Preparation method and application of rosa roxburghii tratt leaf extract for improving sleep

Abstract

The invention discloses a preparation method and application of a rosa roxburghii tratt leaf extract for improving sleep. Roxburgh rose leaves are subjected to heating reflux extraction, ethyl acetate extraction, separation and purification to obtain the roxburgh rose leaf extract. The rosa roxburghii tratt leaf active extract is rich in triterpenes and flavones, has remarkable sleep improvement activity, and can be used for preparing medicines and auxiliary medicines for improving sleep or used as a health food additive; the preparation method is simple and easy to implement, low in cost and easy for industrial production, and has high application value and good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine or natural medicine extraction, and particularly relates to a preparation method and application of a roxburghii leaf extract for improving sleep. Background Art

[0002] Insomnia, as a common chronic sleep disorder, is usually characterized by difficulty falling asleep at night, repeated awakenings, and decreased sleep quality. During the day, it is accompanied by physical fatigue, decreased cognitive function, inattention, depression, and other problems. Insomnia may be caused by a variety of factors, including psychological stress (such as anxiety, depression, etc.), chronic pain, and drug abuse (such as caffeine, etc.). With the development of society, the prevalence of insomnia has gradually increased. It is reported that the prevalence of insomnia in adults worldwide has reached between 30% and 50%. In addition, insomnia is associated with an increased risk of many other diseases, such as inflammatory diseases such as diabetes and neurodegenerative diseases. It is also closely related to mental health problems such as anxiety and depression. If not effectively treated, insomnia will seriously affect the patient's quality of life and may increase the risk of other health problems.

[0003] At present, the treatments for insomnia mainly include cognitive behavioral therapy (CBT-I) and drug therapy. CBT-I is generally considered to be the first choice for the treatment of chronic insomnia in adults, but its applicability is limited and there are certain limitations. The main drug treatments on the market currently include benzodiazepines, antihistamines and antidepressants. Although these drugs are effective in relieving insomnia symptoms, they are usually accompanied by adverse reactions, such as drug tolerance, dependence and sleep disorders. Common side effects include drowsiness, dizziness, fatigue, dysarthria, headache, memory impairment and depression.

[0004] Insomnia is related to neurotransmitters such as γ-aminobutyric acid (GABA), glutamate (Glu), norepinephrine (NE) and 5-hydroxytryptamine (5-HT). GABA is an important inhibitory neurotransmitter that plays a key role in promoting sleep, especially slow-wave sleep (SWS). 5-hydroxytryptamine (5-HT) is involved in sleep regulation and is associated with insomnia. Some studies have also found that orexin receptors are associated with insomnia, and orexin neurons are mainly inhibited through 5-HT neurons. In addition, the locus coeruleus (LC) is the main synthesis site of norepinephrine (NE), and NE is involved in the regulation of wakefulness.

[0005] Alprazolam is a widely used benzodiazepine used to treat anxiety, insomnia, panic disorder and other diseases. It acts as a positive allosteric modulator on GABAA1 and B1 receptors, thereby reducing the excitability of neurons and leading to a hypnotic effect. However, alprazolam has problems such as rapid absorption, short half-life and high abuse potential, so there is an urgent need to find safer and more effective alternative drugs for the treatment of insomnia.

[0006] Rosa roxburghii tratt is a perennial deciduous shrub of the genus Rosa in the family Rosaceae, which is widely distributed in southwest China. The flowers, leaves, fruits and roots of Rosa roxburghii have medicinal value and are rich in active ingredients such as flavonoids, triterpenes and polyphenols. Rosa roxburghii has shown beneficial biological functions in sleep, fertility, anti-aging and anti-inflammation in many studies. In particular, triterpenoid compounds in Rosa roxburghii, such as roxburghii glycosides and rosin acid, are considered to be its main pharmacologically active ingredients. However, the effect of Rosa roxburghii on insomnia has not been systematically studied so far. Therefore, it is necessary to explore the hypnotic effect and mechanism of Rosa roxburghii extract in depth, in order to develop a safer and more effective drug for the treatment of insomnia. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a preparation method and application of a roxburghii leaf extract for improving sleep. The roxburghii leaf extract of the present invention has the effects of alleviating insomnia symptoms and improving sleep.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for preparing a sleep-improving roxburghii leaf extract, comprising the following steps:

