Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Melanogenesis-Inhibitory and Cytotoxic Activities of Triterpene Glycoside Constituents from the Bark of Albizia procera Jie Zhang,†,‡ Toshihiro Akihisa,⊥ Masahiro Kurita,⊥ Takashi Kikuchi,∥ Wan-Fang Zhu,† Feng Ye,† Zhen-Huan Dong,▽ Wen-Yuan Liu,▽ Feng Feng,*,†,‡,§ and Jian Xu*,†
J. Nat. Prod. Downloaded from pubs.acs.org by YORK UNIV on 12/06/18. For personal use only.
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Department of Natural Medicine Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China ‡ Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, People’s Republic of China § Jiangsu Food and Pharmaceutical Science College, Huaian, Jiangsu 223003, People’s Republic of China ⊥ College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku, Tokyo 101-8308, Japan ∥ Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569-1094, Japan ▽ Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *
ABSTRACT: Five oleanane-type triterpene glycosides including three new ones, proceraosides E−G (1−3), were isolated from a MeOH-soluble extract of Albizia procera bark. The structures of 1−3 were determined by use of NMR spectra, HRESIMS, and chemical methods. Compounds 1−5 exhibited inhibitory activities against the proliferation of the A549, SKBR3, AZ521, and HL60 human cancer cell lines (IC50 0.28− 1.8 μM). Additionally, the apoptosis-inducing activity of compound 2 was evaluated by Hoechst 33342 staining and flow cytometry, while the effects of 2 on the activation of caspases-9, -8, and -3 in HL60 cells were revealed by Western blot analysis.
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RESULTS AND DISCUSSION After removal of the lipid components of the A. procera bark by extracting with hexane, the residue was extracted with MeOH to give the soluble hydrophilic extract, which was separated into EtOAc, n-BuOH, and H2O fractions. Then, biological methods were used for evaluating the melanogenesis-inhibitory and cytotoxic activities of these crude extracts and fractions on α-melanocyte-stimulated hormone (α-MSH)-stimulated B16 melanoma cells and several human cancer cell lines, respectively. As compiled in Table S1 (Supporting Information), all crude extracts and initial fractions showed potential melanogenesis-inhibitory activities. Upon evaluation of cytotoxic activities, the hexane and MeOH extracts, as well as nBuOH fraction, exhibited inhibitory effects against the HL60, AZ521, HepG2, HT29, Hela, KB, SKBR3, and A549 cell lines (Table S2, Supporting Information). Among these, the nBuOH fraction exhibited the most promising inhibitory effects (IC50 3.2−20.5 μg/mL). Investigation of the crude n-BuOH fraction using column chromatography and reversed-phase HPLC afforded three undescribed triterpene glycosides, proceraosides E−G (1−3),
lbizia procera (Roxb.) Benth (Leguminosae) is distributed widely in Southeast and South Asia to New Guinea and northern Australia.1−3 This species has been used medicinally in Thailand, especially in the Lanna region for the treatment of several diseases, including cancer.4 The bark of A. procera is used traditionally for alleviating labor pains, stomachache, and ulcers, and this plant part is also used as a fish poison.3,5 The leaves of the A. procera are employed as a forage, which has both high nutritive value and widespread availability.6 A. procera gum can be used as a natural emulsifier and excipient for foods and drugs.7−9 Previous studies revealed that this plant possesses multiple bioactivities, such as antioxidant,10 cytotoxic,11 antiplasmodial,12 analgesic, antibacterial, and central nervous system depressant activities.5 Extensive studies on the phytochemical constituents of A. procera revealed the predominance of phenolic substances and saponins.13−16 To more fully exploit the potential bioactive compounds of A. procera bark, herein are reported the isolation of five oleananetype saponins, including three new triterpene glycosides (1− 3), as well as the evaluation of their cytotoxic activities against lung fibroblast (WI38), breast carcinoma (SKBR3), oral epidermal carcinoma (KB), cervical adenocarcinoma (HeLa), colon adenocarcinoma (HT29), hepatocellular carcinoma (HepG2), lung adenocarcinoma (A549), and promyelocytic leukemia (HL60) human cell lines. © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 2, 2018
