO 3 solution and once comparing chemicalstream. shifts Then, and coupling constants of anomeric proton resonances SMB under nitrogen 100 µL (S)-(+)-2-methylbutyric anhydride and1H-NMR 100ofµLthe of pyridine mL of deuterated acetone. The mixture wasoftransferred toLthe NMRAtube and their spectra D and L -glucose, D and L -arabinose, and D and -xylose. quantity of 1 mg glycoside was with 2 to mLthat H2O. The DCM monosaccharides phase containing the derivatives was concentrated using nitrogen derivative of reference SMBSMB Formixture compounds with under two sugar added to the mixture and incubated at 121 °Cderivative. for 4 h. The was dried nitrogen werewere recorded at 300 MHz, 298 K. The same procedure was applied to reference monosaccharides with hydrolyzed 200 µLdried ofderivatives 2 by M trifluoroacetic (TFA) at 121 °C of for 90evaporation. min in a Wheaton 1 using 1H-NMR stream, then completely the addition ofacid 300 µL of 2-propanol and Preparation substitutions, ofand SMB were was compared the SMB derivative avials stream forofH-NMR about 8h 300 µLAbsolute of toluene addedto toathe residue and evaporated. The of residue the exception hydrolysis reaction. configuration and type of glycoside was confirmed by sealed with Teflon-lined screw cap. After hydrolysis, the solvent was evaporated to complete dryness of samples for NMR measurement was done by dissolving each of the obtained SMB derivatives 0.6 mixture the two in concerning reference sugars. three times with 2 mL of 2 M Na2CO3 solution andin was of dissolved 1shifts mL ofand DCM and extracted once comparing chemicalstream. coupling constants of anomeric proton resonances ofµLthe SMB 1 under nitrogen Then, 100 µL of (S)-(+)-2-methylbutyric anhydride and 100 of pyridine mL of deuterated acetone. The mixture was transferred to the NMR tube and their H-NMR spectra with 2 to mLthat H2O. The DCM monosaccharides phase containing the derivatives was concentrated using nitrogen derivative reference SMBSMB Formixture compounds with under two sugar added toof andK.incubated 121 °Cderivative. for h. The wasmonosaccharides dried nitrogen were recorded atthe 300mixture MHz, 298 The same at procedure was4 applied to reference with 1 1 stream, then completely dried by the addition of 300 µLto ofa2-propanol Preparation substitutions, H-NMR ofand SMB derivatives were was compared H-NMR ofand theevaporation. SMB derivative of a stream for about 8 h 300 µL of toluene added to the residue and evaporated. The residue the exception of hydrolysis reaction. Absolute configuration and type of glycoside was confirmed by
Molecules 2017, 22, 2266
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with 2 mL H2 O. The DCM phase containing the SMB derivatives was concentrated using nitrogen stream, then completely dried by the addition of 300 µL of 2-propanol and evaporation. Preparation of samples for NMR measurement was done by dissolving each of the obtained SMB derivatives in 0.6 mL of deuterated acetone. The mixture was transferred to the NMR tube and their 1 H-NMR spectra were recorded at 300 MHz, 298 K. The same procedure was applied to reference monosaccharides with the exception of hydrolysis reaction. Absolute configuration and type of glycoside was confirmed by comparing chemical shifts and coupling constants of anomeric proton resonances of the SMB derivative to that of reference monosaccharides SMB derivative. For compounds with two sugar substitutions, 1 H-NMR of SMB derivatives were compared to a 1 H-NMR of the SMB derivative of a mixture of the two concerning reference sugars. 4.5. Enzymatic Deglycosidation of Cyclic Diarylheptanoid Glycosides Enzymatic hydrolysis was done using β-glucosidase (almond) to obtain the aglycone by deglycosidation of cyclic diarylheptanoid glycosides [20] with an exception for compounds 12 and 15. The enzyme hydrolysis was conducted as follows: 21.0 mg (0.040 mmol) of a compound was dissolved in 3.0 mL of 0.2 M acetate buffer (0.2 M acetic acid + 0.2 M sodium acetate, pH 4.4). The solution was treated with 40 mg of β-glucosidase and stirred. The mixture was incubated while stirring in the ultrasonic water bath at 38 ◦ C for 2 days. After incubation, the reaction mixture