Rosa roxburghii tratt fruit and gastrodia elata preserved fruit fermentation liquor and preparation method and application thereof

Abstract

The invention relates to the technical field of fruit and vegetable fermented food processing, in particular to roxburgh rose and gastrodia elata preserved fruit fermentation liquor and a preparation method and application thereof.The roxburgh rose and gastrodia elata serve as main raw materials, the gastrodia elata is washed and diced, the roxburgh rose is sliced, seeds are removed, then mixing is conducted, curing and sugaring are conducted, and roxburgh rose and gastrodia elata preserved fruits are prepared and then transferred into a fermentation container to be fermented; according to the present invention, with the preparation process, the effective components of the rosa roxburghii tratt and the gastrodia elata are maintained to the maximum extent, and the rosa roxburghii tratt and the gastrodia elata are subjected to compatibility use in the manner so as to achieve the effect of the preserved fruit; the traditional Chinese medicine composition can play a role in improving mouse insomnia caused by PCPA from the aspects of improving sedative hypnotic and antioxidant capacities, reducing the inflammatory factor level, improving brain injury and the like, and '1 + 1gt; compared with artificially synthesized chemical drugs, the rosa roxburghii tratt and gastrodia elata functional sleep-aiding product has obvious advantages, has wide application value in the field of large health and health-care products, and also provides a certain new way for development of rosa roxburghii tratt and gastrodia elata functional sleep-aiding products.

Description

Technical Field

[0001] The invention relates to the technical field of fruit and vegetable fermented food processing, in particular to a roxburgh roxburgh and gastrodia elata preserved fruit fermentation liquid, and a preparation method and application thereof. Background Art

[0002] Sleep is a spontaneous, cyclical state of rest characterized by relative stillness, a slowed response to external stimuli, and a temporary interruption of consciousness. Good sleep is one of the most direct and effective ways to boost immunity, improve cardiovascular health, and enhance learning and memory. Conversely, sleep disturbances can lead to serious consequences such as depression, anxiety, obesity, and memory loss, and can even contribute to neurological conditions such as degenerative brain diseases, epilepsy, and dementia. Insomnia, a type of sleep disorder, is defined as a condition that prevents the body from achieving normal sleep and impacts normal functioning. Clinically, it manifests as difficulty falling asleep, short sleep duration, shallow sleep, and a restless night, severely impacting daily life. With the accelerated pace of life, increasing work pressure, and a host of unhealthy lifestyle habits, the incidence of insomnia is increasing year by year, and is increasingly affecting younger people. According to the 2023 China Healthy Sleep White Paper, nearly 80% of respondents experience sleep difficulties, making sleep problems a significant health concern. At present, the main drugs used in clinical treatment of insomnia are benzodiazepines, barbiturates, melatonin and antihistamines. Although these drugs have significant therapeutic effects, they have strong toxic side effects. Long-term use can easily increase the risks of drug dependence, addiction, withdrawal, etc. Therefore, it is of profound significance to develop safe and effective natural products and functional foods that can improve sleep.

[0003] Gastrodia elata (Gastrodia elata Bl.), the tuber of the orchid plant Gastrodia elata, has the effects of calming the liver and relieving wind, dispelling wind and unblocking the meridians. As a traditional Chinese medicinal material, it was officially included in the catalog of substances that are both traditional foods and Chinese medicinal materials in November 2023. Studies have shown that Gastrodia elata has anticonvulsant, analgesic, and sedative pharmacological effects, and its active ingredients can promote sleep. Seabuckthorn (Rose roxburghii Tratt), the fruit of the perennial deciduous shrub Rosaceae, is one of Guizhou's most distinctive and advantageous industries and a botanical resource used for both medicinal and edible purposes. It possesses a variety of excellent biological activities, and existing research has shown that seabuckthorn has certain sedative and hypnotic effects.

[0004] At present, researching and developing a green, healthy, naturally derived active substance and product with similar effects to hypnotic drugs but fewer toxic side effects has become a hot topic for current and future research. Sea buckthorn and Gastrodia elata are both advantageous resources of Guizhou's medicinal and edible origins. Both have been widely studied and applied in improving sleep. However, there have been no reports on the combined application of the two to develop a health product for improving sleep disorders, and whether the combined application of the two has a synergistic effect is unknown. Summary of the Invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a fermentation liquid of roxburghii and Gastrodia elata preserved fruit, and a preparation method and application thereof. Specifically, this is achieved by the following technical solutions:

[0006] A method for preparing a fermented liquid of roxburghii and gastrodia elata preserved fruit comprises the following steps:

[0007] A. Wash and dice fresh Gastrodia elata, slice and deseed Rosa roxburghii, mix 10 parts of Rosa roxburghii with 50-200 parts of Gastrodia elata, steam and ripen at 60-90° C., add 60-210 parts of sucrose, and candy at 10-20° C. for 8-10 hours to prepare Rosa roxburghii Gastrodia elata preserved fruit;

[0008] B. transferring the preserved roxburghii and Gastrodia elata fruits obtained in step A into a fermentation tank, adding mixed strains into the fermentation containers, controlling the fermentation temperature to 20-30° C. and aerobically fermenting for 20-50 days, wherein the mixed strains are composed of yeast, lactic acid bacteria, and acetic acid bacteria;

[0009] C. crushing, pressing and filtering the fermented Rosa roxburghii and Gastrodia elata preserved fruits obtained in step B to obtain Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid.

