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A new triglucosylated naphthalene glycoside from Aloe vera L.
Fitoterapia 81 (2010) 59–62
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new triglucosylated naphthalene glycoside from Aloe vera L. Qing-Yun Yang, Chun-Suo Yao, Wei-Shuo Fang ⁎ Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education and Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xiannongtan Street, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 6 April 2009 Accepted in revised form 12 July 2009 Available online 26 July 2009 Keywords: Aloe vera L. Naphthalene glycoside Aloveroside A
a b s t r a c t A new triglucosylated naphthalene derivative, named aloveroside A (1), together with two known anthraquinone dimers and two 6-phenyl-2-pyrone derivatives, was isolated from the Aloe vera ethanolic extracts. The structure of 1 was established as 1-(((4-(1-O-β-D-glucopyranosyl -(1→4)β-D-xylopyranoside)-hydroxymethyl)-1-hydroxy-8-O-α-L-rhamnopyranoside)naphthalene-2yl)-ethanone by means of spectroscopic evidences and chemical methods. All these compounds were tested for their BACE inhibitory activity but no significant activities were found. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aloe vera L. (Liliaceae) is a well-known pharmaceutical herb that has long been used in the traditional Chinese systems of medicine for the treatment of various diseases. It is widely distributed in the semitropical regions and planted in many provinces of China, such as Guangdong, Yunnan and Fujian Provinces. Previous investigations have demonstrated that Aloe species have antivirus [1] and antioxidant activities [2]. Recently, we found it showed potent β-secretase (BACE) inhibitory activity for the first time [3]. BACE is a drug target for the treatment of Alzheimer's disease (AD). Unlike other marketed anti-AD drugs, such as acetylcholine esterase and NMDA inhibitors, BACE inhibitors promised to be a class of disease disrupting rather than symptom relieving agents [4]. Previous studies of our group on A. vera led to the isolation of nine chromone glucosides with bioassay guided fractionations [3]. In search of more bioactive components, further investigation of its ethanol extracts led to the isolation of five compounds. On the basis of spectral analysis, their structures were identified as aloveroside A (1), elgnica dimer A (2) [5], elgnica dimer B (3) [5], p-coumaroyl
⁎ Corresponding author. Institute of Materia Medica, Peking Union Medical College, Beijing 100050, PR China. Tel.: +86 10 63165229; fax: +86 10 63017757. E-mail address: [email protected] (W.-S. Fang). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.07.006
aloenin (4) [6] and aloenin B (5) [6] (Fig. 1) respectively. Here we will report the structure elucidation of the new triglucosylated naphthalene derivative 1 and BACE inhibition test results for all five compounds. 2. Experimental 2.1. General Melting points were determined with a GERMANY-68992 apparatus. Optical rotations were measured with a PE-241 digital polarimeter. EI-MS and HR-FAB-MS were obtained on a QB-200 mass spectrometer. ESI-MS measurements were carried out on an Agilent 1100 series LC/MSD Trap SL mass spectrometer. IR spectra were recorded on an IR-47 spectrometer. NMR spectra were recorded on a Varian MERCURY400 and INOVA-500 spectrometer using TMS as internal standard, and chemical shifts δ were given in ppm. Silica gel (100–200 mesh) for column chromatography and silica gel H for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, Shandong Province, China. Size-exclusion chromatography was performed using Sigma Lipophilic Sephadex LH-20. Preparative HPLC was performed on a Shimadzu liquid chromatography LC-9A instrument with a UV–VIS spectrophotometric detector (SPD-6AV), monitored at 254 nm using an ODS column (Unicorn ODS, 10 μm, 250 × 20 mm).
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Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62
Fig. 1. Structures of compounds 1–5.
