JOURNAL OF MEDICINAL FOOD J Med Food 18 (4) 2015, 403–408 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.3196
Neuroprotective Effect of Prenylated Arylbenzofuran and Flavonoids from Morus alba Fruits on Glutamate-Induced Oxidative Injury in HT22 Hippocampal Cells Kyeong-Hwa Seo,1 Dae-Young Lee,2 Rak-Hun Jeong,1 Dong-Sung Lee,3 Young-Eon Kim,4 Eock-Kee Hong,5 Youn-Chul Kim,3 and Nam-In Baek1 1
Department of Oriental Medicinal Materials and Processing, Graduate School of Biotechnology, Kyung-Hee University, Yongin, Korea. 2 Herbal Crop Utilization Research Team, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea. 3 Hanbang Body-Fluid Research Center, Wonkwang University, Iksan, Korea. 4 Korea Food Research Institute, Sungnam, Korea. 5 School of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Korea.
ABSTRACT A prenylated arylbenzofuran and six flavonoids were isolated from the fruits of Morus alba L. through silica gel, octadecyl silica gel, and Diaion HP-20 column chromatography. Based on the nuclear magnetic resonance, mass spectrometry, and infrared spectroscopic data, the chemical structures of the compounds were determined to be artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7). The isolated compounds were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3 exhibited protective effects with EC50 values of 19.7 – 1.2 and 35.5 – 2.1 lM, respectively. The major compounds 1-3 and 7 were quantified using liquid chromatography/mass spectrometry analysis and were determined to be 1.88 – 2.1, 1.90 – 1.8, 0.78 – 1.5, and 37.29 – 2.2 mg/kg, respectively, in the ethanol extract of M. alba L. fruits.
KEY WORDS: artoindonesianin O HT22 morachalcone A Morus alba L. neuroprotection effect quantitative analysis
effects of the compounds against glutamate-induced neurotoxicity in the mouse hippocampal HT22 cell line. The HT22 mouse hippocampal cell line has been widely used in in vitro models for studying glutamate-induced oxidative stress. These cells lack functional ionotropic glutamate receptors,6 resulting in excessive levels of the neurotransmitter glutamate, which in turn trigger the excitotoxic process of neuronal cell death.7 Fruit from M. alba L. was extracted with aqueous EtOH and successively partitioned with EtOAc, n-BuOH, and H2O. The EtOAc and n-BuOH extracts were applied to repeated column chromatography to yield one prenylated arylbenzofuran 1 and six flavonoids 2-7. The compounds were identified as artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7) based on spectroscopic data. The compounds were evaluated for protection of HT22immortalized hippocampal cells against glutamate-induced oxidative stress and also quantitatively analyzed using LC/MS.
INTRODUCTION
M
orus alba L.(Moraceae) is native to Thailand and is now widely distributed in East Asia, including Korea, China, and Japan. Most parts of this plant have been used in traditional Chinese medicines for pharmacological purposes.1 The bark is used to treat hypertension, while the leaves have been used for antiplatelet effects and for treatment of headaches. Finally, the fruits are often used as a tonic and prophylactic in oriental herbal medication.2 Previous phytochemical studies have resulted in the isolation of secondary metabolites from the leaves and roots of M. alba L.,3–5 such as morin, morusin, mulberrofuran G, calystegines B1, and fagomine. M. alba L. fruits contained a variety of phytochemicals such as tocopherols, sterols, prenylated arylbenzofurans, and flavonoids. However, no report has yet been published on the neuroprotective effects of these fruits. Therefore, the present study focused on the isolation and identification of major secondary metabolites in the fruits as well as an investigation of the neuroprotective
MATERIALS AND METHODS Manuscript received 19 March 2014. Revision accepted 26 September 2014.
Chemicals and instruments
Address correspondence to: Nam-In Baek, PhD, Department of Oriental Medicinal Materials and Processing, Graduate School of Biotechnology, Kyung-Hee University, Yongin 446-701, Republic of Korea, E-mail: [email protected]
Kieselgel 60 (63–200 lm; Merck, Darmstadt, Germany) and LiChroprep RP-18 (46–60 lm; Merck) were used as
403
404
SEO ET AL.
resins for column chromatography (c.c.). Thin layer chromatography (TLC) analysis was carried out using Kieselgel 60 F254 and RP-18 F254s plates (Merck), and the spots on the TLC were detected using a UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, Westbury, NY, USA) and spraying with 10% H2SO4 solution followed by heating. Deuterium solvents for nuclear magnetic resonance (NMR) measurements were purchased from Merck. NMR spectra were recorded on a 400-MHz FT-NMR spectrometer (Varian, Palo Alto, CA, USA), and chemical shifts were calibrated on the solvents used for NMR measurement. IR spectra were obtained from a Perkin Elmer Spectrum One FT-IR spectrometer (model 599B, Waltham, MA, USA). EIMS was recorded on a JMSAX-700 ( JEOL, Tokyo, Japan). The melting point was determined on a Fisher-John’s melting point apparatus (Fisher Scientific, Fremont, CA, USA) and was not corrected. Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT, USA), while L-glutamate, Trolox, MTT, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Rf 0.38, MeOH-H2O [5:1]). Fraction MAE-13 (800 mg, Ve/ Vt 0.821–0.874) was subjected to SiO2 c.c. (F 4.0 · 15 cm) and eluted with CHCl3-MeOH-H2O (16:3:1/12:3:1, 3 L of each), yielding 30 fractions (MAE-13-1 to MAE-13-30) and ultimately yielding compound 4 (MAE-13-15, 24 mg, Ve/Vt 0.186-0.202, TLC [RP-18 F254s] Rf 0.68, MeOH-H2O [3:1]). Fraction MAE-13-22 (42 mg, Ve/Vt 0.380–0.411) was subjected to ODS c.c. (F 2 · 5 cm) and eluted with MeOH-H2O (1:1, 0.3 L), yielding eight fractions (MAE-13-22-1 to MAE13-22-8) along with purified compound 5 (MAE-13-22-5, 2.7 mg, Ve/Vt 0.486-0.502, TLC [RP-18 F254s] Rf 0.52, MeOH-H2O [1:1]). The concentrated n-BuOH fraction (MAB, 247 g) was chromatographed on a column prepared
Plant materials The fruits of M. alba were provided by the Buan Nonghyup, Republic of Korea, in 2011 and identified by Professor Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Republic of Korea. A voucher specimen (KHU-NPCL-110916) was reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Republic of Korea. Extraction and isolation The fresh fruits (15.4 kg) were successively extracted in 100% EtOH (35 L · 1) and 70% EtOH (35 L · 2) at room temperature for 24 h, followed by filtration and concentration in vacuo. The EtOH extracts (2.2 kg) were poured into H2O (1.7 L) and successively extracted with EtOAc (1.7 L · 4) and n-BuOH (1.5 L · 4). Each solvent layer was concentrated to yield EtOAc extract (MAE, 41 g), n-BuOH extract (MAB, 247 g), and water extract (MAH, 1932 g). The EtOAc fraction (MAE, 40 g) was applied to a SiO2 c.c. (F 9.0 · 14 cm) and eluted with n-hexane-EtOAc (10:1/ 8:1/6:1/3:1/1:1, 12 L of each)/CHCl3-MeOH (8:1/ 6:1/4:1/1:1, 10 L of each) and monitored using TLC to provide 14 fractions (MAE-1 to MAE-14). Fraction MAE-9 (670 mg, elution volume/total volume [Ve/Vt] 0.433–0.503) was subjected to ODS c.c. (F 3.5 · 7.0 cm) and eluted with MeOH-H2O (3:1/4:1/5:1/6:1, 1.4 L of each), yielding 28 fractions (MAE-9-1 to MAE-9-28) along with purified compound 1 (MAE-9-7, 7.4 mg, Ve/Vt 0.023-0.028, TLC [RP-18 F254s] Rf 0.44, MeOH-H2O [5:1]) and compound 2 (MAE-9-9, 17.7 mg, Ve/Vt 0.031-0.038, TLC [RP-18 F254s] Rf 0.31, MeOH-H2O [5:1]). Fraction MAE-10 (649 mg, Ve/ Vt 0.503–0.537) was subjected to ODS c.c. (F 4 · 5 cm) and eluted with MeOH-H2O (3:1, 1.3 L), yielding 17 fractions (MAE-10-1 to MAE-10-17) along with purified compound 3 (MAE-10-6, 14.5 mg, Ve/Vt 0.034-0.061, TLC [RP-18 F254s]
FIG. 1. Structures of compounds 1–7. glc, b-D-glucopyranosyl; rhm, a-L-rhamnopyranosyl.
