Folium cortex eucommiae pharmaceutical composition for improving depression as well as preparation method and application of folium cortex eucommiae pharmaceutical composition

Abstract

The invention discloses a folium cortex eucommiae pharmaceutical composition for improving depression as well as a preparation method and application thereof, and relates to the technical field of medicines. The preparation method comprises the following steps: performing liquid nitrogen low-temperature crushing on eucommia ulmoides leaves, performing enzymolysis fermentation on cellulase and pectinase, performing secondary enzymolysis on beta-glucosidase, performing ultrasonic ethanol extraction on filter residues obtained by enzymolysis, performing acetic fermentation, combining secondary enzymolysis liquid and acetic fermentation liquid, and concentrating to obtain eucommia ulmoides leaf extracting liquid; the extracting solution has the effects of improving depression, resisting inflammation and inhibiting HPA axis excitation, can be used as a pharmaceutical composition or a conventional additive for preparing medicines for preparing the medicines for improving depression, resisting inflammation and inhibiting HPA axis excitation, can be prepared into various dosage forms, including but not limited to tablets, capsules, pills, oral solutions, injections and the like, and can be used for preparing medicines for improving depression, resisting inflammation and inhibiting HPA axis excitation. The application value is realized in efficient utilization of the folium cortex eucommiae and depression improvement treatment.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a eucommia leaf medicinal composition for improving depression, and a preparation method and application thereof. Background Art

[0002] Depression is a global mental health problem, and its incidence has increased significantly in recent years. Finding safe and effective antidepressant drugs or food supplements has always been a hot topic of research. As a traditional Chinese medicinal material, Eucommia ulmoides is rich in a variety of compounds with antidepressant activity, such as genipin, chlorogenic acid, flavonoids and polysaccharides. Chinese patent CN201410692645.6 discloses an antidepressant preparation and its preparation method, which discloses a Chinese medicine composition (including: Eucommia ulmoides (salt fried), Psoralea corylifolia (salt fried), walnut kernel (fried), garlic) on the one hand, by affecting the metabolism of glutamate (Glu) and γ-aminobutyric acid (γ-GABA) in the brain, that is, mainly through the Glu / GABA-Glu metabolic loop circulation to exert antidepressant effects; on the other hand, it increases the content of 5-hydroxytryptamine (5-HT), norepinephrine (NE) and GABA in the hippocampus, and reduces the content of Glu to fight depression. However, in this study, there are many ingredients with antidepressant effects, and there is no research on the effect of the active ingredients in Eucommia ulmoides on depression; and this study uses Eucommia ulmoides bark, not Eucommia ulmoides leaves.

[0003] Eucommia leaves are rich in chlorogenic acid, polyphenols, flavonoids, polysaccharides, and essential amino acids such as histidine. The chlorogenic acid content is 5 times higher than that of Eucommia bark, and the content of inorganic elements is also higher than that of Eucommia bark. Compared with bark, flowers and fruits, Eucommia leaves have better antioxidant activity. However, at present, research on the antidepressant effect of Eucommia leaves is still relatively scarce, which leaves a broad space for further scientific exploration. With Eucommia leaves being included in the catalog of medicinal and edible homology, the preparation of substitute tea using Eucommia leaves as raw materials and the development of drugs have become new research hotspots. However, the extraction and utilization of antidepressant active ingredients in Eucommia leaf tea are limited by the cell wall barrier, and the extraction process is prone to lead to the loss of functional ingredients.

[0004] In traditional extraction processes, methods such as hot water extraction, solvent reflux and filter press generally face limitations such as long extraction cycle, high energy consumption and easy damage to heat-sensitive components. In view of these shortcomings, in order to optimize the extraction effect of tea, the industry has increasingly paid attention to exploring and adopting new extraction technologies. In recent years, ultrasonic extraction technology has attracted particular attention in the field of tea processing. With the high-speed, strong cavitation effect and stirring function induced by ultrasound, this technology can effectively destroy the plant cell wall structure, promote the solvent to penetrate into the cell, significantly shorten the extraction cycle and improve the extraction efficiency. Ultrasonic extraction has shown wide application potential in many fields such as biological and plant cell disruption, effective ingredient extraction and low-temperature extraction of Chinese herbal medicine. Its advantages are high efficiency, fast speed, low-temperature operation and wide applicability. However, most of the current research on ultrasonic extraction of Eucommia ulmoides focuses on the optimization of parameters such as temperature, time and solid-liquid ratio, while the influence of two key factors, crushing degree and crushing temperature, on the extraction process of effective ingredients from Eucommia ulmoides leaves has not been given enough attention.