A. vera ethanolic extracts were purchased from Tong Ren Tang Pharmaceutical Stores in 2003. The quality of the plant extracts meets the standards of Aloe in the China Pharmacopoeia 2005 edition, and the original plant was identified by Professor Lin Ma of our institute to be identical to the authenticated sample (No. 18339) of A. vera which is deposited at the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China.
matographed over a Sephadex LH-20 column eluting with MeOH/H2O (20:80) to provide three fractions. Fr. IV-7-1 was purified by RP-C18 column chromatography using CH3OH– H2O (30:70) as eluent to obtain compound 4 (6.0 mg). Compounds 2 (5.0 mg, Rf = 0.65) and 3 (3.4 mg, Rf = 0.50) were isolated from Fr. IV-7-2 using preparative thin-layer chromatography (CHCl3/CH3OH/H2O 70:30:2). Fr. IV-7-3 was further separated on a reversed-phase preparative HPLC [CH3OH/ H2O (32:68), flow rate 3.0 ml/min] to afford compounds 1 (25.0 mg, tR = 46.8 min) and 5 (10.2 mg, tR = 52.6 min).
2.3. Extraction and isolation
2.4. Hydrolysis of aloveroside A
The ethanolic extracts of A. vera (200.0 g) were absorbed to silica gel and eluted with CHCl3/CH3OH/H2O (80:20:2) to provide five fractions (Fr. I–Fr. V). Fr. IV (4.2 g) was divided into eleven subfractions (Fr. IV-1–Fr. IV-11) using a Sephadex LH-20 column chromatography eluting with CH3OH/H2O gradiently (90:10 → 10:90). Fr. IV-7 (1.6 g) was further chro-
Approximately 1.0 mg of compound 1 was dissolved in 5.0 ml of 15% aqueous HCl, and left at 50 °C for about 6 h with constant stirring. The mixture was neutralised with the dropwise addition of 5% aqueous NaOH and then partitioned with EtOAc. The aqueous portion was reduced in vacuo, and the resultant residues were spotted on an analytical silica gel TLC
2.2. Plant material
Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62 Table 1 13 C NMR (100 MHz) and 1H NMR (400 MHz) data of 1 in CD3OD. No.
δC
δH,J (Hz)
δC
δH, J(Hz)
1 2 3 4 5 6 7 8 9 10 11 12 13
153.6 124.0 120.8 138.0 123.9 129.0 110.8 154.6 115.6 135.0 207.6 32.7 69.8
4′ 5′ 7.38 s 6′ xyl 1" 7.42 d (8.0) 2" 7.36 t (8.0) 3" 7.23 d (8.0) 4" 5"
73.3 71.5 18.0 104.0 74.8 76.1 78.5 64.5
glu 1‴ 2‴ 3‴ 4‴
103.0 74.6 77.8 71.7
3.49 t (9.6) 3.20–3.26 m 1.22 d (6.0) 4.22 d (7.2) 3.20–3.26 m 3.41 t (8.8) 3.70 m H-6a′ 3.98 dd (5.2, 11.6) H-6b′ 3.20–3.26 m 4.30 d (7.6) 3.16 m 3.32 m 3.60 m
Rham
1′
2′ 3′
No.
2.52 s 4.63, 4.83 d (13.0) 101.7 5.67 d (1.6) 5‴ 71.6 4.13 m 6‴ 72.5 3.79 m
78.0 3.29 t (5.2, 3.6) 62.6 H-6a‴ 3.83 m H-6b‴ 3.61 m
plate along with D-glucopyranose, L-rhamnopyranose and D-xylopyranose. The plate was developed with n-BuOH– HOAc–H2O (3:1:1), sprayed with 10% aqueous H2SO4 for visualization. The hydrolysate from compound 1 exhibited a dark yellow sport (Rf = 0.38) that was identical with that observed for D-glucopyranose. In addition, the hydrolysate also exhibited a second dark greenish-black spot (Rf = 0.57) and brown spot (Rf = 0.50) that matched L-rhamnopyranose and D-xylopyranose, respectively. Aloveroside A (1): amorphous pale yellow powder; mp 156–157 °C; [α]16 D −46.67°(c 0.06, MeOH); UV (MeOH) λmax (log ε): 226.0 (4.67), 258.0 (4.04 sh), 297.0 (3.78), 318 (3.78) nm; IR (KBr): νmax: 3376, 2924, 2858, 1685, 1635, 1581, 1448, 1365, 1267, 1140, 1070, 1049, 949, 916, 839, 766, 615 cm− 1; 1H NMR and 13C NMR (CD3OD, 400 MHz); see Table 1; ESI-MS (pos.) m/z: 695 [M + Na]+; HR-ESI-MS (pos.) m/z: 695.2157 [M + Na]+(calc. for C30H40O17Na 695.2163). 