Natural Product Sciences 17(3) : 221-224 (2011)
221
Neuroprotective Compounds Isolated from the Methanolic Extract of Lonicera japonica†
Jin Bae Weon1, Hye Jin Yang1, Bohyoung Lee1, Yun Bo-Ra1, Choong Je Ma1,2,*
1Department of Biomaterials Engineering, School of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea
2Research Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea
Abstract −A CH3Cl extract from the flower of Lonicera japonica (Lauraceae) significantly protected primary cultures of rat cortical cells injured by the excitotoxic amino acid, L-glutamate. Loganin (1), secoxyloganin (2), caffeic acid (3) rutin (4), hyperoside (5), quercetin-3-O-glucoside (6), lonicerin (7), kaempferol-3-O-rutinoside (8), luteolin-7-O-b-D-glucopyranoside (9), quercetin (10) and luteolin (11) were isolated by bioactivity-guided fractionation from the CH3Cl fraction and further separated using chromatographic techniques. Caffeic acid, lonicerin, kaempferol-3-O-rutinoside, quercetin and luteolin had significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells at concentrations ranging from 0.1µM to 10.0µM.
Keywords −Lonicera japonica, neuroprotection, lonicerin, glutamate neurotoxicity
Introduction
Glutamate is involved in central excitatory neurotrans- mission, as well as neuronal survival, synaptogenesis, neuronal plasticity and memory (Kubo and Ito, 2004).
However, high concentrations of glutamate can evoke neuronal dysfunction and even damage or death (Cacabelos et al., 1996). Glutamate-mediated neurotoxicity may be involved in several neuropathological disorders such as Alzhemer’s disease, Parkinson’s disease, ischemic stroke, and spinal cord trauma (Heath and Shaw, 2002).
Thus, neuroprotection against glutamate-induced neurotoxicity is a therapeutic strategy to treat neurode- generative disease (Lee et al., 1999). During our search for neuroprotective medicinal plant extracts, we found that an 80% MeOH extract of Lonicera japonica Thunb.
flowers has significant neuroprotective activities against glutamate-induced neurotoxicity in primary cultures of rat cortical cells (Won and Ma, 2009). The flowers and buds of L. japonica are well known for their antiviral, anti- inflammatory and antibacterial activities in traditional Chinese medicine and widely used in the treatment of various diseases, including upper respiratory tract
infections, fever, sores and swelling. Numerous compounds such as alkaloids, cerebrosides, flavonoids, iridoids, polyphenols, and triterpenoid saponins have been reported from various parts of this plant (Jeong et al., 2009;Lin et al., 2008; Qi et al., 2009; Yu et al., 2008). We isolated and identified several bioactive compounds from
L. japonica and investigated their neuroprotective properties.
Experimental
General −The 1H- and 13C-NMR measurements were carried out in a Bruker AMX 400 spectrometer operating at 400 and 100 MHz, respectively. TMS or solvent signals were used as internal standards.
Plant Materials −The flower of Lonicera japonica
was purchased from a commercial supplier in Seoul, Korea and identified by Dr. Young Bae Seo, a professor of the College of Oriental Medicine, Daejeon University (Daejeon, Korea). A voucher specimen (CJ-0001M) has been deposited in the Department of Biomaterials and Bioengineering of Kangwon National University.
Extraction and isolation −The dried flowers of L.
japonica (8 kg) were ground into a powder and extracted 3 times with 80% MeOH using the reflux apparatus.
Upon removal of the solvent in vacuo, the methanolic extract yielded 861 g of material (10.7% by dry weight).
The methanolic extract was suspended in H2O and
†Dedicated to Prof. Young Choong Kim of the Seoul National Univer- sity for her leading works on Pharmacognosy.