405
NEUROPROTECTIVE EFFECTS OF CONSTITUENTS OF MORUS ALBA
with a highly porous polymer, Diaion HP-20 (F 12 · 38 cm) and successively eluted with H2O (8 L)/50% MeOH (4 L)/MeOH (8 L) to yield eight fractions (MAB-1 to MAB-8). Fraction MAB-5 (1.31 g, Ve/Vt 0.522-0.601) was subjected to ODS c.c. (F 4.5 · 10 cm) and eluted with MeOH-H2O (2:3, 1.4 L), yielding 12 fractions (MAB-5-1 to MAB-5-12). Fraction MAB-5-7 (271 mg, Ve/Vt 0.247-0.369) was subjected to SiO2 c.c. (F 2.5 · 15 cm) and eluted with CHCl3-MeOH-H2O (8:3:1, 3.6 L), yielding 12 fractions (MAB-5-7-1 to MAB-5-7-12) along with purified compound 6 (MAB-5-7-6, 11.4 mg, Ve/Vt 0.104-0.185, TLC [RP-18 F254s] Rf 0.35, MeOH-H2O [3:2]) and compound 7 (MAB-57-9, 58 mg, Ve/Vt 0.210-0.275, TLC [RP-18 F254s] Rf 0.28, MeOH-H2O [3:2]). Physical properties and spectroscopic data of compound 1 Compound 1 was colorless crystals (MeOH) with the following properties: melting point 156C; IR (KBr, mmax) 3436, 2920, 1600, 1558 cm - 1; EI/MS m/z 324 [M] + ; 1H-NMR (400 MHz, CD3OD, dH) 7.34 (1H, br.d, J = 8.4 Hz, H-4), 6.86 (1H, d, J = 2.4 Hz, H-7), 6.73 (1H, dd, J = 8.4, 2.4 Hz, H-5), 6.68 (1H, d, J = 2.8 Hz, H-60 ), 6.66 (1H, d, J = 0.8 Hz, H-3), 6.44 (1H, d, J = 2.8 Hz, H-20 ), 5.11 (1H, t, J = 8.0 Hz, H-200 ), 3.78 (3H, s, OCH3-30 ), 3.42 (2H, d, J = 8.0 Hz, H-100 ), 1.63 (3H, s, H-500 ), 1.62 (3H, s, H-400 ); 13C-NMR (100 MHz, CD3OD, dC) 160.3 (C-30 ), 157.3 (C-50 ), 157.0 (C-2), 156.7 (C-6), 155.8 (C-7a), 132.8 (C-10 ), 131.5 (C-300 ), 125.4 (C-200 ), 122.9 (C-40 ), 121.9 (C-4), 120.7 (C-3a), 113.1 (C-5), 108.1 (C-60 ), 105.8 (C-20 ), 100.2 (C-3), 98.3 (C-7), 56.0 (OCH3-30 ), 26.5 (C-100 ), 25.8 (C-500 ), 18.0 (C-400 ). Cell culture Mouse hippocampal HT22 cells, a subclone of the HT4 hippocampal cell line, were obtained from Prof. InheeMook (Seoul National University, Seoul, Korea). The cells were maintained at 5 · 104 cells/mL in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 U/mL), streptomycin (100 mg/mL), and L-glutamine (5 mM) and incubated at 37C in a humidified atmosphere containing 5% CO2 and 95% air. Cytoprotective activity assay Cytoprotective assessment was performed by seeding HT22 cells in 96-well plates at a density of 105 cells/mL. After 24 h, cells were pretreated with either compounds or positive control Trolox for 4 h. The cells were exposed to
glutamate (5 mM) for 12 h. Individual compounds were tested at concentrations of 10, 20, 40, and 80 mM, and each experiment was performed in triplicate. Cell viability was evaluated using the MTT assay.8 Briefly, cells were incubated with MTT (0.5 mg/mL) for 4 h at 37C, the medium was discarded, acidic isopropanol (0.04 N HCl) was added, and after incubating for 30 min, absorbance was measured at 590 nm using a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA). The resulting half maximal effective concentration (EC50) is expressed as the percentage of viable cells versus the control. Data are also expressed as the percentage of protection relative to vehicle-treated control cultures as follows: 100 · [optical density (OD) of glutamate and sample-treated culture - OD of glutamate-treated cultures]/(OD of control cultures - OD of glutamate-treated cultures). Trolox (50 lM) was used as a positive control. Statistical analysis Data are expressed as the mean – standard deviation (SD) of at least three independent experiments. One-way analysis of variance (ANOVA) was used, followed by the Newman– Keuls post hoc test to compare each group and the treatment concentration. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software, Inc., San Diego, CA, USA). Calibration of compounds 1–3 and 7 Calibration curves were constructed by dissolving pure compounds 1–3 and 7 in MeOH and then diluting them into appropriate concentration ranges. Duplicate injections were made at five concentration levels (25, 250, 500, 2500, and 5000 ppm). The calibration curve for each standard was constructed by plotting the peak area versus injection amount. The amount of compounds 1–3 and 7 in the sample was calculated from the corresponding curves. Quantitative analysis of compounds 1–3 and 7 using LC/MS/MS Accurately weighed fresh fruit of M. alba L. (100 g) was added to 150 mL of 100% ethanol and ultrasonically extracted for 4 h. High-performance liquid chromatography (HPLC) (Agilent 6410B, RRLC system; Agilent Technologies, Palo Alto, CA, USA) analysis was carried out on an SB-C18 column (Agilent; 1.8 lm, 21 · 50 mm) with gradient elution using solvents A (acetonitrile) and B (0.1%
Table 1. Protective Effects of Compounds 1–7 from Morus alba L. Fruits on Glutamate-Induced Oxidative Injury in HT22 Cells Compound
1
2
3
4
5
6
7
Trolox
EC50 (lM)
19.7 – 1.2
> 80
35.5 – 2.1
37.2 – 3.6
> 80
> 80
> 80
15.8 – 2.2
The cells were treated with compounds 1–7 and then incubated for 12 h with glutamate (5 mM). The resulting half maximal effective concentration (EC50) is expressed as the percentage of viable cells versus the control. Trolox was used as the positive control. Data represent mean value – standard deviation (SD) of three experiments.
