Anti-anxiety Effect and Mechanism of Passiflora Leaf Extract of Purple Fruit

Abstract Objective To investigate the anxiolytic effect and its mechanism of extracts of Passiflora leaves. Methods Sixty mice were randomly divided into a blank control group, a positive control group and a large, medium and small dose group of extracts. In the large, medium and small doses of the extract, the extract of Passiflora edulis was 800,400,200 mg·kg～·d～, and the positive control group was given diazepam 2 zng·kg～·d～, blank control The group was intragastrically treated with an equal volume of 1% sodium carboxymethylcellulose, and each group was administered continuously for 7 days. The anesthesia activity of the leaves of Passiflora glabra L. was studied by using the elevated cross maze (EPM) experiment as an animal model. The amplified products were analyzed by polymerase chain reaction (PCR). Results The ethanol extract of Passiflora leaves (400,800 rng·kg-.·d-1) can increase the time and frequency of movement in the open arms of mice, and increase the number of times in the central area of ​​the opening; Fluorescence quantitative PCR analysis showed that the ethanol extract of Passiflora edulis leaves (400,800 rng·kg·d_1) can up-regulate GABAA receptor 0 [, subtype and 0 [, subtype] in brain tissue of EPM anxiety mice. mRNA expression level. Conclusion The ethanol extract of Passiflora chinensis leaves has obvious anti-anxiety effect, and its mechanism may be related to the regulation of GABA receptor d2 subtype and only subtype mRNA expression level.

Key words purple passion fruit leaf; anxiety; elevated plus maze; 1 aminobutyric acid type A receptor

Passiflora (P. edu纭Si琊), alias passion fruit, is the genus Passiflora (P0ss坍omce mouth e) Passiflora (several 黜耖%m L.) vine, currently in Taiwan Guangdong, Fujian, Guangxi and Yunnan provinces are heavily cultivated. Passionflower can be used as a medicine for nerve pain, general seizures, rickets, intermittent asthma, menopausal syndrome, arrhythmia, and hypertension. It is widely used as a folk medicine in tropical countries. It is quite famous in Europe and is suitable for treating various diseases such as sleep disorders and anxiety. However, the author has not yet reported the research on the anti-anxiety effect of domestic purple fruit passionflower. Therefore, the author conducted an experimental study on the anxiolytic effect of Passiflora leaves in this study.

1 Materials and methods

1.1 Animal healthy male Kunming mice 60, grade: sPF, initial body weight 20 ~ 25 g, certificate number: scxK (Guangdong) 2008 002, purchased from Guangdong Medical Laboratory Animal Center. The mice were housed in the sPF animal room of the Animal Laboratory of Guangzhou General Hospital of Guangzhou Military Region. Light rhythm 12L: 12D (7:00 ~ 19: oo), room temperature (18 ± 2) ° C, relative humidity of about 50%, animals can take their own standard feed and clean drinking water.

1.2 Drugs and reagents Purple fruit Passiflora leaves were collected from Wuhua County, Meizhou, Guangdong Province, and identified by the Chinese Academy of Plant Research Wei Qian as the Passiflora flower of the Passiflora plant (P∞s汐om) Edu playing Si郴) dry leaves; sodium carboxyl methyl cellulose (cMc); diazepam (Shanghai Xinyi Pharmaceutical Co., Ltd., batch number); Trizol reagent (TaKaRa Code: D9108A); Ester (DEPc, Guangzhou Weijia Technology Co., Ltd.); D12000 DNAMarker (Dalian Bao Biotechnology Co., Ltd.); EB solution (Shanghai Shenneng Gaming Bio Company); M. MLV reverse transcriptase (nvitrogen); sYBRPremix Ex Taq'M (TaKaRa); Loading Bufkr (TaKaRa). Primer synthesis and sequencing were performed by Shanghai Jierui Biotechnology Co., Ltd.

1.3 Instrument mouse elevated cross maze (provided by Shanghai Xinsoft Information Technology Co., Ltd.); NAN0 DROP 2000; constant pressure electrophoresis instrument (Beijing Liuyi Instrument Factory); constant temperature water bath (Shanghai Jinghong Experimental Equipment Company); GeneQ Gene amplification instrument (Hangzhou Bori Technology Co., Ltd.); fluorescence quantitative PcR instrument (Qiage n, company); gel imaging system (universal Hood) II (Bio-Rad company).