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DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
m/z 1685.7393 [(M+Na)−166−146−168]+ (at C-21, loss of an acyl chain comprising two MA moieties and one Qui moiety), 1577.8161 [(M+Na)−(2 × 132)−(2 × 162)]+ (at C3, loss of a sugar chain comprising one Ara moiety, one Xyl moiety, and two Glc moieties), and 1563.8125 [(M+Na)−(2 × 162)−132−146]+ (at C-28, loss of a sugar chain comprising one Rha moiety, one Araf moiety, and two Glc moieties). Upon acid hydrolysis of 1, the sugar moieties were afforded, which were identified by GLC analysis of their trimethylsilyl thiazolidine derivatives. The observed sugar moieties of Glc, Xly, and Qui were confirmed to have the D-configuration, with the Ara, Rha, and Araf moieties present having the Lconfiguration. Therefore, the structure of 21-O-{(6′S)-(2′,6′dimethyl)-6′-O-[4-O-(6″S)-menthiafolyl-β-D-quinovopyranosyl)]-7′-octenoyl]}-3-O-{β-D-xylopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 6)-[β-D-glucopyranosyl]-(1 → 2)-β-Dglucopyranosyl} machaerinic acid 28-O-β-D-glucopyranosyl-(1 → 3)-[α-L-arabinofuranosyl-(1 → 4)]-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl ester (1) were established. Proceraoside F (2) possess a molecular formula of C101H162O49 based on its HRESIMS ([M + Na]+ m/z 2182.0145). In its 1H NMR spectrum, signals for seven tertiary methyls, an olefinic methine, and three secondary oxymethines were observed, and together with selected signals in the 13C NMR spectrum (Table 2) were consistent with those of an acacic acid lactone moiety.19 These NMR spectra suggested that 2 is an analogue of 1, except for an additional signal δH 5.23 (H-16 of the agrycone) of a secondary oxymethine unit in 2. Furthermore, on comparison of the acyl moiety (MA1-Qui-MA2) at C-21 for 2 with that of 1, the resonances of δH 1.45 (m, H-5′), 1.47 (s, H-9′), 5.23, 5.37 (each d, J = 11.5 Hz, H-8′), and 6.32 (dd, J = 17.9, 11.0 Hz, H7′) centering around C-6′ of the inner monoterpenoid acid lactone (MA1) moiety, displayed significant differences in 1H NMR spectrum. This observation was further confirmed by the 13 C NMR spectrum, since the 13C NMR chemical shift of 2 and 1 were almost identical in the aglycon and sugar parts, as
along with two known analogues, proceraoside B (4) and proceraoside D (5).14 The known glycosides were identified by comparison of MS, 1H NMR, and 13C NMR spectroscopic and optical rotation data with the corresponding literature values. Proceraoside E (1) exhibited a molecular ion peak at m/z 2166.0159 [M + Na]+ in the HRESIMS, suggesting a molecular formula of C101H162O48. The 1H and 13C NMR data (Tables 1 and 2) of the aglycone of 1 were in accordance with analogous data for machaerinic acid lactone.17 The anomeric proton and carbon signals observed suggested that compound 1 contains sugar moieties, comprised of four βglucose (Glc1, Glc2, Glc3, and Glc4) units, and a unit each of αarabinose (Ara), β-xylose (Xyl), α-rhamnose (Rha), αarabinofuranose (Araf), and β-quinovose (Qui). Also present were the signals for the vinyl methyls and double bonds of two monoterpenoid acid lactone moieties (MA1 and MA2)14 in the 1D NMR spectra. Moreover, in the 1H NMR spectrum, a pair of resonances of equal intensity at δH 1.45 (s, H-9″), 1.54 (s, H-9′), 5.16, 5.53 (d, J = 10.5, and 16.9 Hz, H-8″), 5.30, 5.44 (each d, J = 10.1, and 17.4 Hz, H-8′), 6.10 (dd, J = 16.9, 10.5 Hz, H-7″), and 6.21 (dd, J = 17.4, 10.1 Hz, H-7′) of MA1 and MA 2 were assigned to the (6′S)- and (6″S)-isomer, respectively.14,18 In the HMBC spectrum of 1, diagnostic long-range correlations were observed between δH 5.08 (H-1 of Xyl) and δC 80.7 (C-2 of Ara), δH 5.16 (H-1 of Ara) and δC 69.1 (C-6 of Glc1), δH 5.30 (H-1 of Glc2) and δC 75.9 (C-2 of Glc1), δH 4.89 (H-1 of Glc1) and δC 88.8 (C-3 of the aglycone), and between δH 6.21 (H-1 of Araf) and δC 79.0 (C4 of Rha), δH 5.25 (H-1 of Glc4) and δC 81.9 (C-3 of Rha), δH 5.92 (H-1 of Rha) and δC 76.9 (C-2 of Glc3), δH 6.01 (H-1 of Glc3) and δC 174.9 (C-28 of the aglycone), as well as between δH 5.37 (H-4 of Qui) and δC 167.7 (C-1″ of MA2), δH 4.84 (H-1 of Qui) and δC 80.2 (C-6′ of MA1), δH 5.15 (H-21 of the aglycone) and δC 176.2 (C-1′ of MA1). This indicated the two sugar chains were located at C-28 and C-3 of the aglycon, and an acyl moiety (MA1-Qui-MA2) at C-21 (Figure 1). The HRESIMS2 experiment of compound 1 showed fragments at B
DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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Table 1. 1H NMR Spectroscopic Data of (δ in ppm, J in Hz, 400 MHz) of Compounds 1−3 in C5D5N position 1 2 3 5 6 7 9 11 12 15 16 18 19 21 22 23 24 25 26 27 29 30 Glc1-1 2 3 4 5 6 Ara (Fuc)-1 2 3 4 5 6 Xly-1 2 3 4 5 Glc2-1 2 3 4 5 6
1 1.58 1.28 1.98 2.21 3.43, 0.89 1.5 1.31 1.75 1.29 1.75 2 5.46, 2.26 2.02 2.13 1.85 3.16, 2.91, 1.37 5.15, 2.70, 2.18 1.24, 1.13, 0.94, 1.11, 1.31, 0.96, 0.98, 4.89, 4.23 4.17 4.15 4.11 4.59 4.18 5.16, 4.51 4.39 4.41 4.4 3.75
2
aglycon 1.62 1.3 1.87 2.32 d (11.0) 3.42 d (11.5) 0.86 1.5 1.31 1.67 1.36 1.87 2.08 brs 5.48, brs 2.01 2.15 5.23, brs d (14.2) t (14.2)
3.21, 2.95, 1.41 d (11.2) 5.13, m 2.7 2.18 s 1.27, s 1.13, s 0.97, s 1.16, s 1.86, s 1.05, s 1.09, sugars-1 d (7.8) 4.86, 4.25 4.16 4.13 4.14 4.61 4.11 d (7.8) 5.16, 4.55 4.42 4.43 4.35 3.76
5.08, d (6.6) 4.05 4 4.1 4.45 3.59 5.30, d (7.8) 4.1 4.13 4.07 3.9 4.39 4.24
d (13.7) t (13.7) d (11.5)
s s s s s s s d (7.7)
d (7.8)
5.04, d (6.5) 4.06 4 4.12 4.43 3.64 5.33, d (7.8) 4.08 4.12 4.06 3.92 4.45 4.35
3 1.6 1.28 1.98 2.24 3.60 d (11.0) 0.88 1.51 1.33 1.71 1.33 1.82 2.01 5.47, brs 2.31 2.01 2.15 1.87 3.20, d (14.0) 2.91, t (14.0) 1.39 5.13, d (11.5) 2.7 2.19 1.30, s 1.17, s 0.98, s 1.21, s 1.37, s 0.95, s 0.98, s 4.90, d (7.3) 4.27 4.16 4.15 4.13 4.6 4.14 5.01, d (7.8) 4.46 4.15 4.03 3.76 1.51, d (6.4) 5.04 d 6.9 4.06 4 4.11 4.45 3.6 5.33, d (7.3) 4.1 4.13 4.07 3.91 4.41 4.28
position Glc3-1 2 3 4 5 6
6.01, 4.11 3.96 4.17 3.97 4.39 4.27 Rha-1 5.92, 2 5.19, 3 4.88 4 4.51 5 4.5 6 1.75, Glc4-1 5.25, 2 4 3 4.18 4 4.31 5 3.98 6 4.32 4.13 Araf-1 6.21, 2 4.95 3 4.78, 4 4.71, 5 4.46 4.07 MA1-Qui-MA2 MA1-2′ 2.36 3′ 1.73 4′ 2.2 5′ 1.69 7′ 6.21, 8′ 5.30, 5.44, 9′ 1.54, 10′ 1.06, Qui-1 4.81, 2 3.77 3 4.19 4 5.37 5 3.65, 6 1.33, MA2-3″ 7.08, 4″ 2.35 5″ 1.77 7″ 6.10, 8″ 5.16, 5.53, 9″ 1.45, 10″ 1.89,
1 d (7.8)
brs d (1.6)
d (6.0) d (7.3)
brs m m
dd (17.4, 10.1) d (10.1) d (17.4) s d (6.9) d (7.3)
dd (6.4, 9.6) d (6.0) t (7.3)
dd (16.9, 10.5) d (10.5) d (16.9) s s
2
3
sugars-2 6.06, d (7.8) 4.14 3.92 4.18 3.97 4.41 4.25 5.87, brs 5.17, d (1.6) 4.96 4.5 4.61 1.79, d (5.5) 5.32, d (7.8) 3.93 4.09 4.27 3.99 4.47 4.3 6.26, brs 4.75 4.83, m 4.99, m 4.47 4.14
6.11, 4.19 3.99 4.25 3.99 4.45 4.34 6.02, 5.22, 4.99 4.58 4.67 1.81, 5.34, 3.95 4.1 4.29 3.99 4.48 4.29 6.24, 4.83 4.81, 4.92, 4.48 4.16
2.65 1.66 1.78 1.45 6.32, 5.23, 5.37, 1.47, 1.10, 4.80, 3.76 4.19 5.36 3.65 1.36, 7.13, 2.46 1.73 6.13, 5.18, 5.56, 1.46, 1.92,
2.38 1.75 2.21 1.69 6.25, 5.32, 5.46, 1.55, 1.06, 4.86, 3.77 4.18 5.36 3.65 1.37, 7.14, 2.46 1.74 6.13, 5.18, 5.58, 1.46, 1.92,
dd (17.9,11.0) d (11.5) d (11.5) s d (7.0) d (7.8)
d (6.0) t (7.3)
dd (16.9, 10.5) dd (11.0, 1.4) dd (17.2, 1.4) s s
d (7.8)
brs d (1.6)
d (5.5) d (7.5)
br s m m
dd (18.1, 11.0) d (11.0) d (17.4) s d (6.9) d (7.8)
d (6.4) t (7.3)
dd (17.4, 10.5) dd (10.8, 1.8) dd (17.1, 1.8) s s
a
Overlapped signals are reported without designating multiplicity. C
DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Table 2.