406
SEO ET AL.
FIG. 2. LC-ESI-MS chromatograms of compounds 1–3, 7, and ethanol extract of Morus alba L. fruits, and mass spectra in multiple reaction monitoring (MRM) scan mode. The tR value and MS of each compound are listed in Table 2. High-performance liquid chromatography analysis was carried out on an SB-C18 column (Agilent; 1.8 lm, 21 · 50 mm) with gradient elution using solvents A (acetonitrile) and B (0.1% formic acid). The concentration of A was 10/90% at 0/25 min, 90/90% at 27 min, and 90/10% at 30 min. The flow rate was 0.2 mL/min and the injection volume was 2 lL. The detection was carried out by LC-ESI-MS/MS. Mass detector settings were as follows: gas temperature: 350C, gas flow: 10 L/min, nebulizer pressure: 45 psi, capillary voltage: 4000 V. Color images available online at www.liebertpub.com/jmf
407
NEUROPROTECTIVE EFFECTS OF CONSTITUENTS OF MORUS ALBA Table 2. Quantitative Analysis of Compounds 1–3 and 7 in the Extracts of the Morus alba L. Fruits Compound 1 2 3 7
tR (min)
Calibration curve
R2
[M + H] +
Content (mg/kg)
23.9 24.6 23.1 8.7
y = 17.576x - 350.75 y = 403.6009x - 2689.4843 y = 37.174x + 165.81 y = 13.708x - 550.43
0.9995 1.0000 0.9989 0.9987
325 325 341 611
1.88 – 2.1 1.90 – 1.8 0.78 – 1.5 37.29 – 2.2
Pure compounds 1–3 and 7 were dissolved in MeOH and diluted into appropriate concentration ranges for the construction of calibration curves. Duplicate injections were made at five concentration levels (25, 250, 500, 2500, and 5000 ppm). The calibration curve of each standard was constructed by plotting the peak area versus injection amount. The amount of compounds 1–3 and 7 in the sample was calculated from the corresponding curves. Data represent mean value – SD of three experiments.
formic acid). The concentration of A was 10/90% at 0/ 25 min, 90/90% at 27 min, and 90/10% at 30 min. The flow rate was 0.2 mL/min, the injection volume was 2 lL, and a photodiode array detector was used at 280 nm, which was connected to an Agilent 6410B triple quadrupole equipped with an Agilent electrospray ion source (ESI). The mass selective detector was used in the multiple reaction monitoring (MRM) mode for the highest possible selectivity and sensitivity. The multimode ion (MMI) source was operated in the positive ESI mode. The mass detector settings were as follows: gas temperature: 350C, gas flow: 10 L/ min, nebulizer pressure: 45 psi, capillary voltage: 4000 V. RESULTS AND DISCUSSION Determining the structure of compound 1 Isolated flavonoids 2–7 often occurred in the plants in this study. Interpretation of NMR and MS data and a comparison of reported values led to identification of the flavonoids as isobavachalcone (2),9 morachalcone A (3),10 quercetin (4),11 astragalin (5),12 isoquercetin (6),12 and rutin (7),12 respectively (Fig. 1). Compound 1 was obtained as colorless crystals from methanol. The spectroscopic data, including IR, MS, and NMR, were very similar as those of artoindonesianin O previously isolated from the bark of Artocarpus gomezianus.13 However, the identification of the structure in the literature was carried out through only 1D NMR data and comparison of the data with those of the related compounds, moracin C14 and albafuran B.15 In this study, 2D-NMR experiments, including gCOSY, gHSQC, and gHMBC, were conducted to confirm the location of the functional groups. In the gHMBC spectrum, there was a cross peak between the methoxy proton signal (dH 3.78, -OCH3) and an oxygenated olefin quaternary carbon signal dC 160.3 (C-30 ), indicating that the methoxy group was linked to the olefin quaternary carbon signal at the C-30 position. The benzylic methylene carbon signal at dC 26.5 (C-100 ) correlated with two oxygenated olefin quaternary carbon signals (dC 160.3, C-30 ; dC 157.3, C-50 ). Therefore, the isoprenyl group was linked to the olefin quaternary carbon at the C-40 position. Artoindonesianin O has been usually isolated from the barks of Artocarpus platns13,16 so far. This is the first report to isolate artoindonesianin O from the fruits of M. alba.
Neuroprotective effects of compounds 1–7 on glutamate-induced toxicity Glutamate typically acts as a major excitatory neurotransmitter in the mammalian central nervous system. Oxidative stress of neuronal cells contributes to delayed neuronal cell death after ischemic injury, Alzheimer’s, and Parkinson’s disease. Glutamate cytotoxicity is mediated by receptor-initiated excitotoxicity and nonreceptor-mediated oxidative stress.6 In addition, the HT22 mouse hippocampal cell line has provided a widely used in vitro model for studying glutamate-induced oxidative stress. These cells lack functional ionotropic glutamate receptors,17 resulting in excessive levels of glutamate, which in turn trigger the excitotoxic process of neuronal cell death.7 The isolated flavonoids from the fruits of M. alba L. were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3–4 exhibited protective effects with EC50 values of 19.7 – 1.2, 35.5 – 2.1, and 37.2 – 3.6 lM, respectively (Table 1). Compound 1 was similar to the positive control, Trolox (Sigma Chemical Co.), which had an EC50 value of 15.8 – 2.2 lM. Previously, compound 2 was reported to inhibit the formation of Ab42 fibrillar aggregates and to exhibit a strong inhibitory effect on ThT fluorescence with IC50 of 25 lM.18 The neuroprotective effects of these compounds are inferred from playing the role of the antioxidant, releasing the antioxidant enzymes, phase II enzymes, or directly inhibiting the apoptosis caused by glutamate. These results suggest that compound 1 may be useful as a neuroprotective agent and this potential should be further explored in future studies. Quantitative analysis of compounds 1–3 and 7 in M. alba L. fruit extracts using LC-MS The quantitative analysis of major compounds 1–3 and 7 in M. alba L. fruit extracts was carried out through LC-ESIMS/MS. HPLC separation was performed using an Agilent 6410B, RRLC system connected with a diode array detector. The column was an SB-C18 column (1.8 lm, 21 · 50 mm) from Agilent Technologies. The eluents were (A) acetonitrile and (B) 0.1% formic acid. Separations were performed by solvent gradient elution; concentration of A was 10/ 90% at 0/25 min, 90/90% at 27 min, and 90/10% at 30 min at a flow rate of 0.2 mL/min. Injection volume was 2 lL and detection was carried out by the total ion
408
SEO ET AL.