2.5. Determination of BACE-1 inhibition by in vitro peptide cleavage assay The ability of the compounds to inhibit BACE-1 cleavage of APP was assessed using a fluorescent resonance energy transfer (FRET) peptide cleavage assay. The FRET peptide substrate is Mca-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys (Dnp)-Arg-Arg-NH (where Mca is (7-methoxycoumarin-4yl)-acetyl, and Dnp is 2, 4-dinitrophenyl) and recombinant human BACE-1 were purchased from R&D Systems, Inc. The assay was carried out according to the supplier's protocol with modifications. Briefly, assays were performed in triplicate in 96-well black plates with a 100 μl of 50 mM sodium acetate buffer (pH 4.0), containing 4 μM substrate, 2 μg/ml recombinant human BACE-1 and different concentrations of inhibitors (dissolved in small volumes of DMSO prior to addition to the buffer). The fluorescence intensity was measured using a SpectraMAX Gemini XS plate reader (Molecular Devices Co., USA) at Ex320/Em405 both at zero time and after 60 min incubation at 25 °C. The inhibition percentage was calculated by the following equation: Inhibition ð%Þ = ½1−ðFS FS0 Þ = ðFC FC0 Þ × 100%
61
where FS0 and FS are the fluorescence of samples at zero time and 60 min, and FC0 and FC are the fluorescence of control at zero time and 60 min, respectively. 3. Results and discussion Compound 1 was obtained as pale yellow amorphous powder. Its positive HR-ESI-MS m/z 695.2157 ([M + Na]+), together with 1H and 13C NMR spectra suggested the molecular formula 1 as C30H40O17. Its IR spectrum indicated the presence of hydroxyl groups (3376 cm− 1), carbonyl group (1685 cm− 1) and aromatic moiety (1581, 1448 cm− 1). The UV spectrum displayed the absorption bands at λmax 226, 258, 297 and 318 nm, suggesting the presence of a strong conjugated system in the molecule. The 1H NMR spectrum of 1 (Table 1) in CD3OD exhibited the presence of one set of ABC system proton signals at δH 7.23 (1H, d, J = 8.0 Hz, H-7), 7.36 (1H, t, J = 8.0 Hz, H-6), 7.42 (1H, d, J = 8.0 Hz, H-5) and one single aromatic proton signal at δH 7.38 (1H, s, H-3). These signals in the 1H NMR spectrum combined with ten corresponding carbon signals at δC 154.6, 153.6, 138.0, 135.0, 129.0, 124.0, 12.9, 120.8, 115.6, 110.8 indicated the presence of naphthalene skeleton. In addition, the signal for methyl group at δH 2.52 (3H, s, H-12) in 1H NMR spectrum, together with the corresponding methyl carbon signal at δC 32.7 and one carbonyl carbon signal at δC 167.6 implied the existence of an acetyl chain. The presence of three additional carbon signals at δC 101.7, 104.0 and 103.4 along with the corresponding proton signals at δH 5.67 (1H, d, J = 1.6 Hz, H-1′), 4.22 (1H, d, J = 7.2 Hz, H-1″) and 4.30 (1H, d, J = 7.6 Hz, H-1″), suggested that compound 1 is a substituted naphthalene triglycoside. HMQC and HMBC experiments established the aglycone portion of this compound. HMBC correlation data (Fig. 2) showed that it possessed a 13-O-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside moiety. Significant HMBC correlations were used to confirm the crucial bond connectivities, including those observed for H-13 (δH 4.63, 4.83) to C-1″ (δC 104.0), and H-1‴ (δH 4.30) to C-4″ (δC 78.5). Further scrutiny of the HMBC data provided for the assignment of an 8-O-rhamnopyranoside linkage to the rutinose moiety based on a correlation from H-1′ (δH 5.67) to C-8 (δC 101.7). Other substituents were also assigned accordingly. Acid hydrolysis of the new naphthalene glycoside also confirmed the presence of D-glucopyranose, L-rhamnopyranose and D-xylopyranose, based on co-TLC with
Fig. 2. Significant HMBC correlations of compound 1.