[0010] Furthermore, the ratios of the raw materials in step A are all by weight.

[0011] Furthermore, in step B, the ratio of yeast, lactic acid bacteria and acetic acid bacteria is 1:1:1 by mass.

[0012] Furthermore, the yeast in step B is one or more of Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces odorifera, Saccharomyces weigensis, Hansenula anomala, Schizosaccharomyces pombe, Rhodotorula glutinosus, Pichia pastoris, Candida glycerogenes and Candida utilis, mixed in any proportion.

[0013] Furthermore, the lactic acid bacteria in step B are one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus gasseri, Bifidobacterium and Lactobacillus reuteri mixed in any proportion.

[0014] Furthermore, the acetic acid bacteria in step B is one or more of Acetobacter orlanii, Acetobacter climbing, Acetobacter film, Acetobacter AS1.41, and Acetobacter Shanghai 1.01, mixed in any proportion.

[0015] Furthermore, the fermentation liquid of the preserved roxburghii and gastrodia elata fruits in step C is also sterilized at an ultra-high temperature of 120°C.

[0016] A fermented liquid of preserved roxburghii and gastrodia elata fruit is prepared by the above-mentioned preparation method. It can be used in the preparation of foods, health products, and pharmaceuticals, particularly in the preparation of health products for improving sleep.

[0017] A roxburgh and gastrodia elata composite fermented oral liquid comprises, by weight, 40-60 parts of the roxburgh and gastrodia elata preserved fruit fermentation liquid, 30-50 parts of purified water, 0.1-0.5 parts of edible essence, 5-10 parts of xylitol, and 0.3-0.8 parts of sodium carboxymethyl cellulose.

[0018] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0019] (1) The present application provides a fermentation liquid of sea buckthorn and gastrodia elata preserved fruit, and a preparation method and application thereof, wherein sea buckthorn and gastrodia elata are combined in a manner such that the preserved fruit is candied and then fermented by microorganisms to develop a health product for improving sleep. The product can improve insomnia caused by PCPA in mice by improving sedative hypnosis and antioxidant abilities, reducing inflammatory factor levels, and improving brain damage. Moreover, the combined use can achieve a "1+1>2" effect, which has obvious advantages over artificially synthesized chemical drugs, and provides a certain new approach for the development of sea buckthorn and gastrodia elata functional sleep-aid products.

[0020] (2) This application uses Rosa roxburghii and Gastrodia elata in combination. Both Rosa roxburghii and Gastrodia elata contain functional ingredients that can calm and hypnotize, reduce the excitability of nerve centers, and improve sleep. They can improve the insomnia caused by PCPA in mice by improving sedation and hypnosis and antioxidant abilities, reducing the level of inflammatory factors, and improving brain damage. The functional substances in them can have an effect on the same symptoms and causes, playing a "superposition" and synergistic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of fermented liquid of preserved fruit of sea buckthorn and Gastrodia elata on the sleep of insomnia mice.

[0022] Figure 2 The effect of fermented liquid of preserved Rosa roxburghii and Gastrodia elata on the behavior of insomnia mice.

[0023] Figure 3 The effect of Rosa roxburghii Gastrodia elata preserved fruit fermentation liquid on the levels of SOD, MDA, IL-6, IL-1β, and TNF-α in mouse serum.

[0024] Figure 4 The effect of Rosa roxburghii Gastrodia elata preserved fruit fermentation liquid on the changes in Glu, GABA, 5-HT and DA contents in mouse brain tissue.

[0025] Figure 5 The hippocampus pathological images of the whole brain of mice in each group (200×). DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0027] Example 1

[0028] A fermented liquid of preserved roxburghii and gastrodia elata fruits is prepared by the following steps:

[0029] A. Wash and dice fresh Gastrodia elata, slice and deseed Rosa roxburghii, mix 1 kg of Rosa roxburghii and 20 kg of Gastrodia elata, steam and ripen at 60°C, add 21 kg of sucrose, and candy at 20°C for 8 hours to prepare Rosa roxburghii Gastrodia elata preserved fruit;

[0030] B. The Rosa roxburghii Gastrodia elata preserved fruit obtained in step A was transferred to a fermentation tank, and mixed strains were added to the fermentation container respectively, and the fermentation temperature was controlled to be 30° C. and aerobically fermented for 20 days, wherein the mixed strains consisted of yeast, lactic acid bacteria and acetic acid bacteria, and the ratio thereof was 1:1:1 by mass;

[0031] C. The fermented Rosa roxburghii and Gastrodia elata preserved fruits obtained in step B are crushed, pressed and filtered to obtain Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid, and then subjected to ultra-high temperature instantaneous sterilization at 120° C. to obtain a finished product.