[0010] (1) Heating reflux extraction: grind the roxburghii leaves through an 80-120 mesh sieve, add the extraction solvent to the roxburghii leaves at a solid-liquid ratio of 1:18-22, heat reflux extraction at 90° C., extract 2-3 times, each extraction for 1-2 hours, filter and combine the filtrates, concentrate under reduced pressure at 45° C. until there is no alcohol smell, add a certain amount of water to the concentrate, and filter to obtain an aqueous solution;

[0011] (2) Ethyl acetate extraction: adding ethyl acetate to the aqueous solution in step (1) at a ratio of 1:1, extracting 2 to 3 times, separating the liquids with a separatory funnel, and concentrating the ethyl acetate layer under low pressure to obtain a brown viscous extract, i.e., the roxburghii leaf extract;

[0012] (3) Separation and purification: Separation technology is used to separate, purify and enrich the sea buckthorn leaf extract.

[0013] Preferably, in step (1), the extraction solvent is water or ethanol.

[0014] More preferably, the extraction solvent is 75% to 80% ethanol.

[0015] Preferably, in step (1), the ratio of roxburghii leaves to the extraction solvent is 1:20.

[0016] Preferably, in step (1), the extraction is performed 3 times, with each extraction lasting 1.5 h.

[0017] Preferably, in step (3), the separation technique is silica gel column chromatography, gel column chromatography or semi-preparative chromatography.

[0018] The invention provides a roxburghii leaf extract prepared by the preparation method described in the above technical scheme.

[0019] The roxburghii leaf extract mainly contains total flavonoids and total triterpenes.

[0020] The total flavonoids content in the roxburghii leaf extract is greater than 1.2%, and the total triterpenoid content is greater than 4.1%.

[0021] The roxburghii leaf extract prepared by the invention is applied in preparing active medicine for improving insomnia.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The present invention provides a roxburghii leaf extract for improving sleep. The extract is obtained by heating and refluxing the roxburghii leaves, extracting with ethyl acetate, and separating and purifying. After testing, it is found that the extract contains effective ingredients such as roxburghii glycoside, rosa glycoside, rosic acid and potentilla acid. After calculation, the total flavonoids content in the roxburghii leaf extract of the present invention is greater than 1.2%, and the total triterpenoid content is greater than 4.1%.

[0024] 2. The active extract of the roxburghii leaves of the present invention contains triterpenes and flavonoid components. The verification experiment provided by the present invention shows that it has significant activity in improving sleep. It can be used to prepare drugs and auxiliary drugs for improving sleep or as a health food additive, and has high application value and good application prospects.

[0025] 3. The preparation process of the roxburghii leaf extract of the present invention is of great significance to the high-value utilization and sustainable development of the roxburghii industry, and also provides an intermediate raw material for drugs for improving sleep. The preparation method is simple and easy, low in cost, and easy to industrialize and produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 HPLC spectra of active monomer (A) and extract (B) of Rosa roxburghii leaves, extract + monomer (C), roxburghii glycoside (1), rosin (2), rosinic acid (3) and potentilla acid (4);

[0027] Figure 2The effect of Rosa roxburghii leaf extract on PCPA-induced insomnia mice body weight (A), during modeling (B), the first week of administration (C), and the second week of administration (D) were measured for circadian food intake;

[0028] Figure 3 For the mine experiment, the movement route (A) and the total distance of the activity (B);

[0029] Figure 4 Effects of Rosa roxburghii leaf extract on the sleep onset rate (A), sleep latency (B) and sleep time (C) of PCPA-induced insomnia mice;

[0030] Figure 5 To investigate the effects of Rosa roxburghii leaf aqueous extract on the levels of γ-aminobutyric acid (GABA) and glutamate (Glu);