[0005] The liquid nitrogen freezing and pulverizing technology not only improves the cell wall breakage rate to increase the extraction rate of antidepressant ingredients, but also provides a low-temperature extraction environment to reduce the loss rate of functional ingredients during the pulverization process. Liquid nitrogen freezing and pulverizing technology freezes the water in the cells of Eucommia ulmoides leaves by rapidly cooling them, thereby destroying the cell wall structure and increasing the release rate of antidepressant ingredients. The low-temperature environment can also reduce the oxidation and degradation of functional ingredients during the pulverization process, maintain their biological activity, and effectively reduce the loss of heat-sensitive active ingredients, thereby increasing the antidepressant active ingredients in the finished drug.

[0006] In addition, in the process of optimizing the extraction of antidepressant active ingredients from Eucommia leaf tea, the enzymatic treatment technology was used to treat Eucommia leaf tea powder with enzyme preparations, which showed significant results. Among them, β-glucosidase is a commonly used enzyme to improve extraction efficiency and flavor. Honey is one of the important natural sources of this enzyme. Using honey rich in high β-glucosidase activity to replace a single β-glucosidase preparation not only effectively breaks the plant cell wall through its enzymatic action, promotes the efficient release of antidepressant ingredients such as genipin (generated by hydrolysis of genipin glycoside), but also gives full play to the antioxidant properties of honey to prevent the oxidative degradation of antidepressant substances. At the same time, the regulatory effect of the polysaccharide components in honey on intestinal microorganisms forms a synergistic effect with the active ingredients in Eucommia leaf tea, further enhancing the antidepressant effect. In addition, as a natural sweetener and prebiotic enhancer, honey not only improves the taste of the drug, but also increases consumer acceptance, achieving dual optimization of functionality and taste.

[0007] Therefore, the research goal of this patent is to provide a preparation process for a Eucommia leaf medicinal composition for improving depression. Liquid nitrogen low-temperature crushing technology is used to take into account both the improvement of cell wall fragmentation rate and the improvement of the retention rate of heat-sensitive functional components. High enzyme activity honey is used in conjunction with complex cellulase to achieve the four-in-one effect of enzyme preparation, antioxidant, sweetener, and prebiotic enhancer. Summary of the invention

[0008] The invention aims to provide a eucommia leaf medicinal composition for improving depression, a preparation method and application thereof, thereby improving the utilization rate of the eucommia leaf and the improving therapeutic effect on depression, and having significant practical value.

[0009] In order to achieve the above object, the present invention provides a method for preparing a Eucommia leaf medicinal composition for improving depression, comprising the following steps:

[0010] (1) The immobilized complex enzyme is prepared by a sodium alginate immobilization method, wherein the enzymes include cellulase and pectinase;

[0011] (2) preparing acetic acid bacteria seed solution;

[0012] (3) freezing the Eucommia leaf tea with liquid nitrogen and then low-temperature crushing it, sieving it to obtain Eucommia leaf tea powder, and enzymolyzing the Eucommia leaf tea powder with an immobilized complex enzyme to obtain an Eucommia leaf enzymolysis solution;

[0013] (4) adding honey with β-glucosidase activity to the Eucommia ulmoides leaf hydrolyzate for secondary enzymolysis, adding 60° C. water after the enzymolysis, and filtering to obtain a secondary enzymolysis solution and a filter residue;

[0014] (5) subjecting the filter residue to secondary ultrasonic ethanol extraction, and filtering to obtain a secondary extract;

[0015] (6) diluting the secondary extract with water to an ethanol concentration of 5%, inoculating acetic acid bacteria seed solution with a volume ratio of 2% to 4% to perform acetic acid fermentation to obtain acetic acid fermentation solution;

[0016] (7) The secondary enzymatic hydrolyzate and the acetic acid fermentation broth are combined and concentrated to obtain an extract which is the Eucommia ulmoides leaf medicinal composition.