[0032] The yeast described in step B is Angel Saccharomyces cerevisiae.

[0033] The lactic acid bacteria described in step B is Lactobacillus plantarum.

[0034] The acetic acid bacteria described in step B is Shanghai Niang 1.01 acetic acid bacteria.

[0035] Example 2

[0036] A fermented liquid of preserved roxburghii and gastrodia elata fruits is prepared by the following steps:

[0037] A. Wash and dice fresh Gastrodia elata, slice and deseed Rosa roxburghii, mix 1 kg of Rosa roxburghii and 10 kg of Gastrodia elata, steam and ripen at 85°C, add 11 kg of sucrose, and candy at 15°C for 9 hours to prepare Rosa roxburghii Gastrodia elata preserved fruit;

[0038] B. The preserved roxburghii and Gastrodia elata fruits obtained in step A were transferred to a fermentation tank, and mixed strains were added to the fermentation containers respectively, and the fermentation temperature was controlled to be 25° C. and aerobically fermented for 30 days, wherein the mixed strains consisted of yeast, lactic acid bacteria and acetic acid bacteria, and the ratio by mass was 1:1:1;

[0039] C. The fermented Rosa roxburghii and Gastrodia elata preserved fruits obtained in step B are crushed, pressed and filtered to obtain Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid, and then subjected to ultra-high temperature instantaneous sterilization at 120° C. to obtain a finished product.

[0040] The yeast described in step B is Angel Saccharomyces cerevisiae.

[0041] The lactic acid bacteria described in step B is Lactobacillus plantarum

[0042] The acetic acid bacteria described in step B is Shanghai Niang 1.01 acetic acid bacteria.

[0043] Example 3

[0044] A fermented liquid of preserved roxburghii and gastrodia elata fruits is prepared by the following steps:

[0045] A. Wash and dice fresh Gastrodia elata, slice and deseed Rosa roxburghii, mix 1 kg of Rosa roxburghii and 15 kg of Gastrodia elata, steam and ripen at 70°C, add 16 kg of sucrose, and candied at 10°C for 10 hours to prepare Rosa roxburghii Gastrodia elata preserved fruit;

[0046] B. The Rosa roxburghii Gastrodia elata preserved fruit obtained in step A was transferred to a fermentation tank, and mixed strains were added to the fermentation container respectively, and the fermentation temperature was controlled to be 30° C. and aerobically fermented for 20 days, wherein the mixed strains consisted of yeast, lactic acid bacteria and acetic acid bacteria, and the ratio thereof was 1:1:1 by mass;

[0047] C. The fermented Rosa roxburghii and Gastrodia elata preserved fruits obtained in step B are crushed, pressed and filtered to obtain Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid, and then subjected to ultra-high temperature instantaneous sterilization at 120° C. to obtain a finished product.

[0048] The yeast described in step B is a flavoring yeast.

[0049] The lactic acid bacteria described in step B is Lactobacillus acidophilus.

[0050] The acetic acid bacteria described in step B is AS1.41 acetic acid bacteria.

[0051] Example 4

[0052] A roxburgh and gastrodia elata composite fermented oral liquid comprises: 6 kg of roxburgh and gastrodia elata preserved fruit fermentation liquid, 3 kg of purified water, 0.05 kg of edible essence, 0.5 kg of xylitol, and 0.08 kg of sodium carboxymethyl cellulose.

[0053] The roxburgh pineapple and gastrodia elata preserved fruit fermentation liquid is the roxburgh pineapple and gastrodia elata preserved fruit fermentation liquid obtained in Example 1.

[0054] Example 5

[0055] A roxburgh and gastrodia elata composite fermented oral liquid comprises, by weight, 5 kg of roxburgh and gastrodia elata preserved fruit fermentation liquid, 4 kg of purified water, 0.03 kg of edible essence, 0.8 kg of xylitol, and 0.05 kg of sodium carboxymethyl cellulose.

[0056] The fermentation liquid of roxburgh roxburgh and gastrodia elata preserved fruit is the fermentation liquid of roxburgh roxburgh and gastrodia elata preserved fruit obtained in Example 2.

[0057] Example 6

[0058] A roxburgh and gastrodia elata composite fermented oral liquid comprises, by weight, 4 kg of roxburgh and gastrodia elata preserved fruit fermentation liquid, 5 kg of purified water, 0.01 kg of edible essence, 1 kg of xylitol, and 0.03 kg of sodium carboxymethyl cellulose.

[0059] The roxburgh pineapple and gastrodia elata preserved fruit fermentation liquid is the roxburgh pineapple and gastrodia elata preserved fruit fermentation liquid obtained in Example 3.