[0031] Figure 6 is the expression level of GABAR1A in the hippocampus of each group of mice after administration of roxburghii leaf extract ( Figure 6 A), protein blot of GABAR1A (6B), GABAR1B expression (6C) and protein blot (6D) (the low, medium and high dose groups of the active fraction of Rosa roxburghii leaf aqueous extract were 50, 100 and 150 mg / kg, respectively);

[0032] Figure 7 Effects of Rosa roxburghii leaf ethanol extract (100 mg / kg) on ​​the sleep onset rate (A), sleep latency (B) and sleep time (C) of PCPA-induced insomnia mice. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail with reference to the accompanying drawings. The production equipment in this application is commonly used in the art, and it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The instruments and reagents used in the following embodiments and experimental examples are:

[0035] Instruments: Hewiett Pakard 110 mass spectrometer (Hewlett-Packard, USA), Bruker AM-600 MHz nuclear magnetic resonance analyzer (Bruker, USA).

[0036] Test drugs and reagents: Rosa roxburghii (origin: Guizhou Guiding Minzi Food Co., Ltd.); ethanol and ethyl acetate were of analytical grade; water was ultrapure water; 200-300 mesh silica gel (Qingdao Marine Biological Company).

[0037] Example 1: Preparation method of roxburghii leaf extract

[0038] 1. Crush the roxburghii leaves through a 100-mesh sieve, add deionized water to the roxburghii leaves at a solid-liquid ratio of 1:20, heat and reflux at 90°C for extraction, extract 3 times, each extraction for 1.5 hours, filter and combine the filtrates, concentrate under reduced pressure at 45°C until there is no alcohol smell, add a certain amount of water to the concentrate, and filter to obtain an aqueous solution;

[0039] 2. Add ethyl acetate to the above aqueous solution at a ratio of 1:1, extract 3 times, separate the liquids with a separatory funnel, and concentrate the ethyl acetate layer under low pressure to obtain a brown viscous extract, i.e., a roxburghii leaf extract;

[0040] 3. Use silica gel column chromatography separation technology to separate, purify and enrich the roxburghii leaf extract to obtain the roxburghii leaf extract.

[0041] HPLC detection of roxburghii leaf extract: (1) Preparation of reference solution: Weigh appropriate amounts of roxburghii glycoside and rosa glycoside reference substances, dissolve them in methanol, and finally dilute to 1 mg / mL with 25% acetonitrile and 75% 0.01% trifluoroacetic acid water; (2) Preparation of test solution: Take appropriate amount of roxburghii leaf extract of the present invention and prepare it in the same way as the reference solution preparation method; (3) Chromatographic analysis conditions: The chromatographic column is Phenomenex, 250*4.6 mm, 5um; phase A is acetonitrile; phase D is 0.01% trifluoroacetic acid water (v / v), the flow rate is 1mL / min, and the gradient elution process is as follows: 0-15min: 25-30% B, 75-70% D; 15-20min: 30-35% B, 70-65% D; 20-25min: 30-35% B, 70-65% D; column temperature: 20℃, wavelength: 203nm, injection volume 10μL.

[0042] Example 1 The test results of the roxburghii extract are shown in Figure 1 , It was calculated that the total flavonoids content in the roxburghii leaf extract prepared in Example 1 was 1.2%, and the total triterpenoid content was 4.1%.

[0043] Example 2: Preparation method of roxburghii leaf extract

[0044] 1. Crush the roxburghii leaves through an 80-mesh sieve, add 75% ethanol to the roxburghii leaves at a solid-liquid ratio of 1:18, heat and reflux at 90°C for extraction, extract twice, each extraction for 1 hour, filter and combine the filtrates, concentrate under reduced pressure at 45°C until there is no alcohol smell, add a certain amount of water to the concentrate, and filter to obtain an aqueous solution;

[0045] 2. Add ethyl acetate to the above aqueous solution at a ratio of 1:1, extract twice, separate the liquids with a separatory funnel, and concentrate the ethyl acetate layer under low pressure to obtain a brown viscous extract, i.e., a roxburghii leaf extract;

[0046] 3. The roxburghii leaf extract was separated, purified and enriched by gel column chromatography to obtain the roxburghii leaf extract.