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C NMR Spectroscopic Data (d in ppm, 100 MHz) of Compounds 1−3 in C5D5N
position
1
2
position
3
aglycon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc1-1 2 3 4 5 6 Ara (Fuc)-1 2 3 4 5 6 Xly-1 2 3 4 5 Glc2-1 2 3 4 5 6
38.9 26.8 88.8 39.8 55.9 18.9 33.4 39.6 48.0 37.0 24.0 112.4 142.8 42.2 35.3 24.7 48.6 41.2 46.6 35.3 75.2 36.3 28.0 16.9 15.8 17.3 25.8 174.9 29.0 18.5 sugars-1 104.9 75.9 78.2 72.5 77.0 69.1 102.3 80.7 72.6 67.1 64.3
39.1 26.8 89.0 40.1 56.0 18.8 33.8 39.8 48.1 37.3 24.1 123.2 143.0 42.4 36.0 74.1 48.8 41.1 47.2 35.4 75.7 36.6 28.3 17.1 16.1 17.5 25.8 174.7 29.5 19.3
39.0 27.0 88.0 39.8 56.0 18.9 33.6 39.9 48.2 37.2 24.0 123.1 143.0 42.4 28.7 24.1 48.8 41.4 46.6 35.4 75.3 36.5 28.2 17.0 15.9 17.5 26.0 175.0 29.0 18.6
106.5 76.1 78.5 72.7 77.1 69.4 102.4 80.7 72.8 67.5 64.4
106.3 75.5 78.1 70.7 67.4 105.7 75.4 77.1 71.7 78.2 62.7
106.1 75.6 78.4 70.7 67.5 105.9 75.9 77.2 71.8 78.4 62.9
106.7 75.9 78.5 71.9 77.0 70.0 103.5 82.5 75.2 72.2 71.4 17.3 107.1 76.1 77.6 70.8 67.3
Glc3-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Glc4-1 2 3 4 5 6 Araf-1 2 3 4 5 MA1-Qui-MA2 MA1-1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ Qui-1 2 3 4 5 6 MA2-1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″
well as the difference (δ2−δ1) were observed only for C-3′ (+0.5 ppm), C-4′ (+0.5 ppm), C-5′ (−0.6 ppm), and C-8′ (−0.7 ppm) were in good agreement with those reported in the literature, 18,19 which were revealed that the MA 1
1
2
3
sugars-2 95.3 76.9 78.8 71.1 79.1 62.0 101.7 70.7 81.9 79.0 68.8 18.8 105.4 75.4 78.0 71.7 78.2 62.3 110.7 84.6 78.2 84.5 64.4
95.8 76.7 78.6 71.3 79.2 62.2 102.1 71.0 82.0 79.2 69.3 19.1 105.1 75.3 78.1 71.9 78.5 62.6 111.1 85.5 78.4 84.7 62.9
95.5 76.7 78.5 71.3 79.1 62.1 101.9 71.0 81.9 79.3 69.0 18.9 105.8 75.5 78.2 71.8 78.5 62.8 111.1 85.0 78.4 84.7 62.5
176.2 40.0 34.5 21.8 42.2 80.2 144.2 115.1 23.9 17.0 99.2 75.4 75.9 77.1 70.1 18.4 167.7 127.8 143.6 23.9 41.4 72.1 146.4 111.8 28.5 12.6
176.6 40.3 35.0 22.3 41.6 80.3 144.5 114.4 23.5 17.3 99.3 75.6 75.8 77.4 70.3 18.6 167.9 128.0 143.8 24.2 41.7 72.3 146.7 111.9 28.8 12.8
176.3 40.0 34.6 21.9 42.2 80.3 144.4 115.1 23.4 17.3 99.5 75.7 75.7 77.3 70.2 18.5 167.9 127.9 143.7 24.2 41.6 72.3 146.7 111.8 28.7 12.7
configuration was in the form of a (6′R)-isomer in 2. The fragments at m/z 1701.7356 [(M+Na)−166−146−168]+ (at C-21, loss of an acyl chain comprising two MA moieties and one Qui moiety), 1593.8024 [(M+Na)−(2 × 132)−(2 × D
DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Selected key COSY (thick black line), and HMBC (→) correlations of compound 1.