[0060] Study on the improving effect of fermented liquid of roxburghii and Gastrodia elata preserved fruit on insomnia mice

[0061] 1. Experimental Materials and Methods

[0062] 1.1 Materials and Instruments

[0063] Rosa roxburghii and Gastrodia elata preserved fruit fermentation broth (prepared by the method of specific embodiment 2 of the present invention); Zao Ren Anshen Liquid (National Medicine Standard No. Z20064008), Jiangsu Jurong Pharmaceutical Group Co., Ltd.; Rosa roxburghii fresh fruit, China National Pharmaceutical Group Guizhou Health Industry Development Co., Ltd.; Gastrodia elata Dejiang Lutong Gastrodia elata Development Co., Ltd.; DL-4-chlorophenylalanine (PCPA), Shanghai Yien Chemical Technology Co., Ltd.; Pentobarbital sodium, China National Pharmaceutical Group Chemical Reagent Co., Ltd.; Superoxide dismutase (SOD), glutamic acid (Glu), malondialdehyde (MDA) biochemical kit, Quanzhou Ruixin Biotechnology Co., Ltd.; Interleukin-1 (IL-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), gamma-aminobutyric acid (GABA) acid, GABA), 5-hydroxytryptamine (5-HT), and dopaminergic (DA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Quanzhou Ruixin Biotechnology Co., Ltd.; yeast Angel Yeast Co., Ltd.; Lactobacillus plantarum (deposit number: CGMCC No.18205) was purchased from Shanenkang Biotechnology Co., Ltd.; Acetobacter (deposit number: BCRC No.10588) was purchased from Tianjin Xiaowei Biotechnology Co., Ltd.; and physiological saline was purchased from Guizhou Kelun Pharmaceutical Co., Ltd. All reagents used in other experiments were of analytical grade, and deionized water was used.

[0064] One hundred and twenty SPF male ICR mice, weighing 20 ± 2 g, were provided by Spefoc Biotechnology (Beijing) Co., Ltd., license number: SCXK(Beijing)2024-0001, quality certificate number: 110324241105756445, animal ethics approval number: 2303331, and animal experimental unit use permit number: SYXK(Guizhou)2023-0002. Animals were housed under the following conditions: temperature: 22 ± 2°C; relative humidity: 55 ± 5%; light on for 12 h per day; and free access to food and water.

[0065] SM-200 magnetic stirrer, Wuhan Seville Biotechnology Co., Ltd.; ZH-OFT mine experiment reaction chamber, Anhui Zhenghua Biological Instrument Equipment Co., Ltd.; Chemray 800 fully automatic biochemical analyzer, Shenzhen Raydu Life Science; SWE-FP low-temperature freeze grinder, Wuhan Seville Biotechnology Co., Ltd.; 5427R high-speed refrigerated centrifuge, Germany's Eppendor; SCI-VS adjustable mixer, Jinan Bohang Biotechnology Co., Ltd.; FA2004N electronic balance, Shanghai Jinghai Instrument Co., Ltd.; VICTORNivo microplate reader, PerkinElmer Enterprise Management Co., Ltd.; UV-1800 UV-visible spectrophotometer, Shimadzu Instrument (Suzhou) Co., Ltd.; GZX-9420MBE electric constant temperature blast drying oven, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; RM2016 pathology slicer, Shanghai Leica Instrument Co., Ltd.

[0066] 1.2 Experimental methods

[0067] 1.2.1 Roseroxburghii fermentation liquid and Gastrodia elata fermentation liquid were prepared with reference to the Roseroxburghii and Gastrodia elata preserved fruit fermentation liquid of the present invention, that is, the Roseroxburghii fresh fruit and Gastrodia elata were washed and dried, pulped, ripened, fermented, filtered and sterilized to obtain Roseroxburghii fermentation liquid (RFL) and Gastrodia elata fermentation liquid (GFL), respectively.

[0068] 1.2.2 Experimental Animal Grouping

[0069] ICR male mice were reared and fed for 7 days before randomization into two experimental groups, A and B. Experiment A determined the suprathreshold dose of sodium pentobarbital to induce sleep in mice; experiment B investigated sleep improvement in mice. The mice were divided into eight groups: control, model, positive, roxburghii fermented liquid (RFL), Gastrodia elata fermented liquid (GFL), and low-, medium-, and high-dose roxburghii and Gastrodia elata preserved fruit fermented liquid groups (RGFL-L, RGFL-M, and RGFL-H), with 10 mice in each group.

[0070] 1.2.3 Determination of suprathreshold dose of pentobarbital sodium

[0071] ICR mice were divided into five groups with 10 mice in each group at doses of 30, 35, 40, 45, and 50 mg / kg, respectively. Sodium pentobarbital was injected intraperitoneally to determine the suprathreshold dose of sodium pentobarbital to induce sleep in mice. After the intraperitoneal injection, the movement state of the mice was observed. When the mice became quiet, they were placed on a soft and flat surface with their abdomen facing upwards. If the mice remained with their abdomen facing upwards within 60 seconds, it was considered that the righting reflex of the mice disappeared. When the mice turned over three times within 30 seconds, it was considered that the righting reflex of the mice was restored

[21] . When the time from the disappearance of the righting reflex to the restoration of the righting reflex of the mice exceeded 10 minutes, it was considered that the mice had successfully fallen asleep. The number of mice that successfully fell asleep in each group was then counted. When all mice in the group fell asleep and the sleep rate reached 100%, the sodium pentobarbital injection dose of the group of mice was the suprathreshold dose of sodium pentobarbital to induce sleep in mice.