[0047] Example 3: Preparation method of roxburghii leaf extract

[0048] 1. Crush the roxburghii leaves through a 120-mesh sieve, add 80% ethanol to the roxburghii leaves at a solid-liquid ratio of 1:22, heat and reflux at 90°C for extraction, extract 3 times, each extraction for 1 hour, filter and combine the filtrates, concentrate under reduced pressure at 45°C until there is no alcohol smell, add a certain amount of water to the concentrate, and filter to obtain an aqueous solution;

[0049] 2. Add ethyl acetate to the above aqueous solution at a ratio of 1:1, extract 3 times, separate the liquids with a separatory funnel, and concentrate the ethyl acetate layer under low pressure to obtain a brown viscous extract, i.e., a roxburghii leaf extract;

[0050] 3. The sea buckthorn leaf extract was separated, purified and enriched using semi-preparative chromatography to obtain the sea buckthorn leaf extract.

[0051] Experimental example:

[0052] The inventors have conducted a large number of experimental studies on the sleep-improving effect of the roxburghii leaf extract of the present invention, some of which are excerpted as follows:

[0053] 1. Experimental animals, drug administration and model evaluation

[0054] Experimental animals: Male BALB / c mice (SPF grade, 18-22g, 6-8 weeks old) were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., Zhejiang Province, China, with an animal production license of (SCX(Zhe)2019-0004). Animal experiments were conducted in the animal room of the research group (a designated pathogen-free room) at a temperature of 23℃±2℃ and a humidity of 55%±5%, and were kept under a 12:12 hour light cycle. During the study, animals had free access to food (standard pelleted food) and drinking water (safe water source free of pathogen infection).

[0055] Drug administration: The mice were adaptively fed for 7 days, and then divided into nine groups according to their body weight. Except for the blank group, which was not modeled, the mice in the other groups were intraperitoneally injected with 400 mg / kg of p-chlorophenylalanine suspension (0.1 mL / 10 g), once a day, for 5 consecutive days. After the modeling, the drugs were all administered by gavage, and the blank group and the model group were given 0.05% sodium carboxymethyl cellulose, namely the blank group, the model group (PCPA group), the positive drug 1 (alprazolam tablets) group (0.15 mg / kg), the positive drug 2 (Shumian capsule) group (216 mg / kg), the three groups of RRTL of roxburghii leaf extract (50, 100 and 150 mg / kg of roxburghii extract containing roxburghii), the roxburghii group (5 mg / kg), the roxburghii group (3.5 mg / kg), 12 mice in each group, for a total of 108 mice.

[0056] p-Chlorophenylalanine modeling: After adaptive feeding, except for the normal control group, which was intraperitoneally injected with an equal volume of saline, the other groups were given a 400 mg / kg (400 mg / ml) PCPA (p-chlorophenylalanine) solution, which was solubilized with 1% gum arabic saline solution [2] (cosolvent) (it should be a milky white liquid after preparation). Each injection was thoroughly shaken and intraperitoneally injected once a day for 5 days.

[0057] Model evaluation: Compared with the normal control group, when the mice showed restlessness, continuous activity, loss of circadian rhythm, decreased appetite, and significant weight loss, it indicated that the model was successfully prepared.

[0058] 3.2. Effects of Rosa roxburghii leaf extract on body weight and food intake of mice during the experiment

[0059] The following experimental results involve the low, medium and high dose groups of the active part of the water extract of roxburghii leaves, which are 50, 100 and 150 mg / kg respectively. The results are expressed as mean ± standard deviation. Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001; compared with the model group * P<0.05, ** P<0.01, *** P<0.001.

