Studies on Anti-Depressant Activity of Four Flavonoids Isolated from Apocynum venetum Linn (Apocynaceae) Leaf in Mice

Purpose: To investigate the anti-depressant activity of kaempferol, quercetin, kaempferol-3-O- β -D-glucose and quercetin-3-O- β -D-glucose isolated from Apocynum venetum Linn. (Apocynaceae) leaf and their mechanisms of action. Methods: The four flavonoids were isolated from Apocynum venetum leaf by chromatography. Mice were divided into vehicle, fluoxetine, kaempferol, quercetin, kaempferol-3-O- β -D-glucose and quercetin-3-O- β -D-glucose groups (n = 10). Forced swimming (FST), tail suspension (TST) and locomotor activity (LAT) tests were used to evaluate the effects of the four flavonoids (0.35 mM/kg) on immobility time, monoamine neurotransmitters, viz, norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT), as well as on the metabolite (5-HIAA) in mice brain and central nervous system (CNS) with the aid of video camera, HPLC-ECD and activity-monitoring system. Results: The four flavonoids significantly (p < 0.05) reduced mice immobility time (72.58 - 90.24; 52.58 - 70.24 s), 5-HIAA levels (940.8 - 1244.7; 880.8 - 1164.1 ng/g) and 5-HIAA/5-HT ratio (1.77 - 4.76; 1.83 - 4.16), but increased NE, DA and 5-HT levels (238.7 - 405.7, 308.4 - 528.1, 261.4 - 531.9; 243.9 -423.6, 296.7 - 534.9, 279.8 - 481.4 ng/g) in FST and TST, compared with control group (146.18, 126.18 s; 1363.4, 1240.9 ng/g; 7.43, 6.16; 138.4, 235.4, 183.4 and 143.7, 218.6, 201.4 ng/g). The effects of the four flavonoids on the above indices were significant (p < 0.05) and positively related to their polarity. They had no CNS-stimulating effects in LAT. Conclusion: The anti-depressant activities of the four flavonoids are positively related to their polarity, and the mechanisms may be due to increased NE, DA and 5-HT and reduced 5-HT metabolism. and Pharmacy Abstracts


INTRODUCTION
Depression is a kind of mental disorders and its major symptoms include the reduced interest and pleasure, lowered mood and metabolic disorder of monoamine neurotransmitters in the central nervous system (CNS) [1].World Health Organization (WHO) predicted that depression will be the second most common disease in 2020 [2].

EXPERIMENTAL Plant material
A. venetum leaves were purchased from Chinese herbal medicine market of Xian in 2013, identified by Jun-Lian Lang.A voucher specimen (voucher no.BJHLGH20130164) was stored in pharmacy department of Beijing Huilongguan Hospital for future reference.

Animals
Male mice (20 ± 2 g) were purchased from the SLRC Laboratory Animal Company (Shanghai, China).The animals were conditioned to standard laboratory conditions (12/12 h light /dark cycle at 25 ± 1 °C) and had free access to food and water.All experiments were strictly in accordance with the international guidelines for care and use of laboratory animals [19].The experiments were carried out with the approval of the Animal Care and Use Committee of Beijing Huilongguan Hospital (protocol no: BJHLGH ACUC2013).

Extraction and isolation
The air-dried A. venetum leaves (10 kg) were finely cut and extracted with 75 % ethanol by percolation method at the speed of 20 mL/min.The 75 % ethanol solvent was combined and concentrated under vacuum to afford a crude extract (1.2 kg), which was then suspended in hot water and successfully partitioned with petroleum ether, ethyl acetate and n-butyl alcohol.The ethyl acetate fraction (180 g) was subjected to column chromatography (CC) over silica gel (74 -149 μm), eluted with chloroformmethanol and chloroform-methanol-water to obtain nine fractions.Fraction 2 (32.2 g) was separated by CC over silica gel (chloroformmethanol) and purified on Sephadex LH-20 (chloroform-methanol) to provide compounds kaempferol (98 mg) and quercetin (173 mg).Fraction 5 (28.9 g) was separated by CC over silica gel (chloroform-methanol-water) and purified on Sephadex LH-20 (chloroformmethanol-water) to provide compounds kaempferol-3-O-β-D-glucose (67 mg) and quercetin-3-O-β-D-glucose (173 mg).The structures of the four compounds were identified by nuclear magnetic resonance (NMR) data, compared with the existing literatures [15,16], and their purities were more than 98 %, verified by area normalization method of high performance liquid chromatography with electrochemical detector (HPLC-ECD).Their chemical structures are shown in Figure 1.