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Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62
Acknowledgment
Table 2 The BACE inhibitory activities of compounds 1–5. Compounds
BACE inhibitory rates (%) a
Compounds
BACE inhibitory rates (%) a
1 3 5
18.68 16.75 37.19
2 4 Aloeresin D b
4.62 32.58 81.24
a b
Measured at the concentration of 100 μg/ml. Positive control (IC50 39.0 μM).
authentic sugar samples. From the above evidences, compound 1, was identified as 1-(((4-(1-O-β-D-glucopyranosyl(1 → 4)-β-D-xylopyranoside)-hydroxymethyl)-1-hydroxy8-O-α-L- rhamnopyranoside)naphthalene-2-yl)-ethanone. It is a new naphthalene glycoside and named as aloveroside A. The BACE inhibitory activities in vitro of compounds 1–5 were tested. Unfortunately none of them was found to have prominent activity at a concentration of 100 μg/ml (Table 2).
The authors acknowledge the financial support from NSFC (Grant Nos. 20432030, 30500647), Drs. G.-H. Du and Y.-H. Wang for the HTS screening tests of plant extracts with BACE inhibitory activity, and the Department of Instrumental Analysis of our institute for the measurement of spectral data. References [1] Dong YM, Chen CS, Zhao H, Lu XM, Duan SL. Natural Product R & D 2001;14:80. [2] Ren D, Wang YF, Yao YX. China Journal of Modern Medicine 1999;9:22. [3] Lv L, Yang QY, Zhao Y, Yao CS, Sun Y, Yang EJ, Song KS, Jung IM. Fang WS. Planta Med 2008;74:540. [4] Cirton M. Trends Pharmacol Sci 2004;25:92. [5] Hyun SK, Sick WW, Sung LS, Sub SC, Sook CH, Kennelly EJ, Douglas KA. J Nat Prod 1997;60:1180. [6] Speranza G, Data G, Lunazzi L. J Nat Prod 1986;45:800.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new triglucosylated naphthalene glycoside from Aloe vera L. Qing-Yun Yang, Chun-Suo Yao, Wei-Shuo Fang ⁎ Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education and Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xiannongtan Street, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 6 April 2009 Accepted in revised form 12 July 2009 Available online 26 July 2009 Keywords: Aloe vera L. Naphthalene glycoside Aloveroside A
a b s t r a c t A new triglucosylated naphthalene derivative, named aloveroside A (1), together with two known anthraquinone dimers and two 6-phenyl-2-pyrone derivatives, was isolated from the Aloe vera ethanolic extracts. The structure of 1 was established as 1-(((4-(1-O-β-D-glucopyranosyl -(1→4)β-D-xylopyranoside)-hydroxymethyl)-1-hydroxy-8-O-α-L-rhamnopyranoside)naphthalene-2yl)-ethanone by means of spectroscopic evidences and chemical methods. All these compounds were tested for their BACE inhibitory activity but no significant activities were found. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Aloe vera L. (Liliaceae) is a well-known pharmaceutical herb that has long been used in the traditional Chinese systems of medicine for the treatment of various diseases. It is widely distributed in the semitropical regions and planted in many provinces of China, such as Guangdong, Yunnan and Fujian Provinces. Previous investigations have demonstrated that Aloe species have antivirus [1] and antioxidant activities [2]. Recently, we found it showed potent β-secretase (BACE) inhibitory activity for the first time [3]. BACE is a drug target for the treatment of Alzheimer's disease (AD). Unlike other marketed anti-AD drugs, such as acetylcholine esterase and NMDA inhibitors, BACE inhibitors promised to be a class of disease disrupting rather than symptom relieving agents [4]. Previous studies of our group on A. vera led to the isolation of nine chromone glucosides with bioassay guided fractionations [3]. In search of more bioactive components, further investigation of its ethanol extracts led to the isolation of five compounds. On the basis of spectral analysis, their structures were identified as aloveroside A (1), elgnica dimer A (2) [5], elgnica dimer B (3) [5], p-coumaroyl