[0060] 2. Effects of Rosa roxburghii leaf extract on body weight and food intake of mice during the experiment

[0061] In order to evaluate the effect of roxburghii extract on the body weight and circadian behavior of insomnia model mice, the body weight and circadian food intake of each group of mice were recorded during the experiment. Figure 2As shown in A, Rosa roxburghii leaf extract at doses of 50, 100, and 150 mg / kg had no significant effect on the body weight of PCPA-induced insomnia mice, and the changes in the daytime and nighttime food intake of each group of mice during the experiment were collected. Figure 2 As shown, the daytime food intake of the experimental group mice increased while the nighttime food intake decreased, and the difference between the two was not statistically significant (P>0.05). After one and two weeks of administration, the food intake of the medication group mice at night increased compared with that during the day (P<0.05), and the changes in feeding behavior indicated that the sea buckthorn leaf extract had an improving effect on the daytime behavioral changes caused by insomnia.

[0062] 3. Sedative effect of Rosa roxburghii leaf water extract on insomnia mice

[0063] The sedative effects of roxburghii leaf extract, roxburghii glycosides and rosin were evaluated by conducting an open field test of spontaneous activity in mice. Compared with the control group, all doses of roxburghii extract showed a significant reduction in total movement distance after administration (P<0.05), see Figure 3 Studies have shown that extracts from prickly pear leaves have a calming effect.

[0064] 4. Sleep rate, sleep latency and sleep duration of mice in the sodium pentobarbital-induced sleep experiment

[0065] The sedative and hypnotic effects of roxburghii leaf extract, roxburghii glycosides, and rosa glycosides were evaluated by sodium pentobarbital hypnosis experiment. 30 mg / kg sodium pentobarbital (subthreshold hypnotic dose) was injected intraperitoneally, as shown in Table 1 and Figure 4 A. The sleep status of each group of experimental mice was recorded. The positive drug (alprazolam) group had the highest rate of 58.3%. For the sleep rate, the PCPA group was 0, and the control group reached 8.3%. Compared with the PCPA group, the sleep rates of the drug-treated groups increased. By injecting PCPA to create a model, the sleep latency of mice increased significantly from 5.3758±1.403min to 10.18±4.818min (P<0.05, P<0.01), and the intervention of roxburghii leaf extract significantly alleviated this situation. In addition, after drug administration, the sleep duration of insomniac mice was also prolonged to varying degrees, as shown in Table 2. Figure 4 B and 4C. Specifically, the treatment effect of 100 mg / kg of roxburghii leaf extract was the best, and the sleep time of mice induced by PCPA was significantly prolonged to 71.626±9.873 min, which was significantly greater than 39.513±3.246 min in the PCPA group (P<0.05, P<0.01). Each dose showed different degrees of improvement, indicating that roxburghii leaf extract had a certain degree of synergistic effect with sodium pentobarbital at a certain dose, and had a certain therapeutic effect on insomnia caused by PCPA.

[0066] Table 1: Effect of sodium pentobarbital (30 mg / kg) on ​​the sleep rate of experimental mice

[0067]

[0068] Table 2: Effects of sodium pentobarbital on sleep latency and sleep time of experimental mice ( n=12±1)

[0069]

[0070] 5. Effects of Rosa roxburghii leaf extract on Glu and GABA in hippocampus

[0071] The effects of roxburghii leaf extract on Glu and GABA in hippocampus are detailed in Figure 5 . The contents of glutamate (Glu) and γ-aminobutyric acid (GABA) in the mouse hippocampus were measured by the corresponding kits. Compared with the control group, the γ-aminobutyric acid (GABA) in the PCPA group was significantly decreased (P<0.001), while the glutamate (Glu) was increased (P<0.01), and the Glu / GABA ratio was significantly increased (P<0.01), indicating that the roxburghii leaf extract may affect the sleep state of insomniac mice by regulating the levels of Glu and GABA.