[0072] 1.2.4 Establishment of insomnia mouse model and drug administration

[0073] Intraperitoneal injection of PCPA is a common method for establishing insomnia models in mice. After adaptive feeding, mice were treated with PCPA. Except for the control group, which received normal saline, all other groups received intraperitoneal injections of 450 mg / kg of PCPA suspension for three consecutive days to establish the insomnia model. After model establishment, mice were administered the corresponding test substance. The control and model groups received distilled water at a dose of 10 mL / kg. The positive group received Zaoren Anshen solution at a dose of 3 mL / kg, serving as a positive control to compare the efficacy of Rosa Roxburghii and Gastrodia elata fermented liquid. The RFL and GFL groups received 10 mL / kg of Rosa Roxburghii and Gastrodia elata fermented liquid. The RGFL-L group received a 4-fold dilution of Rosa Roxburghii and Gastrodia elata fermented liquid, the RGFL-M group received a 2-fold dilution of Rosa Roxburghii and Gastrodia elata fermented liquid, and the RGFL-H group received the original Rosa Roxburghii and Gastrodia elata fermented liquid. The gavage volume for all groups was 10 mL / kg. The above experimental groups were gavaged with the corresponding test substances once a day for 7 consecutive days, and then the mice in each group were subjected to sodium pentobarbital sleep induction test, behavioral test and sample collection.

[0074] 1.2.5 Pentobarbital sodium-induced sleep experiment in mice

[0075] One hour after the last oral gavage, each mouse was injected with the suprathreshold dose of sodium pentobarbital determined in 1.2.3 for a sodium pentobarbital-induced sleep experiment. The mice in each group were observed, and the time it took for the righting reflex to disappear and recover was recorded. The period from the completion of the intraperitoneal injection to the disappearance of the righting reflex was designated as the sleep latency; the time from the disappearance of the righting reflex to the recovery of the righting reflex was designated as the sleep duration. Mice with a sleep duration greater than 10 minutes were considered to have successfully fallen asleep. The sleep onset rate, sleep latency, and sleep duration of each group were calculated and analyzed.

[0076] 1.2.5 Open field test

[0077] Eight hours after the pentobarbital sodium-induced sleep test, the open field test was performed. Mice were placed in the same position in each of the four compartments of the mine reaction box. The device's camera recorded the movement trajectory of each mouse after entering the open field for 4 minutes. The ZH-CSC shuttle video acoustic-photoelectric stimulation analysis system was used to analyze the mouse's movement trajectory and plot the mouse's movement trajectory in the open field. The distance moved and the average movement speed of the mice in the open field were counted and analyzed.

[0078] 1.2.6 Determination of serum SOD, MDA, IL-1, IL-1β, and TNF-α levels

[0079] After the mine experiment, the mice's eyeballs were removed and blood was collected. The blood was placed at room temperature for 2 hours and centrifuged at 3500 r / min for 10 minutes. The upper serum was collected and divided into 50 μL tubes and stored in a -80°C refrigerator for later use. The levels of SOD, MDA, IL-1, IL-1β, and TNF-α were measured according to the requirements of the kit.

[0080] 1.2.7 Detection of Glu, GABA, 5-HT, and DA Contents in Brain Tissue

[0081] After blood collection, the mice were killed, the brain tissue was removed, the excess blood on the surface was wiped dry, and the contents of Glu, GABA, 5-HT, and DA in the brain tissue were determined according to the instructions of the ELISA kit.

[0082] 1.2.8 Pathological observation of whole-brain tissues of mice

[0083] After the mice were killed, the whole brain was removed and fixed in 4% paraformaldehyde tissue fixative for more than 24 hours. The entire hippocampal brain tissue was embedded in paraffin and then cut into 4-5 μm sections, dewaxed and dehydrated, and finally stained with hematoxylin-eosin (HE). The morphological changes of hippocampal neurons in the hippocampus were observed using an optical microscope.