⁎ Corresponding author. Institute of Materia Medica, Peking Union Medical College, Beijing 100050, PR China. Tel.: +86 10 63165229; fax: +86 10 63017757. E-mail address: [email protected] (W.-S. Fang). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.07.006
aloenin (4) [6] and aloenin B (5) [6] (Fig. 1) respectively. Here we will report the structure elucidation of the new triglucosylated naphthalene derivative 1 and BACE inhibition test results for all five compounds. 2. Experimental 2.1. General Melting points were determined with a GERMANY-68992 apparatus. Optical rotations were measured with a PE-241 digital polarimeter. EI-MS and HR-FAB-MS were obtained on a QB-200 mass spectrometer. ESI-MS measurements were carried out on an Agilent 1100 series LC/MSD Trap SL mass spectrometer. IR spectra were recorded on an IR-47 spectrometer. NMR spectra were recorded on a Varian MERCURY400 and INOVA-500 spectrometer using TMS as internal standard, and chemical shifts δ were given in ppm. Silica gel (100–200 mesh) for column chromatography and silica gel H for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, Shandong Province, China. Size-exclusion chromatography was performed using Sigma Lipophilic Sephadex LH-20. Preparative HPLC was performed on a Shimadzu liquid chromatography LC-9A instrument with a UV–VIS spectrophotometric detector (SPD-6AV), monitored at 254 nm using an ODS column (Unicorn ODS, 10 μm, 250 × 20 mm).
60
Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62
Fig. 1. Structures of compounds 1–5.
A. vera ethanolic extracts were purchased from Tong Ren Tang Pharmaceutical Stores in 2003. The quality of the plant extracts meets the standards of Aloe in the China Pharmacopoeia 2005 edition, and the original plant was identified by Professor Lin Ma of our institute to be identical to the authenticated sample (No. 18339) of A. vera which is deposited at the Herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China.
matographed over a Sephadex LH-20 column eluting with MeOH/H2O (20:80) to provide three fractions. Fr. IV-7-1 was purified by RP-C18 column chromatography using CH3OH– H2O (30:70) as eluent to obtain compound 4 (6.0 mg). Compounds 2 (5.0 mg, Rf = 0.65) and 3 (3.4 mg, Rf = 0.50) were isolated from Fr. IV-7-2 using preparative thin-layer chromatography (CHCl3/CH3OH/H2O 70:30:2). Fr. IV-7-3 was further separated on a reversed-phase preparative HPLC [CH3OH/ H2O (32:68), flow rate 3.0 ml/min] to afford compounds 1 (25.0 mg, tR = 46.8 min) and 5 (10.2 mg, tR = 52.6 min).
2.3. Extraction and isolation
2.4. Hydrolysis of aloveroside A
The ethanolic extracts of A. vera (200.0 g) were absorbed to silica gel and eluted with CHCl3/CH3OH/H2O (80:20:2) to provide five fractions (Fr. I–Fr. V). Fr. IV (4.2 g) was divided into eleven subfractions (Fr. IV-1–Fr. IV-11) using a Sephadex LH-20 column chromatography eluting with CH3OH/H2O gradiently (90:10 → 10:90). Fr. IV-7 (1.6 g) was further chro-
Approximately 1.0 mg of compound 1 was dissolved in 5.0 ml of 15% aqueous HCl, and left at 50 °C for about 6 h with constant stirring. The mixture was neutralised with the dropwise addition of 5% aqueous NaOH and then partitioned with EtOAc. The aqueous portion was reduced in vacuo, and the resultant residues were spotted on an analytical silica gel TLC
2.2. Plant material
Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62 Table 1 13 C NMR (100 MHz) and 1H NMR (400 MHz) data of 1 in CD3OD. No.