[0072] 6. Effects of Rosa roxburghii leaf extract on 5-HT and NE in the hypothalamus

[0073] The effects of roxburghii leaf extract on 5-HT and NE in the hypothalamus are detailed in Figure 6 . Effect of Rosa Rosa Leaf Extract on Monoamine Neurotransmitters in Insomnia Mice By measuring the contents of NE and 5-HT in the hypothalamus of mice, the PCPA group showed a significant decrease in 5-HT levels (P<0.01) and a significant increase in NE levels (P<0.01) compared with the control group. Compared with the PCPA group, the three doses of Rosa Rosa Leaf Extract, Alprazolam treatment group, Su Mian capsule treatment group, and Rosa Rosa glycoside group mice increased 5-HT to varying degrees (P<0.05, 0.01), and reduced the level of NE in the hypothalamus (P<0.05), while the effect of Rosa Rosa glycoside (3.5 mg / kg) on ​​5-HT and NE was not significantly different from that of the PCPA group (P>0.05). Therefore, based on the experimental results, it is speculated that the symptom relief of Rosa Rosa Rosa Leaf Extract on PCPA insomnia mice may be regulated by monoamine neurotransmitters such as 5-HT and NE.

[0074] 7. Histological evaluation of experimental mouse brain tissue using H&E staining

[0075] In the control group, the neurons in the CA3 region of the hippocampus were arranged in an orderly manner and evenly distributed, with no obvious pathological damage. However, in the PCPA group, the hippocampal neurons were arranged in a disordered manner, and even the normal morphology of the neurons disappeared. After PCPA treatment, there were obvious changes in brain morphology compared with the control group, and the number of neurons in the CA3 region of the hippocampus of the experimental mice was significantly reduced (see Figure 7 A), while the hypothalamus (see Figure 7 B) Neurons showed morphological abnormalities and nuclear condensation, resulting in uneven distribution of neurons and decreased number. In addition, some new pathological changes were found, such as proliferation of glial cells in the brain. Although its therapeutic effect was not as good as the positive control alprazolam, sea buckthorn leaf extract could reverse brain damage to a certain extent.

[0076] Although the present invention has been described in detail above by means of general description, specific implementation methods and experiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a sleep-improving Rosa roxburghii leaf extract, characterized in that: The following steps are involved: (1) Heating reflux extraction: grind the roxburghii leaves through an 80-120 mesh sieve, add the extraction solvent to the roxburghii leaves at a solid-liquid ratio of 1:18-22, heat reflux extraction at 90° C., extract 2-3 times, each extraction for 1-2 hours, filter and combine the filtrates, concentrate under reduced pressure at 45° C. until there is no alcohol smell, add a certain amount of water to the concentrate, and filter to obtain an aqueous solution; (2) Ethyl acetate extraction: adding ethyl acetate to the aqueous solution in step (1) at a ratio of 1:1, extracting 2 to 3 times, separating the liquids with a separatory funnel, and concentrating the ethyl acetate layer under low pressure to obtain a brown viscous extract, i.e., the roxburghii leaf extract; (3) Separation and purification: Separation technology is used to separate, purify and enrich the sea buckthorn leaf extract.

2. The method for preparing a sleep-improving Rosa roxburghii leaf extract according to claim 1, characterized in that: In the step (1), the extraction solvent is water or ethanol.

3. The method for preparing a sleep-improving Rosa roxburghii leaf extract according to claim 2, characterized in that: The extraction solvent is 75% to 80% ethanol.

4. The method for preparing a sleep-improving Rosa roxburghii leaf extract according to claim 1, characterized in that: In the step (1), the ratio of the roxburghii leaves to the extraction solvent is 1:

20.

5. The method for preparing a sleep-improving Rosa roxburghii leaf extract according to claim 1, characterized in that: In the step (1), the extraction is performed 3 times, and each extraction lasts for 1.5 hours.

6. The method for preparing a sleep-improving Rosa roxburghii leaf extract according to claim 1, characterized in that: In the step (3), the separation technique is silica gel column chromatography, gel column chromatography or semi-preparative chromatography.

7. A Rosa roxburghii leaf extract for improving sleep, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 6.

8. The sleep-improving Rosa roxburghii leaf extract according to claim 7, characterized in that: It mainly contains total flavonoids and total triterpenes.

9. The sleep-improving Rosa roxburghii leaf extract according to claim 8, characterized in that: The total flavonoid content is greater than 1.2%, and the total triterpenoid content is greater than 4.1%.

10. Use of the roxburghii leaf extract according to any one of claims 7 to 9 in the preparation of an active drug for improving insomnia.

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