[0084] 2. Test results and analysis

[0085] 2.1 Determination of suprathreshold dose of pentobarbital sodium

[0086] Sodium pentobarbital belongs to the barbiturate class of drugs, which selectively depress the central nervous system and produce potent sedative and hypnotic effects. The threshold dose of pentobarbital for sedation and hypnosis varies across species. This study used ICR mice as experimental subjects, setting up five concentration gradients of pentobarbital to determine the suprathreshold dose. Ten mice were injected intraperitoneally with varying doses of pentobarbital, and the sleep-inducing effects of each group were observed. The results are shown in Table 1. When 40 mg / kg of pentobarbital was injected intraperitoneally, 8 out of 10 mice successfully fell asleep, achieving a sleep-onset rate of 80%. When the injection dose was increased to 45 mg / kg, all 10 mice successfully fell asleep, achieving a sleep-onset rate of 100%. Based on the 100% sleep-onset rate, the suprathreshold dose for sleep-inducing mice with intraperitoneal injection of pentobarbital was determined to be 45 mg / kg.

[0087] Table 1 Determination of the suprathreshold dose of sodium pentobarbital to induce sleep in mice (n=10)

[0088]

[0089] Effects of RGFL on sleep in insomnia mice

[0090] Compared with the expensive EEG detection technology, the sodium pentobarbital sleep induction test is a common method for preliminary screening of sleep effects. Sodium pentobarbital is used together with RGFL to comprehensively judge the effect of RGFL on mouse sleep from aspects such as the number of mice falling asleep, sleep duration, and sleep latency. Through the above test, it was determined that the upper threshold dose of sodium pentobarbital to induce sleep in mice is 45 mg / kg. Therefore, 45 mg / kg of sodium pentobarbital was injected intraperitoneally into each group of mice. The results are as follows: Figure 1As shown, all mice in the Control group, Positive, RFL, GFL, RGFL-L, RGFL-M, and RGFL-H fell asleep, with a sleep rate of 100%, while only 7 mice in the Model group successfully fell asleep, with a sleep rate of 70%, indicating that the injection of PCPA affected the central nervous system of the mice and made the mice manic; at the same time, compared with the Control group (3.0±0.82min), the sleep latency of the mice in the Model group (6.33.0±1.0min) was significantly increased (P<0.01), and the sleep duration of the mice in the Model group (23.20±17.13min) was significantly decreased compared with the sleep duration of the mice in the Control group (63.44±24.51min) (P<0.01). After intraperitoneal injection of PCPA suspension, the sleep rate of mice was reduced, the sleep duration was shortened, and the sleep latency was prolonged, indicating that the insomnia mouse model was successfully established. After continuous gavage of the corresponding test substances for 7 days, the sleep latency of mice in the Positive group (4.10±0.32min), RGFL-L (3.82±2.23min), RGFL-M (2.25±0.46min), and RGFL-H (3.29±1.98min) groups was significantly reduced (P<0.01), while the sleep duration of mice in the Positive group (88.9±12.29min), RGFL-L (43.89±10.98min), RGFL-M (74.0±14.45min), and RGFL-H (82.0±19.64min) was significantly increased (P<0.05). It is worth noting that under the synergistic effect of sodium pentobarbital, the sleep time of mice in the RGFL-H group and the Positive group was even significantly higher than that of mice in the normal control group (P<0.01). It can be seen that RFL and GFL intervention can effectively improve the sleep condition of PCPA insomnia mice and RGFL can synergistically enhance the improvement effect on insomnia mice. This is because sodium pentobarbital is injected into the mice in advance, which plays the first role of sedation and hypnosis. Secondly, sea buckthorn and gastrodia contain functional ingredients that can calm and hypnotize and reduce central nervous system excitability. When the effect of sodium pentobarbital is about to expire, sea buckthorn and gastrodia play a second role, prolonging the sedation and hypnosis time of mice and jointly improving the insomnia of mice. Note Figure 1 In Chinese: ##: indicates extremely significant difference compared with the Control group (P<0.01), *: indicates significant difference compared with the Model group (P<0.05), **: indicates extremely significant difference compared with the Model group (P<0.01), the same below.

[0091] 2.3 Effects of RGFL on the behavior of insomnia mice

[0092] The open field test is to evaluate the effect of drugs on the activities of mice by placing them in a mine box and using a camera to track their movement distance, movement speed, number of activities, etc. in the open field within a specified time. Figure 2 The total distance traveled by mice in the Model group (2664.08±243.17 mm) was significantly increased compared with the Control group (2126.73±51.31 mm) (P<0.01). The average movement speed of mice in the Model group (11.02±1.10 mm / s) was significantly faster than that in the Control group (8.35±0.82 mm / s) (P<0.01). Furthermore, the number of rotations in the open field in the Model group (64.5±20.20 times) was significantly increased compared with that in the Control group (46.5±3.83 times) (P<0.01). After 7 consecutive days of intervention with the corresponding test substances, each group was able to significantly reduce the movement distance, average speed and number of activities of mice. This may be because Positive, RFL, GFL, and RGFL all contain ingredients that inhibit the excitatory nerve center and can have a sedative and hypnotic effect, but the RGFL group showed a better effect, and the movement distance (1262.64±328.14mm), average speed (4.79±1.31mm / s) and number of activities (20.67±2.42 times) of the RGFL-H group were the lowest among all groups. Behavioral results showed that the combined oral administration of RFL and GFL showed a better sedative effect than oral administration of either alone. Note Figure 2 Middle: (A) Trajectory diagram of mice in each group; (B) Behavioral indicators of mice in each group.