*Author for correspondence
Tel: +82-33-250-6565; E-mail: cjma@kangwon.ac.kr
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partitioned successively with CHCl3. The CHCl3 fraction (97 g) which showed significant neuroprotective activity, was subjected to column chromatography (cc) over silica gel (12×80 cm) eluted with n-hexane-EtOAc (80:1, 40:1, 20:1, 5:1, 2:1, 1:1, EtOAc, MeOH) to afford ten fractions (F01-F10). F03 was further subjected to silica gel cc (3.5×45 cm) eluting with n-hexane-EtOAc (20:1, 10:1, 5:1, 2:1, 1:1, EtOAc, MeOH), affording twelve fractions (F03-I-F03-X). F03-V was subjected to a Sephadex LH- 20 column eluting with MeOH to afford compound 1
(12.8 mg), 2 (6.3 mg), 3 (7.2 mg) and 4 (6.6 mg).
Compound 5 (2.9 mg), 6 (12.0 mg) and 7 (5.8 mg) were isolated from F04-V by the Sepahdex LH-20 column eluting with MeOH. Compound 8 (6.9 mg) and 9 (10.5
mg) were isolated from F05-III by the Sepahdex LH-20 column eluting with MeOH. Also, compound 10
(11.9 mg) and 11 (24.1 mg) were isolated from F08-VI by HPLC system eluting with MeOH:Water, 60:40. Eleven
Compounds (1-11; Fig. 1) were identified based on comparisons with previously reported spectroscopic data (Choi et al., 2007; Jeong et al., 2009; Kumar et al., 2005;
Lin et al., 2008; Qi et al., 2009; Tang et al., 2008; Yu et al., 2008).
Evaluation of neuroprotective activity −Female Sprague-Dawley rats (housed at 20 - 23oC with a 12 h light cycle from 09:00 to 21:00, given food [Agribrand Purinar Korea] and water ad libitum) were provided by the Laboratory Animal Center, Kangwon National
Fig. 1. Chemical structures of compounds 1 - 11 isolated from the CHCl3 fraction of L. japonica.
Vol. 17, No. 3, 2011 223
University. All experiments were conducted according to the guidelines of the Committee on Care and Use of Laboratory Animals of the Kangwon National University.
Primary cultures of mixed cortical cells containing both neuronal and glial cells were prepared from late fetal SD rats (17-19-days gestation in utero) as described previously (Won and Ma, 2009). All compounds were dissolved in DMSO (final culture concentration, 0.1%). Preliminary studies indicated that the solvent had no effect on cell viability at the concentration used (data not shown). Two known glutamate receptor antagonists, MK-801 (dizocilpine maleate, a non-competitive antagonist of the NMDA receptor) and CNQX (6-cyano-7-nitroquinoxaline-2,3- dione, a non-NMDA receptor antagonist) were used as positive controls for the assessment of neuroprotective activity (Foster et al., 1987; Sheardown, 1988). Cortical cell cultures were washed with DMEM and incubated with the test compounds for 1 h. The cultures were then exposed to 100µM glutamate. After 24 h incubation, the cultures were assessed for the extent of neuronal damage.
Neuronal viability and integrity were quantified by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and/or LDH (lactate dehydrogenase) assay
described in our previous report (Won and Ma, 2009).
Data are expressed as the percentage of protection relative to control cultures.
Statistical analysis −Data were evaluated for statistical significance by a Student’s t-test using a computerized statistical package. The confidence level for statistical significance was set at a probability value of 0.05.
Results and Discussion
As a continuation of our search for neuroprotective compounds from natural resources, the methanolic extract of the flower of Lonicera japonica was found to significantly protect primary cultures of rat cortical cells against glutamate-induced neurotoxicity. Subsequent phytochemical studies coupled with our bioassay were performed to elucidate the active principles responsible for the neuroprotective activity of L. japonica (Won and Ma, 2009).