[0017] Preferably, the preparation method of the above-mentioned immobilized complex enzyme is: take a sodium alginate solution with a mass fraction of 4%, a complex enzyme solution with a volume fraction of 2:1:1, and mix them evenly, and drip them drop by drop into 1% CaCl2, replace them with 1% CaCl2 after forming small balls, and store them in a refrigerator at 4°C for 2h. After filtering, soak the filter residue with glutaraldehyde with a volume fraction of 0.5% for 4h, and then wash the filter residue with distilled water several times, and then dry it to obtain the immobilized complex enzyme; wherein the enzyme activity ratio of cellulase and pectinase in the complex enzyme solution is 1:1.

[0018] Preferably, the concentration of the acetic acid bacteria seed solution is OD600=0.4-0.6.

[0019] Preferably, the above-mentioned screening is through an 80-mesh sieve.

[0020] Preferably, the enzymatic hydrolysis treatment in step (3) above is specifically to take eucommia leaf tea powder and immobilized complex enzyme at a mass ratio of 1:0.0345, add disodium hydrogen phosphate-citric acid buffer at a material-liquid ratio of 1:20, adjust the pH to 4.5, and perform enzymatic hydrolysis at 55°C for 4.5 hours.

[0021] Preferably, in the above step (4), the amount of honey added is 1 / 10 of the weight of the Eucommia leaf tea powder, the β-glucosidase activity in the honey is 40.9 mU / g; the pH of the secondary enzymatic hydrolysis is 4.2, the enzymatic hydrolysis temperature is 60°C, and the enzymatic hydrolysis time is 2.5 h; the ratio of added water to Eucommia leaf tea powder is 40:1 mL / g.

[0022] Preferably, the ultrasonic ethanol secondary extraction has an ultrasonic time of 50 min, an ultrasonic temperature of 60° C., an ethanol concentration of 65%, and a solid-liquid ratio of 1:35 g / mL.

[0023] Preferably, the acetic acid fermentation is carried out at a fermentation temperature of 30° C., a stirring speed of 400 r / min, and a fermentation time of 72 h.

[0024] The present invention also provides a Eucommia leaf pharmaceutical composition prepared by any of the above-mentioned preparation methods, which can be prepared into a variety of dosage forms, including but not limited to tablets, capsules, pills, oral preparations, injections, etc. The Eucommia leaf pharmaceutical composition can be used in any of the following, including efficient utilization of Eucommia leaves and improved treatment of depression.

[0025] The present invention has the following advantages:

[0026] The present invention discloses for the first time the use of Eucommia ulmoides leaves in improving depression, and at the same time proposes a method for preparing an Eucommia ulmoides leaves pharmaceutical composition for improving depression with high extraction efficiency, synergistic enhancement, rich nutrition and improved taste, and finally obtains an Eucommia ulmoides leaves pharmaceutical composition with a higher depression improvement effect than an Eucommia ulmoides leaves water-extracted beverage. The extraction rate of antidepressant components in Eucommia ulmoides leaves is significantly improved by combining β-glucosidase in honey and liquid nitrogen freezing and crushing technology. In the technical solution provided by the present invention, the polysaccharides and antioxidants in honey synergize with the active ingredients in Eucommia ulmoides leaves to enhance the antidepressant effect, and the practical value is significant. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0029] Example 1

[0030] This embodiment 1 provides a method for preparing a Eucommia leaf medicinal composition for improving depression, comprising the following steps:

[0031] Step (1): prepare immobilized complex enzyme. Adopt sodium alginate fixation method, take 20mL of sodium alginate solution with a mass fraction of 4%, add 10mL of 25mg / mL complex enzyme solution, stir, add 10mL of glutaraldehyde with a volume fraction of 5%, stir well, use a syringe to drip into 1% CaCl2 drop by drop to form a small ball, replace 1% CaCl2 and soak, store in a refrigerator at 4℃ for 2h, filter and soak in glutaraldehyde with a volume fraction of 0.5% for 4h, take out and wash with distilled water several times, dry and store in a refrigerator at 4℃ for standby use. The complex enzyme solution is a mixture of cellulase and pectinase, and the enzyme activity ratio is 1:1.