Forced swimming test (FST)
FST was carried out on mice according to the existing method with some modifications [18,20,21].Mice were individually placed into glass cylinders (height 25 cm, diameter 10 cm), which were filled with water (temperature: 25 ± 1 °C; depth: 18 cm).Twenty-four hours after the pretest, mice were forced to swim in the glass cylinders for 6 min.After 2 min swimming, the duration of immobility was recorded during the remaining 4 min using a video camera.Mice were judged to be immobile when no additional activity was observed other than that those movements necessary to keep its head above the water.Additionally, the observer recording the immobility of mice was blinded to the drug treatments and after each test, the glass cylinders was refilled with fresh water.

Tail suspension test (TST)
TST was carried out on mice according to the existing method with some modifications [22].Briefly, each mouse was individually suspended by the tail (20 mm from the end) for 6 min in a box (250 × 250 × 300 mm 3 ) with the head 50 mm from the bottom.Testing was carried out in a darkened room with minimal background noise.The duration of immobility was recorded during the final 4 min of the test using a video camera.Mice were judged to be immobile only when they were passively hanged and completely motionless.Additionally, the observer recording the immobility of mice was blinded to the drug treatments.

Locomotor activity test (LAT)
To access the effects of drugs on locomotor activity, mice were individually placed into cylinders (height 25 cm, diameter 15 cm) at the same time 60 min after drug administration and then the total locomotor activity of each mouse was automatically measured and recorded in 5 min separately by an activity-monitoring system (Institute of Materia Medica, Chinese Academy of Medical Sciences) [1].

Determination of monoamine neurotransmitter and metabolite
The neurotransmitter concentrations in mice brain were simultaneously determined according to the existing method with some modifications [23].The vehicle group (negative control group) without any stress was added as vehicle-stress group in this part.After the FST and TST were completed, mice were sacrificed by decapitation in the animal laboratory, and their brain tissues were immediately obtained on ice and stored at -80 °C until biochemistry assay.The brain tissue of each mouse was homogenized in ice-cold 0.02 M perchloric acid including 1.0 μM DHBA (internal standard).Then the homogenates were centrifuged at 14000 rpm for 15 min at 4 °C, and the supernatant was separated and filtered through a 0.22 μm membrane filter prior to HPLC analysis.The HPLC analysis was carried out on Agilent 1200 (Ageilent, USA), equipped with ECD (Model 5300A CoulArray Detector) and connected to an Agilent ChemStation.The chromatography was performed on a Diamonil C18 column (4.6 mm × 25 mm, 5 μm).The mobile phase consisted of 11% methanol and 0.1 M NaH2PO4 aqueous solution including 0.5 mM EDTA•Na 2 and 0.85 mM OSA.The pH of mobile phase was adjusted to 3.4 using phosphate acid.Then mobile phase were filtered through 0.22 μm membrane prior to HPLC analysis.The flow rate, injection volume, column temperature and working potential were 0.8 mL/min, 20 μL, 25 °C and +80 mV, respectively.The quantifications of NE, DA, 5-HT and 5-HIAA in sample were analyzed by standard curve method.Additionally, the levels of NE, DA, 5-HT and 5-HIAA were represented as ng/g per brain wet weight.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM, n = 10).One-way ANOVA (LSD test) was used to analyze differences among different groups on SPSS (version 20.0).Differences were recognized as statistically significant at p < 0.05.