chromatogram (TIC) in the MRM mode of MS. The standards for compounds 1–3 and 7 were chromatographed to determine their retention times (tR) and mass data. The corresponding TIC and MS/MS spectrum of each compound is shown in Figure 2 and the tR, calibration curves, R2 values, and molecular ion mass are listed in Table 2. The peak of artoindonesianin O (1) appeared at 23.9 min, indicating a molecular ion at m/z 325 and a product ion at m/z 257 arising from the loss of 68 Da, representing the loss of an isoprenyl moiety (C5H9) unit and a fragment ion at m/z 69 of the isoprene (C5H8) moiety. The peak of isobavachalcone (2) appeared at 24.6 min, indicating a fragment ion at m/z 269 arising from the loss of 56 Da, representing a loss of the C4H7 chain unit connected to the isoprenyl moiety. A fragment ion at m/z 149 was caused by an additional loss of a parahydroxylphenylethenyl (C8H7O) unit, and a fragment ion at m/z 69 was due to an isoprene (C5H8) moiety. The peak of morachalcone A (3) appeared at 23.1 min, indicating a fragment ion at m/z 267 arising from the loss of 74 Da, representing a loss of the C4H7 chain unit of the isoprenyl group and one hydroxyl (OH) moiety. A fragment ion at m/z 149 was caused by a loss of the C4H7 chain unit of the isoprenyl group and 3,4-dihydroxylphenylethenyl (C8H7O) moiety. The peak of rutin (7) appeared at 8.7 min, indicating a molecular ion at m/z 611 and a product ion at m/z 465 arising from the loss of 146 Da, representing a loss of a rhamnosyl (C6H10O4) unit. A fragment ion at m/z 303 was from a characteristic peak of the aglycone, quercetin, and moiety, which arose from the product ion at m/z 465 due to a loss of 162 Da, a glucosyl (C6H10O5) moiety. The TIC of the extract from M. alba L. fruits is shown in Figure 2. M. alba L. fruits (100 g) were extracted with ethanol by sonication for 4 h. A portion of the extract was filtered through a 0.2-lm membrane filter, 2 lL of which was injected. The mass spectrum and tR of the peaks on the TIC were compared with the standard compounds. The contents of the compounds were calculated from the corresponding regression curves. Data are presented as mean – SD (n = 3). The contents of compounds 1–3 and 7 in the ethanol extract were determined as 1.88 – 2.1, 1.90 – 1.8, 0.78 – 1.5, and 37.29 – 2.2 mg/kg, respectively (Table 2).
ACKNOWLEDGMENT This work was supported by the (311025-03-2-SB010) project from the Agricultural Research & Development Promotion Center, Republic of Korea. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Fukai T, Satoh K, Nomura T, Sakagami H: Antinephritis and radical scavenging activity of prenylflavonoids. Fitoterapia 2003;74:720–724.
2. Jeong BS, Shin MK: Phanerogamae. In: Medicinal Botany Hyangyak Dictionary. Younglimsa, Seoul, 2002, pp. 545–548. 3. Yang JY, Lee HS: Evaluation of antioxidant and antibacterial activities of morin isolated from mulberry fruits (Morus alba L.). J Korean Soc Appl Biol Chem 2012;55:485–489. 4. Geng C, Yao S, Xue D, Zuo A, Zhang X, Jiang Z, et al.: New isoprenylated flavonoid from Morus alba. China J Chin Mater Med 2010;35:1560–1565. 5. Asano N, Yamashita T, Yasuda K, Ikeda K, Kizu H, Kameda Y, et al.: Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). J Agric Food Chem 2001;49:4208–4213. 6. Maher P, Davis JB: The role of monoamine metabolism in oxidative glutamate toxicity. Neuroscience 1996;16:6394–6401. 7. Meldrum B, Garthwaite J: Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci 1990;11: 379–387. 8. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Method 1983;65:55–63. 9. Pistelli L, Spera K, Flamini G, Mele S, Morelli I: Isoflavonoids and chalcones from Anthyllis hermanniae. Phytochemistry 1996;42:1455–1458. 10. Monache GD, De Rosa MC, Scurria R, Vitali A, Cuteri A, Monacelli B, et al.: Comparison between metabolite productions in cell culture and in whole plant of Maclura pomifera. Phytochemistry 1995;39:575–580. 11. Jung SJ, Kim DH, Hong YH, Lee JH, Song HN, Rho YD, et al.: Flavonoids from the flower of Rhododendron yedoense var. poukhanense and their antioxidant activities. Arch Pharm Res 2007;30:146–150. 12. Han JT, Bang MH, Chun OK, Kim DO, Lee CY, Baek NI: Flavonol glycosides from the aerial parts of Aceriphyllum rossii and their antioxidant activities. Arch Pharm Res 2004;27:390– 395. 13. Hakim EH, Ulinnuha UZ, Syah YM, Ghisalberti EL: Artoindonesianins N and O, new prenylated stilbene and prenylated arylbenzofuran derivatives from Artocarpus gomezianus. Fitoterapia 2002;73:597–603. 14. Takasugi M, Hagao S, Ueno S, Masamune T, Shirata A, Takahashi K: Moracin C and D, new phytoalexins from diseased mulberry. Chem Lett 1978;11:1239–1240. 15. Christensen LP, Jorgen L: Excelsaoctaphenol, a stilbene dimer from Chlorophora excelsa. Phytochemistry 1989;28:917– 918. 16. Yang ZG, Matsuzaki K, Takamatsu S, Kitanaka S: Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW 264.7 cells. Molecules 2011;16:6010–6022. 17. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT: Glutamate toxicity in a neuronal cell line involves inhibition of cysteine transport leading to oxidative stress. Neuron 1989;2: 1547–1558. 18. Chen XM, Yang YF, Zhang YT: Isobavachalcone and bavachinin from Psoraleae Fructus modulate Ab42 aggregation process through different mechanisms in vitro. FEBS Lett 2013;18:2930– 2935.