[0093] Effects of RGFL on the levels of SOD, MDA, IL-6, IL-1β, and TNF-α in mouse serum

[0094] Sleep is closely related to inflammatory factors and antioxidant damage. Studies have found that during sleep, the level of inflammatory factors in the body decreases. However, on the contrary, when animals are subjected to insomnia models and sleep deprivation, the level of inflammatory factors in their blood increases. It can be seen that sleep deprivation is an important factor leading to a decline in the body's immunity. Figure 3As shown in the results, compared with the Control group, the SOD level in the Model group was significantly (P<0.01) decreased by 53.87%, and the MDA, IL-6, IL-1β, and TNF-α levels were significantly (P<0.01) increased by 140.50%, 74.10%, 52.95%, and 56.93%, respectively. This indicates that after PCPA modeling, the sleep of mice was disturbed, the antioxidant enzymes in serum decreased, and the oxidative damage products and inflammatory factors were activated. However, after oral administration of the corresponding test substances, the inflammatory factors and antioxidant enzymes in the serum of each group were significantly increased. The levels of SOD, MDA, IL-6, IL-1β and TNF-α in serum were all improved, and the effect of RGFL-H was more significant, which significantly (P<0.01) increased SOD by 50.18%, and significantly (P<0.01) decreased MDA, IL-6, IL-1β and TNF-α by 45.74%, 34.87%, 24.13% and 25.72% respectively. Moreover, the regulatory effect of RGFL group on SOD, MDA, IL-6, IL-1β and TNF-α in serum was significantly better than that of RFL and GFL groups (P<0.05), showing better improvement effect.

[0095] Effects of 2.5RGFL on the Contents of Glu, GABA, 5-HT and DA in Mouse Brain Tissue

[0096] The neurotransmitters involved in the regulation of sleep and wakefulness are mainly 5-HT, DA, GABA, NE and ACh, so the measurement of neurotransmitters involved in the regulation of sleep in brain tissue can assess the sleep status of the body. Figure 4 As shown in the data, compared with the Control group, the Glu content in the Model group was significantly increased (P<0.01), while the GABA, 5-HT and DA contents were significantly decreased (P<0.01). The Glu content increased by 113.29%, and the GABA, 5-HT and DA contents decreased by 58.50%, 55.26% and 60.94%, respectively, indicating that insomnia leads to disorders in the neurotransmitters regulating sleep-wakefulness. After administration, the Glu in each group was downregulated, and the GABA, 5-HT and DA contents began to increase. The effect of the RGFL group was better than that of RFL and GFL alone. The RGFL groups showed a certain dose-effect relationship with the effect of improving sleep. The RGFL-H group had the best effect, downregulating Glu by 39.62% and increasing the GABA, 5-HT and DA contents by 66.84%, 63.37% and 88.06%, respectively. Therefore, the combined application of RGFL can better regulate the content of sleep-related neurotransmitters in the body.

[0097] Effects of 2.6RGFL on pathological damage of whole brain tissue in mice

[0098] Insomnia is related to damage to neurons in the brain. When hippocampal neurons are damaged and atrophied, rapid eye movement (REM) sleep is affected. In addition, extensive damage to the hypothalamus may also lead to complete insomnia. Figure 5 It can be seen that the neuronal cells in the hippocampus of the Control group are arranged regularly and orderly, the cells are arranged tightly, and the cell nuclei are not coagulated; compared with the Control group, it is found that the neuronal cells in the hippocampus of the Model group are arranged irregularly, the cells are loosely arranged, edema occurs, the cell nuclei are coagulated and vacuolated, indicating that intraperitoneal injection of PCPA can cause sleep disorders in mice, destroy neuronal cells in the mouse brain tissue, and damage hippocampal neurons; after the corresponding drug intervention, the loose arrangement of neuronal cells in the hippocampus, edema, cell nuclei coagulation and vacuolation in each group were alleviated and improved to varying degrees. In the groups where RFL and GFL were administered alone and RGFL was combined with oral gavage, the improvement effect of RGFL was better, showing a synergistic effect. These results indicate that the combined use of RGFL can reverse brain damage to a certain extent.