[0032] Step (2): Activation of acetic acid bacteria. Use an inoculation loop to pick a few acetic acid bacteria (CICC 20441) colonies from the original bacterial species slant (or safekeeping tube) under sterile conditions, streak them on a plate culture medium, and then place them at 30°C for 48 hours. Let them stand for culture, and check by smear and staining that there are no foreign bacteria, the bacterial bodies grow neatly, and the colonies are plump when observed with the naked eye. Pick a single colony and place it in a liquid culture medium, control the liquid culture medium to 20% of the triangular flask, and then place them at 30°C for 48 hours. Culture them on a shaking table with the shaking table speed set to 200 rpm to obtain acetic acid bacteria seed liquid, and adjust the bacterial liquid concentration to OD (A600 nm) = 0.4-0.6. The acetic acid bacteria can also be selected from other different acetic acid strains.

[0033] Step (3): freezing the Eucommia leaf tea with liquid nitrogen for 10 minutes, wherein the Eucommia leaf tea is a substitute tea prepared from Eucommia leaves using a conventional method;

[0034] Step (4): placing the Eucommia leaf tea obtained in step (3) in a low-temperature pulverizer for pulverization;

[0035] Step (5): sieving the Eucommia leaf tea powder obtained in step (4), preferably through a 80-mesh sieve;

[0036] Step (6): Weigh 2 g of the Eucommia leaf tea powder obtained in step (5) for enzymatic hydrolysis. Add 0.0690 g of the immobilized complex enzyme obtained in step (1), add disodium hydrogen phosphate-citric acid buffer to a solid-liquid ratio of 1:20, adjust the pH to pH = 4.5 with citric acid and sodium citrate, and perform enzymatic hydrolysis at 55°C for 4.5 hours to obtain an Eucommia leaf hydrolyzate.

[0037] Step (7): Add 0.2 g of high enzyme activity honey having a β-glucosidase activity of 40.9 mU / g to the Eucommia leaf hydrolysate obtained in step (6), adjust the pH to pH = 4.2 using citric acid and sodium citrate, set the hydrolysis temperature to 60° C., and the hydrolysis time to 2.5 h. At the end of the hydrolysis, add 80 mL of 60° C. water, and filter to obtain a secondary hydrolysate and a filter residue.

[0038] Step (8): subjecting the filter residue obtained in step (7) to secondary ultrasonic ethanol extraction: ultrasonic time 50 min, ultrasonic temperature 60° C., ethanol concentration 65%, solid-liquid ratio 1:35 g / mL, and centrifugation to obtain a secondary extract.

[0039] Step (9): dilute the secondary extract obtained in step (8) to an ethanol concentration of 5% by adding distilled water, inoculate acetic acid bacteria seed liquid at a volume ratio of 2%-4%, control the temperature to 30° C., stir at a speed of 400 r / min, and carry out the acetic acid fermentation stage for a total of 72 hours. After 72 hours, obtain the acetic acid fermentation liquid.

[0040] Step (10): combining the obtained secondary enzymatic hydrolysate with the acetic acid fermentation broth, and concentrating the broth to 1 / 6 of the original volume to obtain a high-purity extract, which is the Eucommia leaf medicinal composition.

[0041] Comparative Example 1

[0042] The comparative example 1 is different from the example 1 only in step (3), that is, the Eucommia leaf tea is not frozen by liquid nitrogen, but a normal temperature pulverizer is used, and the other steps are the same.

[0043] Comparative Example 2

[0044] The comparative example 2 is different from the example 1 only in step (4), that is, the eucommia leaf tea is not subjected to a pulverization process, and the other steps are the same.

[0045] Comparative Example 3

[0046] Compared with Example 1, the only difference in this comparative example 3 is step (7), in which 0.2 g of high enzyme activity honey is replaced by 0.2 g of fructose syrup, and the other steps are the same.

[0047] Comparative Example 4

[0048] Compared with Example 1, the only difference in this comparative example 4 is step (8), in which ultrasonic ethanol extraction is not performed, but ethanol of the same mass and concentration is directly added, and the other steps are the same.