Effects of four flavonoids on duration of immobility
As shown in Figures 2 and 3, after treatment with fluoxetine at dose of 0.2 mM/kg, the duration of immobility (58.9 and 38.59 sec) in FST and TST was significantly (p < 0.05) reduced, compared with the vehicle group (146.18 and 126.18 sec), and the results indicated that the FST and TST models were successfully established.After treatment with kaempferol, quercetin, kaempferol-3-O-β-D-glucose or quercetin-3-O-β-D-glucose at dose of 0.35 mM/kg, the duration of immobility (72.58 -90.24 and 52.58 -70.24 sec) was significantly (p < 0.05) reduced in FST and TST, compared with the vehicle group (146.18 and 126.18 sec).Moreover, the inhibitory effects of the four flavonoids on the duration of immobility in FST and TST were significantly (p < 0.05

Effects of four flavonoids on locomotor activity
As shown in Figure 4   The effects of drug treatments on the levels of NE, DA, 5-HT and 5-HIAA in FST and TST are presented in Tables 1 and 2. The effects of drug treatments on the ratio of 5-HIAA/5-HT in FST and TST are shown in Figures 6 and 7. Compared with the vehicle-stress group, the levels of NE, DA, 5-HT of vehicle group were significantly (p < 0.05) reduced in FST and TST, and the level of 5-HIAA and the ratio of 5-HIAA/5-HT of vehicle group were significantly (p < 0.05) increased in FST and TST.After treatment with fluoxetin at dose of 0.2 mM/kg, the levels of NE, DA and 5-HT were significantly (p < 0.05) increased in FST and TST, compared with the vehicle group, and the level of 5-HIAA and the ratios of 5-HIAA/5-HT were significantly (p < 0.05) reduced in FST and TST, compared with the vehicle group.The results of the vehiclestress, vehicle and fluoxetin groups indicated that the FST and TST models were successfully established.After treatment with kaempferol, quercetin, kaempferol-3-O-β-D-Glucose or quercetin-3-O-β-D-glucose at dose of 0.35 mM/kg, the levels of NE, DA and 5-HT were significantly (p < 0.05) increased in FST and TST, compared with the vehicle group, and the level of 5-HIAA and the ratios of 5-HIAA/5-HT were significantly (p < 0.05) reduced in FST and TST, compared with the vehicle group.Moreover, the reversed effects of the four flavonoids on the levels of NE, DA, 5-HT and 5-HIAA and the ratio of 5-HIAA/5-HT were significantly (p < 0.05) different in FST and TST (kaempferol < quercetin < kaempferol-3-O-β-Dglucose < quercetin-3-O-β-D-glucose).The monoamine theory is a widely admitted explanation for depression, which can lead to the impairment of monoaminergic functions and reduce the levels of monoamine neurotransmitters (NE, DA and 5-HT) [26].The symptom of depression can be alleviated by upregulating the levels of monoamine neurotransmitters (NE, DA and 5-HT) in the CNS [27].The tricyclic antidepressants, monoamine oxidase inhibitors and selective 5-HT reuptake inhibitors are now widely used in the clinic, and fluoxetine, a kind of selective 5-HT reuptake inhibitor, are more popular because of its lower toxicity [28,29].So, the fluoxetine was selected as the positive control in the study.The ratio of neurotransmitter/its metabolite can be considered as an index of neurotransmitter metabolism, and the reduction of the ration indicates the reduction of neurotransmitter metabolism [29,30].As shown in Tables 1 and 2 and Figures 6 and 7, kaempferol, quercetin, kaempferol-3-O-β-D-glucose and quercetin-3-Oβ-D-glucose significantly increased the levels of NE, DA and 5-HT and reduced the levels of 5-HIAA and the ratio of 5-HIAA/5-HT in the mice brain of FST and TST.Namely, their possible mechanisms of anti-depressant activity were related to the increase of NE, DA and 5-HT as well as reduction of 5-HT metabolism in mice brain.Moreover, the reversed effects of the four flavonoids on the levels of NE, DA, 5-HT and 5-HT metabolism were positively related to their polarity.

CONCLUSION
Kaempferol, quercetin, kaempferol-3-O-β-Dglucose and quercetin-3-O-β-D-glucose from A. venetum leaf have significant anti-depressant activity that is positively related to their polarity.The mechanism may be related to the increase of NE, DA and 5-HT as well as reduction of 5-HT metabolism.The study provides the evidence to support that A. venetum leaf has anti-depressant activity and the reference for studying the structure-function relationship of flavonoids on anti-depressant activity.