The flowers and buds of L. japonica has been well known as an antiviral, anti-inflammatory, and antibacterial agent in traditional Chinese medicine and widely used in the treatment of various diseases, including upper
Table 1. Protective activities of compounds isolated from the CHCl3 fraction of L. japonica against glutamate-induced neurotoxicity in primary cultures of rat cortical cells
Protection (%)
0.1µM 1.0µM 10.0µM
Control 100.0±0.55
Glutamate-treated 550.0±0.65
loganin (1) 515.3±5.44 17.5±6.18 14.0±1.24
secoxyloganin (2) 516.7±1.05 14.1±3.32 18.0±4.15
caffeic acid (3) 531.6±2.74* 34.0±4.29** 36.9±2.57**
rutin (4) 518.9±2.54 18.0±3.33 18.3±5.55
hyperoside (5) 514.1±3.10 19.8±2.20 21.6±3.49
quercetin-3-O-glucoside (6) 515.6±1.46 15.0±3.79 15.8±5.13
lonicerin (7) 535.3±3.57** 36.1±1.86*** 39.0±4.15***
kaempferol-3-O-rutinoside (8) 521.4±2.64* 23.5±3.19* 25.1±2.15* luteolin-7-O-β-D-glucopyranoside (9) 518.4±2.84 19.7±3.64 21.8±2.88
quercetin (10) 534.9±1.88** 47.5±1.18*** 34.1±2.24**
luteolin (11) 516.7±1.05 35.1±1.32** 34.0±1.15**
MK-801 65.8±4.55***
CNQX 57.4±2.95***
a LDH released from control and glutamate-treated cultures was 106.8±4.2 and 185.3±5.2 mU/ml, respectively. Cell viability (%) was calculated as 100×(LDH released from glutamate-treated−LDH released from glutamate + test compound) / (LDH released from glutamate-treatedb −LDH released from control).
Glutamate-treated values differ significantly from the untreated controls (p < 0.001).
c MK-801: dizocilpine maleate, a non-competitive antagonist of the NMDA receptor.
d CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione, non-NMDA receptor antagonist.
The values are expressed as means±SD of triplicate experiments. *p < 0.05, **p < 0.01and ***p < 0.001 levels of significance from glutamate-treated cells.
224 Natural Product Sciences
respiratory tract infections, fever, sores and swelling.
However, to our knowledge to date, no studies relating to any neuroprotective activity have been linked to this plant. In the present report, we describe neuroprotective compounds of L. japonica flower against glutamate- induced neurotoxicity as measured in vitro.
Bioassay-guided fractionation of the methanolic extract of L. japonica flower revealed that the CHCl3 fraction was the most active one (58.2% protection against glutamate-induced toxicity at 1µg/ml, p < 0.01). Further fractionation and separation of the CHCl3 fraction by several chromatographic methods yielded eleven com- pounds. Compounds were identified as loganin (1), secoxyloganin (2), caffeic acid (3) rutin (4), hyperoside (5), quercetin-3-O-glucoside (6), lonicerin (7), kaempferol- 3-O-rutinoside (8), luteolin-7-O-β-D-glucopyranoside (9), quercetin (10) and luteolin (11) by the direct comparison of their physicochemical and spectroscopic data with those previously reported [6-12].
Among them, five compounds (3, 7, 8, 10, 11) showed significant neuroprotective activities at concentrations ranging from 0.1µM to 10.0µM (Table 1; MTT assay showed the same trend as the LDH assay; data not shown). Of the five neuroprotective compounds, compound
7 and 10 showed the most potent activity against glutamate-induced neurotoxicity. The potency of the two compounds is about 70% to that of MK-801 or CNQX, the positive controls. The neuroprotective activities of caffeic acid, lonicerin and kaempferol-3-O-rutinoside were slightly increased in a dose-dependent manner. At high concentrations, however, quercent and luteolin did not show the improvement in the cell survival due to inherent cytotoxicity. To assess a more relevant relationship between the structure and activity, more derivatives should be assessed for their neuroprotective activity.
Furthermore, the mechanism of action of the neuropro- tective activities of flavonoids will be assessed in primary cultures of rat cortical cells against glutamate-induced neurotoxicity in our laboratory.
Acknowledgement
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST). (KRF-2008-331- E00452)
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Received April 18, 2011 Revised June 22, 2011 Accepted June 24, 2011