was mixed with 10 mL of absolute EtOH and evaporated to dryness by using a vacuum rotary evaporator at 40 ◦ C. The residues were dissolved in CHCl3 /H2 O (1 + 1), thoroughly mixed, left to settle and finally the two phases were separated. The obtained upper and lower phase were dried and separately purified by semi-preparative HPLC method eluted with water (A) and MeOH (B), flow rate: 2 mL/min, gradient: 60% B (10 min) 60–100% B (1 min) → 100% B (4 min). The collected peaks from HPLC purification of the upper and lower phase were dried under nitrogen stream and subjected to 1 H-NMR measurement. Prior to 1 H-NMR measurement, the samples were dissolved in 0.6 mL deuterated methanol, filled in the NMR tubes followed by measurement at 300 MHz, 298 K. The recorded 1 H-NMR spectra data of the obtained peaks were compared with the existing data to identify which peak stands particularly for the aglycone. 4.6. Circular dichroism Spectra: Measurement and Simulation A quantity of 0.6 mg of the isolated compounds and the obtained aglycone from enzymatic deglycosydation were dissolved in 10 mL methanol, and their CD spectra were recorded. Recorded CD spectra data were used during electronic CD spectra simulation using time-dependent density functional theory (TDDFT) quantum mechanics. Molecular models of the 11R-enantiomer of myricanol were generated with the software package Molecular Operating Environment (MOE, CCG, Montréal, Canada). After a low-mode-dynamics conformational search (default settings), three conformations were obtained within an energy window of 3 kcal/mol of which conformer 1 corresponded to the 11R,Ra and conformers 2 and 3 to two slightly different 11R,Sa forms. The 3D structures were exported to the software Gaussian 03W and completely energy-minimized using the B3LYP density functional and the 6-31D (d,p) basis set. According to these calculations, conformer 3 was the energetically most favorable form: conformer 1 had a value of 2.01, and conformer 2 was even 3.31 kcal/mol higher in energy. The Boltzmann distribution calculated with these energy differences indicates that conformer 3 would strongly dominate (97%) in a conformational equilibrium, while only 2.7% and 0.3% would be contributed by conformers 1 (11R,Ra) and 2 (11R,Sa), respectively. The geometry of the most favorable conformer 3 is in very good agreement with the crystal structure of myricanol, as published by Begley et al. [19]. Electronic CD spectra were simulated for all three conformers by performing a time-dependent DFT (TDDFT) calculation for the first 30 electronic transitions of each of the three conformers using the same basis set as mentioned above. The resulting transition vectors (R, length) were used to simulate the CD spectra for each form by multiplying them with Gaussian functions of width of 0.1 eV and summing the resulting curves
Molecules 2017, 22, 2266
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up over the whole energy/wavelength scale. Furthermore, an averaged spectrum was generated for the theoretical equilibrium mixture corresponding to 97% R,Sa (conformer 3) and 3% R,Ra-myricanol (conformer 1). The resulting spectra were compared with the experimental CD spectrum of myricanol and the obtained results were used to confirm the absolute configuration of the aglycone myricanol and other isolated diarylheptanoids. Supplementary Materials: Supplementary materials are available online. Acknowledgments: This research was financially supported by MoEVT, DAAD, and NM-AIST, who gave a grant to E. M. Fritz Kastner is kindly acknowledged for performing the NMR-experiments. Thanks are given to Josef Kiermaier and Wolfgang Söllner for acquiring the HR-ESI-MS data (all Zentrale Analytik, Fakultät Chemie und Pharmazie, Universität Regensburg). Guido Jürgenliemk is acknowledged for fruitful discussions (Pharmazeutische Biologie, Universität Regensburg). Author Contributions: E.M. isolated compounds and performed structure elucidation; T.J.S. performed the ECD simulations; and B.K. and J.H. designed the project. All authors read and approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–16 are available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