δC
δH,J (Hz)
δC
δH, J(Hz)
1 2 3 4 5 6 7 8 9 10 11 12 13
153.6 124.0 120.8 138.0 123.9 129.0 110.8 154.6 115.6 135.0 207.6 32.7 69.8
4′ 5′ 7.38 s 6′ xyl 1" 7.42 d (8.0) 2" 7.36 t (8.0) 3" 7.23 d (8.0) 4" 5"
73.3 71.5 18.0 104.0 74.8 76.1 78.5 64.5
glu 1‴ 2‴ 3‴ 4‴
103.0 74.6 77.8 71.7
3.49 t (9.6) 3.20–3.26 m 1.22 d (6.0) 4.22 d (7.2) 3.20–3.26 m 3.41 t (8.8) 3.70 m H-6a′ 3.98 dd (5.2, 11.6) H-6b′ 3.20–3.26 m 4.30 d (7.6) 3.16 m 3.32 m 3.60 m
Rham
1′
2′ 3′
No.
2.52 s 4.63, 4.83 d (13.0) 101.7 5.67 d (1.6) 5‴ 71.6 4.13 m 6‴ 72.5 3.79 m
78.0 3.29 t (5.2, 3.6) 62.6 H-6a‴ 3.83 m H-6b‴ 3.61 m
plate along with D-glucopyranose, L-rhamnopyranose and D-xylopyranose. The plate was developed with n-BuOH– HOAc–H2O (3:1:1), sprayed with 10% aqueous H2SO4 for visualization. The hydrolysate from compound 1 exhibited a dark yellow sport (Rf = 0.38) that was identical with that observed for D-glucopyranose. In addition, the hydrolysate also exhibited a second dark greenish-black spot (Rf = 0.57) and brown spot (Rf = 0.50) that matched L-rhamnopyranose and D-xylopyranose, respectively. Aloveroside A (1): amorphous pale yellow powder; mp 156–157 °C; [α]16 D −46.67°(c 0.06, MeOH); UV (MeOH) λmax (log ε): 226.0 (4.67), 258.0 (4.04 sh), 297.0 (3.78), 318 (3.78) nm; IR (KBr): νmax: 3376, 2924, 2858, 1685, 1635, 1581, 1448, 1365, 1267, 1140, 1070, 1049, 949, 916, 839, 766, 615 cm− 1; 1H NMR and 13C NMR (CD3OD, 400 MHz); see Table 1; ESI-MS (pos.) m/z: 695 [M + Na]+; HR-ESI-MS (pos.) m/z: 695.2157 [M + Na]+(calc. for C30H40O17Na 695.2163). 2.5. Determination of BACE-1 inhibition by in vitro peptide cleavage assay The ability of the compounds to inhibit BACE-1 cleavage of APP was assessed using a fluorescent resonance energy transfer (FRET) peptide cleavage assay. The FRET peptide substrate is Mca-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys (Dnp)-Arg-Arg-NH (where Mca is (7-methoxycoumarin-4yl)-acetyl, and Dnp is 2, 4-dinitrophenyl) and recombinant human BACE-1 were purchased from R&D Systems, Inc. The assay was carried out according to the supplier's protocol with modifications. Briefly, assays were performed in triplicate in 96-well black plates with a 100 μl of 50 mM sodium acetate buffer (pH 4.0), containing 4 μM substrate, 2 μg/ml recombinant human BACE-1 and different concentrations of inhibitors (dissolved in small volumes of DMSO prior to addition to the buffer). The fluorescence intensity was measured using a SpectraMAX Gemini XS plate reader (Molecular Devices Co., USA) at Ex320/Em405 both at zero time and after 60 min incubation at 25 °C. The inhibition percentage was calculated by the following equation: Inhibition ð%Þ = ½1−ðFS FS0 Þ = ðFC FC0 Þ × 100%
61
where FS0 and FS are the fluorescence of samples at zero time and 60 min, and FC0 and FC are the fluorescence of control at zero time and 60 min, respectively. 3. Results and discussion Compound 1 was obtained as pale yellow amorphous powder. Its positive HR-ESI-MS m/z 695.2157 ([M + Na]+), together with 1H and 13C NMR spectra suggested the molecular formula 1 as C30H40O17. Its IR spectrum indicated the presence of hydroxyl groups (3376 cm− 1), carbonyl group (1685 cm− 1) and aromatic moiety (1581, 1448 cm− 1). The UV spectrum displayed the absorption bands at λmax 226, 258, 297 and 318 nm, suggesting the presence of a strong conjugated system in the molecule. The 1H NMR spectrum of 1 (Table 1) in