1.4 Preparation of the solution Take the purple fruit passionflower leaf powder 160 g, and extract it with 15 times 60% ethanol at 80 ° C for 3 times, each time for 2 h, the filtrate is combined, and the filtrate is dried by rotary evaporation to obtain purple. The fruit extract of Passiflora leaves was dissolved with 1% CMC before use. Diazepam is formulated with 1% CMC. 125 mg·mL '1 solution, stored at 4 ° C, shake well before use.

1.5 Animal grouping and administration methods Mice were randomly divided into blank control group, positive control group and purple, passion fruit extract large, medium and small dose groups. Among them, the large, medium and small doses of Passiflora extract were 800, 400, 200 mg·k ~·d. 1 Administration by intragastric administration ̈1, the positive control group was given diazepam 2 mg·kg～·d～, and the blank control group was given the same volume of 1% cMc. Each group was administered continuously for 7 days, and the behavioral test was performed on the 7th day after 1 hour of the last administration, 8:00 to 16:00.

1.6 Mouse elevated maze (EMP) experimental elevated plus maze consists of two open arms (length 30 cm, width 5 cm) and two closed arms (length 30 cm, width 5 cm, height 15 cm) from the ground 50 Cm. Before the labyrinth test, each mouse was placed in a 50 cm × 50 cm × 40 cm field box, and it was quickly placed on the central platform of the EPM after 5 minutes of free exploration, so that its head was open to one of the heads. Arm, record the number of times the mouse entered the open arm, the number of closed arms and the central area of ​​the maze within 5 min and the movement time of the open arm and the closed arm. The percentage of the number of open arms and the total number of input arms and the percentage of exercise time in the open arms and the total time of the human arm represent the anxiolytic effect indicators. The entire behavioral process is analyzed in real time using Supermaze software for real-time monitoring. After each test, wipe the maze with a damp cloth, remove the feces, wipe with a dry cloth, and then test the next mouse.

1.7 Rats with 1 aminobutyric acid type A (GABA) receptor in brain tissue only 2 and only, subtype mRNA expression was detected immediately after the end of EPM behavioral test in mice, 5 small in each group The brain tissue of the mouse was isolated under aseptic conditions. The brain tissue was placed in a pre-cooled mortar and mashed into powder under liquid nitrogen conditions. The total RNA of brain tissue was extracted with TRIz01 reagent according to the kit instructions. After % agarose gel electrophoresis to identify RNA integrity, GABA was detected by semi-quantitative RT-PcR method. Receptor d: subtype and 0 [, subtype mRNA level expression, B-actin as an internal reference, only 2 subtype upstream primer 5'-GcTAcGC吼~C-ACAACCTC-3', downstream bow I 5 7 A TCA7ITGAC711ACACT-C1Trcc. 3';o[, subtype upstream primer 5'. CAACTAC rITIICACcAAGCGG. 3 7, downstream bow l 5'. CGGGAAATrITGTCAACCT-37; PcR reaction conditions: pre-denaturation at 94 °C for 5 min, 94 °C for 30 s, 53.6 °C for 30 s, 72 °C for 45 s, amplification for 38 cycles, 72 °C for 7 min, PcR product for 3% agar Sugar gel electrophoresis. Real-timePcR analysis: only: subtype upstream primer 5 7 AATcT-GGACTCCTGA'rACCTTC. 3', downstream bow I 5 7-CATTCGGcTTGGAcTGTAAG 3.7; d, subtype upstream primer 5'-AAGT-GCACAGGATGGCTCACG-3', downstream bow 1'ACATGG-CAAGTAGGTCTGGATGAC-3 7; Real-time PCR reaction After the end, the results of the reaction were analyzed using a PcR instrument software to detect c in each sample. Value to Delta-delta c. The method was used to analyze each group of GABA. Receptors only have 2 subtypes and instruments, and subtypes have relative expression levels.

1.8 statistical methods applied sPSsl8. The o version for windows statistical software package statistical analysis of the data, the experimental data is expressed by mean ± standard deviation (孑 ± s), using one-way ANOVA for one-way ANOVA. P

3 Discussion
Passiflora in Brazil is used as a medicine for the treatment of anxiety disorders. Literature studies have shown that the difference between the extract of Passiflora edulis and diazepam is that it does not block the memory process while resisting anxiety. In this study, RT-PCR and Real-time PCR were used to determine whether the extract of Passiflora leaves passed through GABA. Type receptors only: subtypes and only subtypes to exert anxiolytic effects, the results showed that 800 and 400 mg·kg ~ · d of purple fruit passionflower leaf extract relative to the blank control group, only: There was a statistically significant difference in the relative expression of subtype and instrumental subtype mRNA (P<0.01), suggesting that the anxiolytic effect of Passiflora edulis extract and GABA. Type receptor related.

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