Neuroprotective Effect of Prenylated Arylbenzofuran and Flavonoids from Morus alba Fruits on Glutamate-Induced Oxidative Injury in HT22 Hippocampal Cells Kyeong-Hwa Seo,1 Dae-Young Lee,2 Rak-Hun Jeong,1 Dong-Sung Lee,3 Young-Eon Kim,4 Eock-Kee Hong,5 Youn-Chul Kim,3 and Nam-In Baek1 1
Department of Oriental Medicinal Materials and Processing, Graduate School of Biotechnology, Kyung-Hee University, Yongin, Korea. 2 Herbal Crop Utilization Research Team, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea. 3 Hanbang Body-Fluid Research Center, Wonkwang University, Iksan, Korea. 4 Korea Food Research Institute, Sungnam, Korea. 5 School of Biotechnology and Bioengineering, Kangwon National University, Chuncheon, Korea.
ABSTRACT A prenylated arylbenzofuran and six flavonoids were isolated from the fruits of Morus alba L. through silica gel, octadecyl silica gel, and Diaion HP-20 column chromatography. Based on the nuclear magnetic resonance, mass spectrometry, and infrared spectroscopic data, the chemical structures of the compounds were determined to be artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7). The isolated compounds were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3 exhibited protective effects with EC50 values of 19.7 – 1.2 and 35.5 – 2.1 lM, respectively. The major compounds 1-3 and 7 were quantified using liquid chromatography/mass spectrometry analysis and were determined to be 1.88 – 2.1, 1.90 – 1.8, 0.78 – 1.5, and 37.29 – 2.2 mg/kg, respectively, in the ethanol extract of M. alba L. fruits.
KEY WORDS: artoindonesianin O HT22 morachalcone A Morus alba L. neuroprotection effect quantitative analysis
effects of the compounds against glutamate-induced neurotoxicity in the mouse hippocampal HT22 cell line. The HT22 mouse hippocampal cell line has been widely used in in vitro models for studying glutamate-induced oxidative stress. These cells lack functional ionotropic glutamate receptors,6 resulting in excessive levels of the neurotransmitter glutamate, which in turn trigger the excitotoxic process of neuronal cell death.7 Fruit from M. alba L. was extracted with aqueous EtOH and successively partitioned with EtOAc, n-BuOH, and H2O. The EtOAc and n-BuOH extracts were applied to repeated column chromatography to yield one prenylated arylbenzofuran 1 and six flavonoids 2-7. The compounds were identified as artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7) based on spectroscopic data. The compounds were evaluated for protection of HT22immortalized hippocampal cells against glutamate-induced oxidative stress and also quantitatively analyzed using LC/MS.
INTRODUCTION
M
orus alba L.(Moraceae) is native to Thailand and is now widely distributed in East Asia, including Korea, China, and Japan. Most parts of this plant have been used in traditional Chinese medicines for pharmacological purposes.1 The bark is used to treat hypertension, while the leaves have been used for antiplatelet effects and for treatment of headaches. Finally, the fruits are often used as a tonic and prophylactic in oriental herbal medication.2 Previous phytochemical studies have resulted in the isolation of secondary metabolites from the leaves and roots of M. alba L.,3–5 such as morin, morusin, mulberrofuran G, calystegines B1, and fagomine. M. alba L. fruits contained a variety of phytochemicals such as tocopherols, sterols, prenylated arylbenzofurans, and flavonoids. However, no report has yet been published on the neuroprotective effects of these fruits. Therefore, the present study focused on the isolation and identification of major secondary metabolites in the fruits as well as an investigation of the neuroprotective
MATERIALS AND METHODS Manuscript received 19 March 2014. Revision accepted 26 September 2014.
Chemicals and instruments
Address correspondence to: Nam-In Baek, PhD, Department of Oriental Medicinal Materials and Processing, Graduate School of Biotechnology, Kyung-Hee University, Yongin 446-701, Republic of Korea, E-mail: [email protected]
Kieselgel 60 (63–200 lm; Merck, Darmstadt, Germany) and LiChroprep RP-18 (46–60 lm; Merck) were used as
403
404
SEO ET AL.
resins for column chromatography (c.c.). Thin layer chromatography (TLC) analysis was carried out using Kieselgel 60 F254 and RP-18 F254s plates (Merck), and the spots on the TLC were detected using a UV lamp Spectroline Model ENF-240 C/F (Spectronics Corporation, Westbury, NY, USA) and spraying with 10% H2SO4 solution followed by heating. Deuterium solvents for nuclear magnetic resonance (NMR) measurements were purchased from Merck. NMR spectra were recorded on a 400-MHz FT-NMR spectrometer (Varian, Palo Alto, CA, USA), and chemical shifts were calibrated on the solvents used for NMR measurement. IR spectra were obtained from a Perkin Elmer Spectrum One FT-IR spectrometer (model 599B, Waltham, MA, USA). EIMS was recorded on a JMSAX-700 ( JEOL, Tokyo, Japan). The melting point was determined on a Fisher-John’s melting point apparatus (Fisher Scientific, Fremont, CA, USA) and was not corrected. Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT, USA), while L-glutamate, Trolox, MTT, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Rf 0.38, MeOH-H2O [5:1]). Fraction MAE-13 (800 mg, Ve/ Vt 0.821–0.874) was subjected to SiO2 c.c. (F 4.0 · 15 cm) and eluted with CHCl3-MeOH-H2O (16:3:1/12:3:1, 3 L of each), yielding 30 fractions (MAE-13-1 to MAE-13-30) and ultimately yielding compound 4 (MAE-13-15, 24 mg, Ve/Vt 0.186-0.202, TLC [RP-18 F254s] Rf 0.68, MeOH-H2O [3:1]). Fraction MAE-13-22 (42 mg, Ve/Vt 0.380–0.411) was subjected to ODS c.c. (F 2 · 5 cm) and eluted with MeOH-H2O (1:1, 0.3 L), yielding eight fractions (MAE-13-22-1 to MAE13-22-8) along with purified compound 5 (MAE-13-22-5, 2.7 mg, Ve/Vt 0.486-0.502, TLC [RP-18 F254s] Rf 0.52, MeOH-H2O [1:1]). The concentrated n-BuOH fraction (MAB, 247 g) was chromatographed on a column prepared
Plant materials The fruits of M. alba were provided by the Buan Nonghyup, Republic of Korea, in 2011 and identified by Professor Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Republic of Korea. A voucher specimen (KHU-NPCL-110916) was reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Republic of Korea. Extraction and isolation The fresh fruits (15.4 kg) were successively extracted in 100% EtOH (35 L · 1) and 70% EtOH (35 L · 2) at room temperature for 24 h, followed by filtration and concentration in vacuo. The EtOH extracts (2.2 kg) were poured into H2O (1.7 L) and successively extracted with EtOAc (1.7 L · 4) and n-BuOH (1.5 L · 4). Each solvent layer was concentrated to yield EtOAc extract (MAE, 41 g), n-BuOH extract (MAB, 247 g), and water extract (MAH, 1932 g). The EtOAc fraction (MAE, 40 g) was applied to a SiO2 c.c. (F 9.0 · 14 cm) and eluted with n-hexane-EtOAc (10:1/ 8:1/6:1/3:1/1:1, 12 L of each)/CHCl3-MeOH (8:1/ 6:1/4:1/1:1, 10 L of each) and monitored using TLC to provide 14 fractions (MAE-1 to MAE-14). Fraction MAE-9 (670 mg, elution volume/total volume [Ve/Vt] 0.433–0.503) was subjected to ODS c.c. (F 3.5 · 7.0 cm) and eluted with MeOH-H2O (3:1/4:1/5:1/6:1, 1.4 L of each), yielding 28 fractions (MAE-9-1 to MAE-9-28) along with purified compound 1 (MAE-9-7, 7.4 mg, Ve/Vt 0.023-0.028, TLC [RP-18 F254s] Rf 0.44, MeOH-H2O [5:1]) and compound 2 (MAE-9-9, 17.7 mg, Ve/Vt 0.031-0.038, TLC [RP-18 F254s] Rf 0.31, MeOH-H2O [5:1]). Fraction MAE-10 (649 mg, Ve/ Vt 0.503–0.537) was subjected to ODS c.c. (F 4 · 5 cm) and eluted with MeOH-H2O (3:1, 1.3 L), yielding 17 fractions (MAE-10-1 to MAE-10-17) along with purified compound 3 (MAE-10-6, 14.5 mg, Ve/Vt 0.034-0.061, TLC [RP-18 F254s]
FIG. 1. Structures of compounds 1–7. glc, b-D-glucopyranosyl; rhm, a-L-rhamnopyranosyl.