CD3OD exhibited the presence of one set of ABC system proton signals at δH 7.23 (1H, d, J = 8.0 Hz, H-7), 7.36 (1H, t, J = 8.0 Hz, H-6), 7.42 (1H, d, J = 8.0 Hz, H-5) and one single aromatic proton signal at δH 7.38 (1H, s, H-3). These signals in the 1H NMR spectrum combined with ten corresponding carbon signals at δC 154.6, 153.6, 138.0, 135.0, 129.0, 124.0, 12.9, 120.8, 115.6, 110.8 indicated the presence of naphthalene skeleton. In addition, the signal for methyl group at δH 2.52 (3H, s, H-12) in 1H NMR spectrum, together with the corresponding methyl carbon signal at δC 32.7 and one carbonyl carbon signal at δC 167.6 implied the existence of an acetyl chain. The presence of three additional carbon signals at δC 101.7, 104.0 and 103.4 along with the corresponding proton signals at δH 5.67 (1H, d, J = 1.6 Hz, H-1′), 4.22 (1H, d, J = 7.2 Hz, H-1″) and 4.30 (1H, d, J = 7.6 Hz, H-1″), suggested that compound 1 is a substituted naphthalene triglycoside. HMQC and HMBC experiments established the aglycone portion of this compound. HMBC correlation data (Fig. 2) showed that it possessed a 13-O-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside moiety. Significant HMBC correlations were used to confirm the crucial bond connectivities, including those observed for H-13 (δH 4.63, 4.83) to C-1″ (δC 104.0), and H-1‴ (δH 4.30) to C-4″ (δC 78.5). Further scrutiny of the HMBC data provided for the assignment of an 8-O-rhamnopyranoside linkage to the rutinose moiety based on a correlation from H-1′ (δH 5.67) to C-8 (δC 101.7). Other substituents were also assigned accordingly. Acid hydrolysis of the new naphthalene glycoside also confirmed the presence of D-glucopyranose, L-rhamnopyranose and D-xylopyranose, based on co-TLC with
Fig. 2. Significant HMBC correlations of compound 1.
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Q.-Y. Yang et al. / Fitoterapia 81 (2010) 59–62
Acknowledgment
Table 2 The BACE inhibitory activities of compounds 1–5. Compounds
BACE inhibitory rates (%) a
Compounds
BACE inhibitory rates (%) a
1 3 5
18.68 16.75 37.19
2 4 Aloeresin D b
4.62 32.58 81.24
a b
Measured at the concentration of 100 μg/ml. Positive control (IC50 39.0 μM).
authentic sugar samples. From the above evidences, compound 1, was identified as 1-(((4-(1-O-β-D-glucopyranosyl(1 → 4)-β-D-xylopyranoside)-hydroxymethyl)-1-hydroxy8-O-α-L- rhamnopyranoside)naphthalene-2-yl)-ethanone. It is a new naphthalene glycoside and named as aloveroside A. The BACE inhibitory activities in vitro of compounds 1–5 were tested. Unfortunately none of them was found to have prominent activity at a concentration of 100 μg/ml (Table 2).
The authors acknowledge the financial support from NSFC (Grant Nos. 20432030, 30500647), Drs. G.-H. Du and Y.-H. Wang for the HTS screening tests of plant extracts with BACE inhibitory activity, and the Department of Instrumental Analysis of our institute for the measurement of spectral data. References [1] Dong YM, Chen CS, Zhao H, Lu XM, Duan SL. Natural Product R & D 2001;14:80. [2] Ren D, Wang YF, Yao YX. China Journal of Modern Medicine 1999;9:22. [3] Lv L, Yang QY, Zhao Y, Yao CS, Sun Y, Yang EJ, Song KS, Jung IM. Fang WS. Planta Med 2008;74:540. [4] Cirton M. Trends Pharmacol Sci 2004;25:92. [5] Hyun SK, Sick WW, Sung LS, Sub SC, Sook CH, Kennelly EJ, Douglas KA. J Nat Prod 1997;60:1180. [6] Speranza G, Data G, Lunazzi L. J Nat Prod 1986;45:800.