[0099] 3 Conclusion

[0100] In this study, a fermented liquid of Rosa roxburghii and Gastrodia elata fruit was prepared using microbial fermentation technology. A sleep-deprived mouse model was established by intraperitoneal injection of PCPA. The effects of the fermented liquid on insomnia in mice were comprehensively evaluated by performing a pentobarbital-induced sleep experiment and an open-field test, measuring changes in serum and brain tissue parameters, and examining pathological changes. The pentobarbital-induced sleep experiment showed that the fermented liquid significantly (P<0.01) shortened the latency period (3.29±1.98 min), prolonged sleep duration (82.0±19.64 min), and reduced the number of activity times (20.67±2.42) and movement status (distance traveled: 1262.64±328.14 mm; average speed: 4.79±1.31 mm / s), demonstrating a sedative and hypnotic effect. In addition, the fermented liquid of sea buckthorn and Gastrodia elata preserved fruit can improve the levels of inflammatory factors and antioxidants in serum, significantly (P<0.01) increasing SOD by 50.18%, and significantly (P<0.01) decreasing MDA, IL-6, IL-1β, and TNF-α levels by 45.74%, 34.87%, 24.13%, and 25.72% respectively; it can regulate the content of sleep-related neurotransmitters in the body, decreasing Glu by 39.62%, and increasing GABA, 5-HT, and DA levels by 66.84%, 63.37%, and 88.06%, respectively, which can reverse brain damage and improve sleep to a certain extent.

[0101] This study found that combined treatment with RGFL significantly reduced the distance, speed, and number of activities in an open field in insomniac mice. The levels of related neurotransmitters were significantly increased, and inflammatory factors were significantly reduced, indicating that RGFL can improve insomnia in mice. Traditional Chinese medicine theory emphasizes "mutual synergy," meaning that the combined efficacy of two drugs is superior to that of either drug alone. Therefore, this study used a PCPA-induced insomnia model in mice, with RFL and GFL alone in the control group, and a combined RGFL group. The aim was to investigate the sleep-improving effects of RFL and GFL, and whether their combination could exert a synergistic effect. The results showed that the combined use of Rosa Roxburghii and Gastrodia elata improved insomnia in PCPA-induced mice by enhancing their sedative, hypnotic, and antioxidant capacities, reducing inflammatory factors, and ameliorating brain damage. This is primarily due to the fact that both Rosa Roxburghii and Gastrodia elata contain functional components that are sedative, hypnotic, reduce central nervous system excitability, and improve sleep. When used together, these functional components can exert an additive and synergistic effect on the same symptoms and underlying causes, resulting in a synergistic effect. The combined use of the two can achieve the effect of "1+1>2", which has obvious advantages compared to artificially synthesized chemical drugs. It will have broad application value in the field of big health and health care products in the future, and also provide a certain experimental basis for the development of sea buckthorn and Gastrodia functional sleep aid products.

[0102] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a fermentation liquid of roxburghii and gastrodia elata preserved fruit, characterized in that: The steps include: A. Wash and dice fresh Gastrodia elata, slice and deseed Rosa roxburghii, mix 10 parts of Rosa roxburghii with 50-200 parts of Gastrodia elata, steam and ripen at 60-90° C., add 60-210 parts of sucrose, and candy at 10-20° C. for 8-10 hours to prepare Rosa roxburghii Gastrodia elata preserved fruit; B. transferring the preserved roxburghii and Gastrodia elata fruits obtained in step A into a fermentation tank, adding mixed strains into the fermentation containers, controlling the fermentation temperature to 20-30° C. and aerobically fermenting for 20-50 days, wherein the mixed strains are composed of yeast, lactic acid bacteria, and acetic acid bacteria; C. crushing, pressing and filtering the fermented Rosa roxburghii and Gastrodia elata preserved fruits obtained in step B to obtain Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid.

2. The method for preparing the Rosa roxburghii and Gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein The ratios of the raw materials in step A are all by weight.

3. The method for preparing the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein In the step B, the ratio of yeast, lactic acid bacteria and acetic acid bacteria is 1:1:1 by mass.

4. The method for preparing the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein The yeast in step B is one or more of Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces odorifera, Saccharomyces weigensis, Hansenula anomala, Schizosaccharomyces pombe, Rhodotorula glutinosus, Pichia pastoris, Candida glycerogenes and Candida utilis, mixed in any proportion.

5. The method for preparing the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein The lactic acid bacteria in step B are one or more of Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, Lactobacillus gasseri, Bifidobacterium and Lactobacillus reuteri, mixed in any proportion.

6. The method for preparing the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein The acetic acid bacteria in step B is one or more of Acetobacter orlanii, Acetobacter climbing, Acetobacter film, Acetobacter AS1.41, and Acetobacter Shanghai 1.01, mixed in any proportion.

7. The method for preparing the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 1, wherein The fermented liquid of the preserved roxburghii and gastrodia elata fruits in step C is also sterilized at an ultra-high temperature of 120° C.

8. A fermentation liquid of preserved roxburghii and gastrodia elata, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the roxburghii and gastrodia elata preserved fruit fermentation liquid according to claim 8 in the preparation of foods, health products and medicines.

10. A Rosa Roxburghii and Gastrodia elata composite fermented oral liquid, characterized in that: The roxburgh and gastrodia elata composite fermented oral liquid comprises, by weight: 40-60 parts of the roxburgh and gastrodia elata preserved fruit fermentation liquid according to claim 8, 30-50 parts of purified water, 0.1-0.5 parts of edible flavoring, 5-10 parts of xylitol, and 0.3-0.8 parts of sodium carboxymethyl cellulose.

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