405
NEUROPROTECTIVE EFFECTS OF CONSTITUENTS OF MORUS ALBA
with a highly porous polymer, Diaion HP-20 (F 12 · 38 cm) and successively eluted with H2O (8 L)/50% MeOH (4 L)/MeOH (8 L) to yield eight fractions (MAB-1 to MAB-8). Fraction MAB-5 (1.31 g, Ve/Vt 0.522-0.601) was subjected to ODS c.c. (F 4.5 · 10 cm) and eluted with MeOH-H2O (2:3, 1.4 L), yielding 12 fractions (MAB-5-1 to MAB-5-12). Fraction MAB-5-7 (271 mg, Ve/Vt 0.247-0.369) was subjected to SiO2 c.c. (F 2.5 · 15 cm) and eluted with CHCl3-MeOH-H2O (8:3:1, 3.6 L), yielding 12 fractions (MAB-5-7-1 to MAB-5-7-12) along with purified compound 6 (MAB-5-7-6, 11.4 mg, Ve/Vt 0.104-0.185, TLC [RP-18 F254s] Rf 0.35, MeOH-H2O [3:2]) and compound 7 (MAB-57-9, 58 mg, Ve/Vt 0.210-0.275, TLC [RP-18 F254s] Rf 0.28, MeOH-H2O [3:2]). Physical properties and spectroscopic data of compound 1 Compound 1 was colorless crystals (MeOH) with the following properties: melting point 156C; IR (KBr, mmax) 3436, 2920, 1600, 1558 cm - 1; EI/MS m/z 324 [M] + ; 1H-NMR (400 MHz, CD3OD, dH) 7.34 (1H, br.d, J = 8.4 Hz, H-4), 6.86 (1H, d, J = 2.4 Hz, H-7), 6.73 (1H, dd, J = 8.4, 2.4 Hz, H-5), 6.68 (1H, d, J = 2.8 Hz, H-60 ), 6.66 (1H, d, J = 0.8 Hz, H-3), 6.44 (1H, d, J = 2.8 Hz, H-20 ), 5.11 (1H, t, J = 8.0 Hz, H-200 ), 3.78 (3H, s, OCH3-30 ), 3.42 (2H, d, J = 8.0 Hz, H-100 ), 1.63 (3H, s, H-500 ), 1.62 (3H, s, H-400 ); 13C-NMR (100 MHz, CD3OD, dC) 160.3 (C-30 ), 157.3 (C-50 ), 157.0 (C-2), 156.7 (C-6), 155.8 (C-7a), 132.8 (C-10 ), 131.5 (C-300 ), 125.4 (C-200 ), 122.9 (C-40 ), 121.9 (C-4), 120.7 (C-3a), 113.1 (C-5), 108.1 (C-60 ), 105.8 (C-20 ), 100.2 (C-3), 98.3 (C-7), 56.0 (OCH3-30 ), 26.5 (C-100 ), 25.8 (C-500 ), 18.0 (C-400 ). Cell culture Mouse hippocampal HT22 cells, a subclone of the HT4 hippocampal cell line, were obtained from Prof. InheeMook (Seoul National University, Seoul, Korea). The cells were maintained at 5 · 104 cells/mL in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 U/mL), streptomycin (100 mg/mL), and L-glutamine (5 mM) and incubated at 37C in a humidified atmosphere containing 5% CO2 and 95% air. Cytoprotective activity assay Cytoprotective assessment was performed by seeding HT22 cells in 96-well plates at a density of 105 cells/mL. After 24 h, cells were pretreated with either compounds or positive control Trolox for 4 h. The cells were exposed to
glutamate (5 mM) for 12 h. Individual compounds were tested at concentrations of 10, 20, 40, and 80 mM, and each experiment was performed in triplicate. Cell viability was evaluated using the MTT assay.8 Briefly, cells were incubated with MTT (0.5 mg/mL) for 4 h at 37C, the medium was discarded, acidic isopropanol (0.04 N HCl) was added, and after incubating for 30 min, absorbance was measured at 590 nm using a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA). The resulting half maximal effective concentration (EC50) is expressed as the percentage of viable cells versus the control. Data are also expressed as the percentage of protection relative to vehicle-treated control cultures as follows: 100 · [optical density (OD) of glutamate and sample-treated culture - OD of glutamate-treated cultures]/(OD of control cultures - OD of glutamate-treated cultures). Trolox (50 lM) was used as a positive control. Statistical analysis Data are expressed as the mean – standard deviation (SD) of at least three independent experiments. One-way analysis of variance (ANOVA) was used, followed by the Newman– Keuls post hoc test to compare each group and the treatment concentration. Statistical analysis was performed using GraphPad Prism software version 3.03 (GraphPad Software, Inc., San Diego, CA, USA). Calibration of compounds 1–3 and 7 Calibration curves were constructed by dissolving pure compounds 1–3 and 7 in MeOH and then diluting them into appropriate concentration ranges. Duplicate injections were made at five concentration levels (25, 250, 500, 2500, and 5000 ppm). The calibration curve for each standard was constructed by plotting the peak area versus injection amount. The amount of compounds 1–3 and 7 in the sample was calculated from the corresponding curves. Quantitative analysis of compounds 1–3 and 7 using LC/MS/MS Accurately weighed fresh fruit of M. alba L. (100 g) was added to 150 mL of 100% ethanol and ultrasonically extracted for 4 h. High-performance liquid chromatography (HPLC) (Agilent 6410B, RRLC system; Agilent Technologies, Palo Alto, CA, USA) analysis was carried out on an SB-C18 column (Agilent; 1.8 lm, 21 · 50 mm) with gradient elution using solvents A (acetonitrile) and B (0.1%
Table 1. Protective Effects of Compounds 1–7 from Morus alba L. Fruits on Glutamate-Induced Oxidative Injury in HT22 Cells Compound
1
2
3
4
5
6
7
Trolox
EC50 (lM)
19.7 – 1.2
> 80
35.5 – 2.1
37.2 – 3.6
> 80
> 80
> 80
15.8 – 2.2
The cells were treated with compounds 1–7 and then incubated for 12 h with glutamate (5 mM). The resulting half maximal effective concentration (EC50) is expressed as the percentage of viable cells versus the control. Trolox was used as the positive control. Data represent mean value – standard deviation (SD) of three experiments.