162)]+ (at C-3, loss of a sugar chain comprising two Glc moieties, one Xyl moiety, and one Ara moiety), and 1579.8006 [(M+Na)−(2 × 162)−132−146]+ (at C-28, loss of a sugar chain comprising two Glc moieties, one Araf moiety, and one Rha moiety) in the HRESIMS2 of 2 were observed. Acid hydrolysis of 2 afforded L-rhamnose, D-glucose, D-quinovose, Dxylose, L-arabinofuranose, and L-arabinose as the sugar moieties, as determined after derivatization by GLC analysis. Taken together, the structure of 2 was defined as 21-O-{(6′R)(2′,6′-dimethyl)-6′-O-[4-O-(6″S)-menthiafolyl-β-D-quinovopyranosyl)]-7′-octenoyl]}-3-O-{β-D-xylopyranosyl-(1→2)-α-Larabinopyranosyl-(1→6)-[β-D-glucopyranosyl]-(1→2)-β-D glucopyranosyl} acacic acid 28-O-β-D-glucopyranosyl-(1→3)[α-L-arabinofuranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)β-D-glucopyranosyl ester. Proceraoside G (3) has a molecular formula of C102H164O48, which was deduced from the [M + Na]+ ion peak at m/z 2180.0326 in the HRESIMS. The 1H and 13C NMR spectra (Tables 1 and 2) of the aglycone resonances of 3 were essentially the same as those of 1. However, a difference occurred in the 1D NMR spectrum, which showed a tertiary methyl signal at δH 1.51 (d, J = 6.4 Hz, Fuc-6) and an anomeric signal at δH 5.01 (d, J = 7.8 Hz, Fuc-1), along with a tertiary methyl signal at δC 17.3 due to the presence of a fucose (Fuc) moiety. This observation was further clarified from the HMBC spectrum, in which correlations between δH 4.90 (H-1 of Glc1) and δC 88.0 (C-3 of the aglycone), δH 5.01 (H-1 of Fuc) and δC 70.0 (C-6 of Glc1), and δH 5.04 (H-1 of Xyl) and δC 82.5 (C-2 of Fuc) revealed that the fucose moiety replaced an arabinose moiety in the sugar chain at C-3 of aglycone. Its HRESIMS2 exhibited fragments at m/z 1699.7562 [(M+Na)− 166−146−168]+ (at C-21, loss of an acyl chain comprising two MA moieties and one Qui moiety), 1577.8196 [(M+Na)− 132−146−(2 × 162)]+ (at C-3, loss of a sugar chain comprising two Glc moieties, one Xyl moiety, and one Fuc moiety, or at C-28, loss of a sugar chain comprising two Glc moieties, one Araf moiety, and one Rha moiety). Upon acid hydrolysis, 3 furnished the same aglycone moiety of 1, as well as D-quinovose, D-fucose, D-xylose, D-glucose, L-rhamnose, and
L-arabinofuranose.
Hence, the structure of 3 was proposed as 21-O-{(6′S)-(2′,6′-dimethyl)-6′-O-[4-O-(6″S)-menthiafolyl-βD-quinovopyranosyl)]-7′-octenoyl]}-3-O-{β-D-xylopyranosyl(1→2)-β-D-fucopyranosyl-(1→6)-[β-D-glucopyranosyl]-(1→ 2)-β-D-glucopyranosyl} machaerinic acid 28-O-β-D-glucopyranosyl-(1→3)-[α-L-arabinofuranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl ester. It is noteworthy that triterpenoid glycosides have been encountered frequently in Albizia species.14,20−22 Acacic acid lactone-type and echinocystic acid-type triterpene glycosides were also isolated from A. procera, Albizia inundata, and Acacia ligulata,15,16,23,24 while oleanolic acid-type triterpene glycosides were isolated from Albizia anthelmintica.25,26 Some of these naturally occurring triterpene glycosides may also contain Nacetylglucosamine, with such compounds exhibiting antiproliferative activities.27,28 The melanogenesis inhibitory activity and safety of compounds 1−5 were determined in α-MSH-stimulated B16 melanoma cells and B16 melanoma cells, respectively. The risk/benefit ratio of each compound was calculated as in previous literature.29 The results as shown in Table 3, all isolated triterpene glycosides were proved to be lower-risk melanogenesis inhibitors (42.5−89.2% melanin content, and 100.1−110.2% cell viability), and exhibited superior melanogenesis inhibitory activities than the positive control, arbutin. Therefore, compounds 1−5 might be responsible, in part, for the melanogenesis-inhibitory activities of A. procera bark extracts. Moreover, compounds 1−5 were evaluated for their cytotoxic activities against the HL60, AZ521, SKBR3, and A549 human cancer cell lines. The results (Table 4) showed that these compounds possessed potential cytotoxicities against all cell lines with IC50 values in the range of 0.28− 2.6 μM. Moreover, WI38 normal cells were also used for the evaluation of selectivity index, and compounds 1−5 displayed high selectivities for WI38/A549 (SI 1.8−16.9). Hoechst 33342 staining and cell cycle analysis were used for exposing the cytotoxicity mechanism of 2 on HL60 cells.31 As shown in Figure 2, the typical morphological features of E
DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
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used to measure melting points and optical rotations, respectively. UV and IR spectra were obtained on a JASCO V-630Bio spectrophotometer and a JASCO FTIR-300 E spectrometer, respectively. A JEOL ECX-400 spectrometer and an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) system were used to record NMR spectra and HRESIMS, respectively. GLC: Shimadzu GC-2014 instrument on a DB-17 fused silica glass capillary column (Agilent Technologies, Inc., Santa Clara, CA, USA). Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), ODS (100−200 mesh; Fuji Silysia Chemical, Ltd., Aichi, Japan), and Silica gel (230−400 mesh; Merck) were employed for column chromatography (CC). Reversed-phase preparative HPLC with a refractive index detector was carried out on ODS columns (Senshu Scientific Co., Ltd., Tokyo, Japan); on a Pegasil ODS SP100 column (250 × 10 mm i.d.) with CH3CN− H2O−HCOOH [39:61:0.2 (HPLC system II)] or with CH3CN− H2O−HCOOH [41:59:0.2 (HPLC system III)] at the flow rate of 3.0 mL·min−1; or on a Pegasil ODS column (250 × 20 mm i.d.) with CH3CN−H2O−HCOOH [40:60:0.2 (HPLC system I)] at the flow rate of 10.0 mL·min−1. Plant Material. The A. procera bark sample was collected at Lampang, Thailand, in May, 2015. Dr. W. Kitdamrongthama (Faculty of Pharmacy, Chiang Mai University) identified this plant material. A voucher specimen (No. 20150050) was deposited at the Faculty of Pharmacy, Chiang Mai University, Thailand. Extraction and Isolation. The whole bark was oven-dried at 60 °C over 48 h and decorticated, and crushed into a powder. The pulverized sample (2110.5 g) was extracted successively with hexane (3 × 6 L) and MeOH (3 × 6 L) under reflux to yield hexane extract (6.7 g) and MeOH extract (296.8 g), respectively. The MeOH extract was suspended in H2O, and partitioned sequentially with EtOAc and n-BuOH. The n-BuOH partition (105.0 g) was applied to CC [Diaion HP-20 (1000 g)] eluted with MeOH−H2O (0:1 → 1:0) to afford six pooled fractions, A−F. Fraction E (32.1 g) was separated by an ODS CC [650 g; MeOH−H2O (0:1 → 7:3)] to give five subfractions, E1−E5. Sunfraction E3 (19.5 g) was purified by silica gel CC [360 g; CHCl3− MeOH (19:1 → 0:1)] to afford six additional subfractions, E3−1− E3−6. HPLC (system II) of fraction E3−2 yielded compounds 4 (9.6 mg, tR = 32.5 min) and 5 (23.8 mg, tR = 26.5 min). Further HPLC (system I) of fraction E3−3 yielded compound 2 (24.2 mg, tR = 30.0 min; purity >95%) and an impure mixture, which were purified by HPLC (system III) to afford compound 1 (60.0 mg, tR = 42.5 min; purity >95%). Compound 3 (9.2 mg, tR = 65.5 min; purity >95%) was also obtained by HPLC (system I). Proceraoside E (1). White, amorphous powder, mp 206−208 °C; [α]25D − 10.2 (c 1.21, MeOH); UV (MeOH) λmax (log ε) 205 (3.98), 254 (2.06) nm; IR (KBr) vmax 3417, 2937, 1715, 1460, 1367, 1075 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 2166.0159 [M + Na]+ (calcd for C101H162O48Na, 2166.0133). HRESIMS2 m/z 1685.7393 [(M+Na)-MA2-Qui-MA1]+, 1577.8161 [(M+Na)-Xyl-Ara-Glc1-Glc2]+, 1563.8125 [(M+Na)-Glc4-Araf-RhaGlc3]+, 1097.5421 [(M+Na)-Xyl-Ara-Glc1-Glc2-MA2-Qui-MA1]+, 1083.5385 [(M+Na)-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+, 975.6253
Table 3. Melanogenesis Inhibitory Activities of Compounds 1−5a melanin content (%) 10 μM
100 μM
89.2 ± 0.7 80.9 ± 6.3 86.5 ± 2.3 74.3 ± 0.7 86.2 ± 0.5 92.7 ± 4.6
75.2 ± 0.5 50.7 ± 4.4 79.6 ± 1.9 42.5 ± 0.5 80.0 ± 0.3 81.5 ± 1.3
compound 1 2 3 4 5 arbutinc
A/C ratiob
cell viability (%) 10 μM 110.2 0.9 103.2 3.0 109.8 3.3 107.4 0.9 106.8 2.5 102.3 1.5
± ± ± ± ± ±
100 μM 102.1 0.2 100.1 6.7 103.8 3.6 102.3 0.2 104.2 0.3 101.0 6.3
10 μM
100 μM
±
0.81
0.74
±
0.78
0.51
±
0.79
0.77
±
0.69
0.42
±
0.81
0.77
±
0.91
0.81
a
Melanin content and cell viability were determined based on the absorbances at 405 and 570 (test wavelength)−630 (reference wavelength) nm, respectively, by comparison with those for DMSO (100%). Each value represents the mean ± SD (n = 3). Concentration of DMSO in the sample solution was 2 μL·mL−1. bActivity-tocytotoxicity (A/C) ratio, which was obtained by dividing the melanin content (%) by the cell viability (%). cPositive control.