406
SEO ET AL.
FIG. 2. LC-ESI-MS chromatograms of compounds 1–3, 7, and ethanol extract of Morus alba L. fruits, and mass spectra in multiple reaction monitoring (MRM) scan mode. The tR value and MS of each compound are listed in Table 2. High-performance liquid chromatography analysis was carried out on an SB-C18 column (Agilent; 1.8 lm, 21 · 50 mm) with gradient elution using solvents A (acetonitrile) and B (0.1% formic acid). The concentration of A was 10/90% at 0/25 min, 90/90% at 27 min, and 90/10% at 30 min. The flow rate was 0.2 mL/min and the injection volume was 2 lL. The detection was carried out by LC-ESI-MS/MS. Mass detector settings were as follows: gas temperature: 350C, gas flow: 10 L/min, nebulizer pressure: 45 psi, capillary voltage: 4000 V. Color images available online at www.liebertpub.com/jmf
407
NEUROPROTECTIVE EFFECTS OF CONSTITUENTS OF MORUS ALBA Table 2. Quantitative Analysis of Compounds 1–3 and 7 in the Extracts of the Morus alba L. Fruits Compound 1 2 3 7
tR (min)
Calibration curve
R2
[M + H] +
Content (mg/kg)
23.9 24.6 23.1 8.7
y = 17.576x - 350.75 y = 403.6009x - 2689.4843 y = 37.174x + 165.81 y = 13.708x - 550.43
0.9995 1.0000 0.9989 0.9987
325 325 341 611
1.88 – 2.1 1.90 – 1.8 0.78 – 1.5 37.29 – 2.2
Pure compounds 1–3 and 7 were dissolved in MeOH and diluted into appropriate concentration ranges for the construction of calibration curves. Duplicate injections were made at five concentration levels (25, 250, 500, 2500, and 5000 ppm). The calibration curve of each standard was constructed by plotting the peak area versus injection amount. The amount of compounds 1–3 and 7 in the sample was calculated from the corresponding curves. Data represent mean value – SD of three experiments.
formic acid). The concentration of A was 10/90% at 0/ 25 min, 90/90% at 27 min, and 90/10% at 30 min. The flow rate was 0.2 mL/min, the injection volume was 2 lL, and a photodiode array detector was used at 280 nm, which was connected to an Agilent 6410B triple quadrupole equipped with an Agilent electrospray ion source (ESI). The mass selective detector was used in the multiple reaction monitoring (MRM) mode for the highest possible selectivity and sensitivity. The multimode ion (MMI) source was operated in the positive ESI mode. The mass detector settings were as follows: gas temperature: 350C, gas flow: 10 L/ min, nebulizer pressure: 45 psi, capillary voltage: 4000 V. RESULTS AND DISCUSSION Determining the structure of compound 1 Isolated flavonoids 2–7 often occurred in the plants in this study. Interpretation of NMR and MS data and a comparison of reported values led to identification of the flavonoids as isobavachalcone (2),9 morachalcone A (3),10 quercetin (4),11 astragalin (5),12 isoquercetin (6),12 and rutin (7),12 respectively (Fig. 1). Compound 1 was obtained as colorless crystals from methanol. The spectroscopic data, including IR, MS, and NMR, were very similar as those of artoindonesianin O previously isolated from the bark of Artocarpus gomezianus.13 However, the identification of the structure in the literature was carried out through only 1D NMR data and comparison of the data with those of the related compounds, moracin C14 and albafuran B.15 In this study, 2D-NMR experiments, including gCOSY, gHSQC, and gHMBC, were conducted to confirm the location of the functional groups. In the gHMBC spectrum, there was a cross peak between the methoxy proton signal (dH 3.78, -OCH3) and an oxygenated olefin quaternary carbon signal dC 160.3 (C-30 ), indicating that the methoxy group was linked to the olefin quaternary carbon signal at the C-30 position. The benzylic methylene carbon signal at dC 26.5 (C-100 ) correlated with two oxygenated olefin quaternary carbon signals (dC 160.3, C-30 ; dC 157.3, C-50 ). Therefore, the isoprenyl group was linked to the olefin quaternary carbon at the C-40 position. Artoindonesianin O has been usually isolated from the barks of Artocarpus platns13,16 so far. This is the first report to isolate artoindonesianin O from the fruits of M. alba.
Neuroprotective effects of compounds 1–7 on glutamate-induced toxicity Glutamate typically acts as a major excitatory neurotransmitter in the mammalian central nervous system. Oxidative stress of neuronal cells contributes to delayed neuronal cell death after ischemic injury, Alzheimer’s, and Parkinson’s disease. Glutamate cytotoxicity is mediated by receptor-initiated excitotoxicity and nonreceptor-mediated oxidative stress.6 In addition, the HT22 mouse hippocampal cell line has provided a widely used in vitro model for studying glutamate-induced oxidative stress. These cells lack functional ionotropic glutamate receptors,17 resulting in excessive levels of glutamate, which in turn trigger the excitotoxic process of neuronal cell death.7 The isolated flavonoids from the fruits of M. alba L. were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3–4 exhibited protective effects with EC50 values of 19.7 – 1.2, 35.5 – 2.1, and 37.2 – 3.6 lM, respectively (Table 1). Compound 1 was similar to the positive control, Trolox (Sigma Chemical Co.), which had an EC50 value of 15.8 – 2.2 lM. Previously, compound 2 was reported to inhibit the formation of Ab42 fibrillar aggregates and to exhibit a strong inhibitory effect on ThT fluorescence with IC50 of 25 lM.18 The neuroprotective effects of these compounds are inferred from playing the role of the antioxidant, releasing the antioxidant enzymes, phase II enzymes, or directly inhibiting the apoptosis caused by glutamate. These results suggest that compound 1 may be useful as a neuroprotective agent and this potential should be further explored in future studies. Quantitative analysis of compounds 1–3 and 7 in M. alba L. fruit extracts using LC-MS The quantitative analysis of major compounds 1–3 and 7 in M. alba L. fruit extracts was carried out through LC-ESIMS/MS. HPLC separation was performed using an Agilent 6410B, RRLC system connected with a diode array detector. The column was an SB-C18 column (1.8 lm, 21 · 50 mm) from Agilent Technologies. The eluents were (A) acetonitrile and (B) 0.1% formic acid. Separations were performed by solvent gradient elution; concentration of A was 10/ 90% at 0/25 min, 90/90% at 27 min, and 90/10% at 30 min at a flow rate of 0.2 mL/min. Injection volume was 2 lL and detection was carried out by the total ion
408
SEO ET AL.