apoptosis, such as fragmentation of nuclei and chromatin condensation, in the experimental cells were obvious after treatment with compound 2 (1.0 and 3.0 μM) for 24 h. In Figure 3, after treatment with compound 2 (3.0 μM) in HL60 cells for 24 h, the early and late apoptotic rate of cells were 19.0% and 22.7%, with 2.4% and 3.0% of negative control, respectively; while the early and late apoptotic rate of cells were increased to 38.2% and 42.2%, with 0.8% and 1.9% of negative control, respectively. These results revealed that compound 2 can induce apoptotic HL60 cell death. It is well-known that the caspase pathway plays a key role in apoptosis.30 To further clarify the cytotoxicity mechanism of 2, the effects of this active molecule on the activation of caspases3, -8, and -9 in HL60 cells were analyzed by Western blotting. As shown in Figure 4, after treatment with compound 2 (3.0 μM) for 24 and 48 h, the levels of procaspases-3, -8, and -9 in HL60 cells were significantly down-regulated, while the levels of the cleaved caspases-3, -8, and -9 were remarkably upregulated. The results clearly indicated that compound 2 possesses potential cytotoxic activity, as a result of activating the mitochondrial apoptotic pathway.
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EXPERIMENTAL SECTION
General Experimental Procedures. A Yanagimoto micromelting point apparatus and a JASCO P-2200 polarimeter were
Table 4. Cytotoxic Activities (IC50 ± S.D., μM) of Compounds 1−5 cell lines,a IC50 ± SD (μM) compound 1 2 3 4 5 cisplatin
HL60 0.5 0.3 0.6 0.9 0.9 4.2
± ± ± ± ± ±
0.04 0.01 0.06 0.11 0.09 1.12
AZ521 0.4 0.5 0.6 1.7 1.2 9.5
± ± ± ± ± ±
0.01 0.04 0.08 0.24 0.13 0.52
SKBR3 1.1 ± 1.3 ± 2.6 ± 0.4 ± 0.8 ± >10
0.04 0.30 0.54 0.02 0.06
SIb A549 0.66 ± 0.2 0.3 ± 0.02 1.8 ± 0.11 1.3 ± 0.28 1.0 ± 0.08 >10
WI38 2.2 ± 5.1 ± 4.8 ± 2.5 ± 8.3 ± >10
0.47 0.41 0.65 0.02 0.12
(wi38/A549) 3.4 16.9 2.7 1.8 8.2 1.1
Cell lines were treated with compounds (1 × 10−4 to 1 × 10 −6 M) for 48 h, and cell viability was analyzed by the MTT assay. IC50 Values based on triplicate five points. bSelectivity index. cPositive control.
a
F
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Figure 2. Detection of cell shrinkage induced by compound 2, and fragmentation by nuclear staining with Hoechst 33342 in HL60 cells, which were cultured for 24 h.
Figure 3. Induction of apoptosis against HL60 cells treated with compound 2 (PI: propidium iodide; N: negative control; C: compound 2). (A) HL60 cells were cultured with 2 (3.0 μM) for 0, 24, and 48 h. Each value is the mean of three experiments. (B) Early apoptosis of HL60 cells. (C) Late apoptosis of HL60 cells. Significantly different (***p < 0.001) compared with control model group. [(M+Na)-Xyl-Ara-Glc1-Glc2-Glc4-Araf-Rha-Glc3]+, 495.3413 [(M +Na)-Xyl-Ara-Glc1-Glc2-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+. Proceraoside F (2). White, amorphous powder, mp 194−196 °C; [α]25D − 16.6 (c 1.25, MeOH); UV (MeOH) λmax (log ε) 205 (4.11), 262 (2.61) nm; IR (KBr) vmax 3414, 2935, 1710, 1455, 1386, 1075 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 2182.0145 [M + Na]+ (calcd for C101H162O49Na, 2182.0082). HRESIMS2 m/z 1701.7356 [(M+Na)-MA2-Qui-MA1]+, 1593.8024
[(M+Na)-Xyl-Ara-Glc1-Glc2]+, 1579.8006 [(M+Na)-Glc4-Araf-RhaGlc3]+, 1113.5284 [(M+Na)-Xyl-Ara-Glc1-Glc2-MA2-Qui-MA1]+, 1099.5248 [(M+Na)-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+, 991.5916 [(M+Na)-Xyl-Ara-Glc1-Glc2-Glc4-Araf-Rha-Glc3]+, 511.3176 [(M +Na)-Xyl-Ara-Glc1-Glc2-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+. Proceraoside G (3). White, amorphous powder, mp 204−207 °C; [α]25D − 13.1 (c 1.15, MeOH); UV (MeOH) λmax (log ε) 205 (4.37), 250 (2.14) nm; IR (KBr) vmax 3416, 2936, 1712, 1458, 1373, 1072 G
DOI: 10.1021/acs.jnatprod.8b00167 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 4. Western blot analysis of caspases-3, -8, and -9 in HL60 cells with compound 2 (3.0 μM) for 24 and 48 h. The results are from one representative experiment among three runs, showed similar patterns. Significantly different (***p < 0.001) compared with the initial model group.
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cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 2180.0326 [M + H]+ (calcd for C102H164O48Na, 2180.0290). HRESIMS2 m/z 1699.7562 [(M+Na)-MA2-Qui-MA1]+, 1577.8196 [(M+Na)-Xyl-Fuc-Glc1-Glc2]+ or [(M+Na)-Glc4-Araf-Rha-Glc3]+, 1097.5485 [(M+Na)-Xyl-Fuc-Glc1-Glc2-MA2-Qui-MA1]+ or [(M +Na)-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+, 975.6187 [(M+Na)-XylFuc-Glc1-Glc2-Glc4-Araf-Rha-Glc3]+, 495.3349 [(M+Na)-Xyl-FucGlc1-Glc2-Glc4-Araf-Rha-Glc3-MA2-Qui-MA1]+. Acid Hydrolysis. The sugar residues of compounds (1−3) were obtained using an acid hydrolysis method.34 Briefly, after each of the pure compounds (3.0 mg) was hydrolyzed in the mixed solution comprising 2 M aq. CF3COOH (11.0 mL) and H2O (2.0 mL), the mixture was extracted with EtOAc, and the aqueous residue was then evaporated to give total sugar residues, which were compared with standard sugars using a TLC method. Then, GLC analysis of the chiral trimethylsilyl thiazolidine derivatives of sugar residues were employed to confirm the absolute configuration of these sugar residues.35 The details were shown in S26, Supporting Information. Melanogenesis-Inhibitory Assay. The cell culture conditions were following the previous literature.31−33 The melanin levels in experimental cells were determined as described in the previous literature.36 Briefly, the B16F10 cells were incubated in 24-well plates. The cells in the model group were stimulated with 0.10 μM α-MSH for 48 h, and the cells in the treatment group were cotreated with the test samples and 0.10 μM α-MSH for 48 h. Then, 100 μL of 2 N NaOH-10% DMSO solution were used for solubilize the melanin in harvested cells, and a microplate reader (Tecan Japan Co., Ltd., Kawasaki, Japan) was employed for recording the absorbance value at 405 nm. Cytotoxicity Assay. After the treatment of HL60 cells with the test compound for 48 h, the cell viability were determined by a MTT method.32 Hoechst Staining. After the treatment with compound 2 for 24 h, the HL60 cells were collected and fixed with 4% paraformaldehyde. Subsequently, the cell nuclei were stained with Hoechst 33342 and observed with a fluorescence microscope (Olympus Optical Co., Ltd. Tokyo, Japan).37,38 Apotosis Detection. After the treatment with compound 2, the HL60 cells were stained with propidium iodide (PI) and annexin Vfluorescein isothiocyanate (FITC) and then analyzed using a Cell Lab Quanta SC flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).37,38 Western Blotting. HL60 cells were harvested and lysed by RIPA buffer after treatment with 3.0 μM of compound 2 for 24 and 48 h. The total cellular proteins were obtained by centrifugation. A BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) were employed for determining the protein concentrations. The electrophoresis and immunoblotting Western blotting procedures were following the previous literature.39
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00167.
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Acid hydrolysis, MS and NMR spectrum of new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Tel: +86 25-86185197. *E-mail:
[email protected]. Tel: +86 25-86185418. ORCID
Jie Zhang: 0000-0003-1779-1075 Jian Xu: 0000-0001-7033-8574 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Jiangsu Province (grant no. BK20170742), Youth Science Fund Project of National Natural Science Foundation of China (grant no. 81703383), and the General Program of National Natural Science Foundation of China (grant no. 81573557). We thank Dr. W. Kitdamrongthama (Faculty of Pharmacy, Chiang Mai University, Thailand) for collecting the Albizia procera plant material.
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REFERENCES
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