chromatogram (TIC) in the MRM mode of MS. The standards for compounds 1–3 and 7 were chromatographed to determine their retention times (tR) and mass data. The corresponding TIC and MS/MS spectrum of each compound is shown in Figure 2 and the tR, calibration curves, R2 values, and molecular ion mass are listed in Table 2. The peak of artoindonesianin O (1) appeared at 23.9 min, indicating a molecular ion at m/z 325 and a product ion at m/z 257 arising from the loss of 68 Da, representing the loss of an isoprenyl moiety (C5H9) unit and a fragment ion at m/z 69 of the isoprene (C5H8) moiety. The peak of isobavachalcone (2) appeared at 24.6 min, indicating a fragment ion at m/z 269 arising from the loss of 56 Da, representing a loss of the C4H7 chain unit connected to the isoprenyl moiety. A fragment ion at m/z 149 was caused by an additional loss of a parahydroxylphenylethenyl (C8H7O) unit, and a fragment ion at m/z 69 was due to an isoprene (C5H8) moiety. The peak of morachalcone A (3) appeared at 23.1 min, indicating a fragment ion at m/z 267 arising from the loss of 74 Da, representing a loss of the C4H7 chain unit of the isoprenyl group and one hydroxyl (OH) moiety. A fragment ion at m/z 149 was caused by a loss of the C4H7 chain unit of the isoprenyl group and 3,4-dihydroxylphenylethenyl (C8H7O) moiety. The peak of rutin (7) appeared at 8.7 min, indicating a molecular ion at m/z 611 and a product ion at m/z 465 arising from the loss of 146 Da, representing a loss of a rhamnosyl (C6H10O4) unit. A fragment ion at m/z 303 was from a characteristic peak of the aglycone, quercetin, and moiety, which arose from the product ion at m/z 465 due to a loss of 162 Da, a glucosyl (C6H10O5) moiety. The TIC of the extract from M. alba L. fruits is shown in Figure 2. M. alba L. fruits (100 g) were extracted with ethanol by sonication for 4 h. A portion of the extract was filtered through a 0.2-lm membrane filter, 2 lL of which was injected. The mass spectrum and tR of the peaks on the TIC were compared with the standard compounds. The contents of the compounds were calculated from the corresponding regression curves. Data are presented as mean – SD (n = 3). The contents of compounds 1–3 and 7 in the ethanol extract were determined as 1.88 – 2.1, 1.90 – 1.8, 0.78 – 1.5, and 37.29 – 2.2 mg/kg, respectively (Table 2).
ACKNOWLEDGMENT This work was supported by the (311025-03-2-SB010) project from the Agricultural Research & Development Promotion Center, Republic of Korea. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist. REFERENCES 1. Fukai T, Satoh K, Nomura T, Sakagami H: Antinephritis and radical scavenging activity of prenylflavonoids. Fitoterapia 2003;74:720–724.
2. Jeong BS, Shin MK: Phanerogamae. In: Medicinal Botany Hyangyak Dictionary. Younglimsa, Seoul, 2002, pp. 545–548. 3. Yang JY, Lee HS: Evaluation of antioxidant and antibacterial activities of morin isolated from mulberry fruits (Morus alba L.). J Korean Soc Appl Biol Chem 2012;55:485–489. 4. Geng C, Yao S, Xue D, Zuo A, Zhang X, Jiang Z, et al.: New isoprenylated flavonoid from Morus alba. China J Chin Mater Med 2010;35:1560–1565. 5. Asano N, Yamashita T, Yasuda K, Ikeda K, Kizu H, Kameda Y, et al.: Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). J Agric Food Chem 2001;49:4208–4213. 6. Maher P, Davis JB: The role of monoamine metabolism in oxidative glutamate toxicity. Neuroscience 1996;16:6394–6401. 7. Meldrum B, Garthwaite J: Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci 1990;11: 379–387. 8. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Method 1983;65:55–63. 9. Pistelli L, Spera K, Flamini G, Mele S, Morelli I: Isoflavonoids and chalcones from Anthyllis hermanniae. Phytochemistry 1996;42:1455–1458. 10. Monache GD, De Rosa MC, Scurria R, Vitali A, Cuteri A, Monacelli B, et al.: Comparison between metabolite productions in cell culture and in whole plant of Maclura pomifera. Phytochemistry 1995;39:575–580. 11. Jung SJ, Kim DH, Hong YH, Lee JH, Song HN, Rho YD, et al.: Flavonoids from the flower of Rhododendron yedoense var. poukhanense and their antioxidant activities. Arch Pharm Res 2007;30:146–150. 12. Han JT, Bang MH, Chun OK, Kim DO, Lee CY, Baek NI: Flavonol glycosides from the aerial parts of Aceriphyllum rossii and their antioxidant activities. Arch Pharm Res 2004;27:390– 395. 13. Hakim EH, Ulinnuha UZ, Syah YM, Ghisalberti EL: Artoindonesianins N and O, new prenylated stilbene and prenylated arylbenzofuran derivatives from Artocarpus gomezianus. Fitoterapia 2002;73:597–603. 14. Takasugi M, Hagao S, Ueno S, Masamune T, Shirata A, Takahashi K: Moracin C and D, new phytoalexins from diseased mulberry. Chem Lett 1978;11:1239–1240. 15. Christensen LP, Jorgen L: Excelsaoctaphenol, a stilbene dimer from Chlorophora excelsa. Phytochemistry 1989;28:917– 918. 16. Yang ZG, Matsuzaki K, Takamatsu S, Kitanaka S: Inhibitory effects of constituents from Morus alba var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW 264.7 cells. Molecules 2011;16:6010–6022. 17. Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT: Glutamate toxicity in a neuronal cell line involves inhibition of cysteine transport leading to oxidative stress. Neuron 1989;2: 1547–1558. 18. Chen XM, Yang YF, Zhang YT: Isobavachalcone and bavachinin from Psoraleae Fructus modulate Ab42 aggregation process through different mechanisms in vitro. FEBS Lett 2013;18:2930– 2935.