[0049] Experimental Example 1 Extract Analysis

[0050] The functional components of the extracts obtained in Example 1 and Comparative Examples 1-4 were analyzed, including the contents of flavonoids, chlorogenic acid, polysaccharides and genipin. The results are shown in Table 1 below.

[0051] Table 1 Content of functional components in the extract (based on dry weight of Eucommia ulmoides leaf tea)

[0052]

[0053] Experimental Example 2: Medicinal Analysis of the Extract

[0054] This experiment was conducted in accordance with the "Chinese Laboratory Animal Management Standards". 72 Balb / c male mice (8 weeks old, weighing 18g to 22g) were placed in an animal facility that meets the requirements of specific pathogen-free (SPF) (room temperature 20-22°C, humidity 45-55%, 12h light / dark cycle), with unrestricted access to standard laboratory diet and water.

[0055] After 7 days of adaptive feeding, the mice were randomly divided into groups of 8 mice each, and were set and recorded as a control group (group B), a model group (group M), an experimental group of Example 1 (group S1), an experimental group of comparative example 1 (group D1), an experimental group of comparative example 3 (group D3), an experimental group of comparative example 4 (group D4), a positive drug control group (group Y), a polysaccharide control group (group C), a flavonoid control group (group H), a chlorogenic acid control group (group L), and a genipin control group (group J). Among them, due to the low extraction rate of comparative example 2 group, the treatment of comparative example 2 group and the obtained product were abandoned. The control group was untreated mice, and the mice in the remaining groups except the control group were subcutaneously injected with CORT (40 mg / kg) solution for 28 consecutive days to induce chronic depression.

[0056] If a person takes 10g of Eucommia tea every day, based on a human body weight of 60kg, it is equivalent to 0.17g / kg (Eucommia tea mass / human body weight) per person per day. Multiplying this by the human-to-mouse conversion ratio of 9:1, the daily extract usage for mice is 1.5g / kg.

[0057] The processing of the above groups is as follows:

[0058] Control group (Group B): gavage 0.4 mL of normal saline every day;

[0059] Model group (M group): gavage with 0.4 mL of normal saline every day;

[0060] Experimental group (S1 group) of Example 1: The extract prepared by the parameters of Example 1 was administered by intragastric administration, and the extract of Eucommia leaf tea (converted to the original Eucommia tea) was administered; the intragastric administration volume was 20 mL / kg BW, i.e., 0.4 mL was administered per day;

[0061] Comparative Example 1 Experimental Group (D1 Group): The Eucommia Tea Extract prepared by the parameters of Comparative Example 1 was intragastrically administered, 0.4 mL per day;

[0062] Comparative Example 3 Experimental Group (D3 Group): The Eucommia Tea Extract prepared by the parameters of Comparative Example 3 was intragastrically administered, 0.4 mL per day;

[0063] Comparative Example 4 Experimental Group (D4 Group): The Eucommia Tea Extract prepared by the parameters of Comparative Example 4 was intragastrically administered, 0.4 mL per day;

[0064] Positive drug control group (Group Y): intragastric administration of fluoxetine hydrochloride at 10 mg / kg / d;

[0065] Polysaccharide control group (Group C): 100 mg / kg / d by gavage;

[0066] Flavonoid control group (H group): 100 mg / kg / d by gavage;

[0067] Chlorogenic acid control group (L group): 100 mg / kg / d by gavage;

[0068] Genipin control group (Group J): 100 mg / kg / d was administered by oral gavage.

[0069] All groups of mice also underwent behavioral studies during the 4-week treatment period, including the following:

[0070] (1) Weight changes

[0071] After one week of adaptive feeding, the mice were divided into groups and weighed. The weights were recorded every 7 days after treatment, and the weight change rate was calculated based on the initial weight of the mice. The results are shown in Table 2.

[0072] Table 2 Weight change rate of mice

[0073]

[0074]

[0075] As shown in Table 2, the weight of mice in group S1 was significantly higher than that in group M, D1, D3, and D4, indicating that after extraction by the present method, eucommia leaf tea can effectively improve the phenomenon of loss of appetite and weight loss in depression model mice. The weight of group S1 was also significantly higher than that of group C, group H, group L, and group L, indicating that the eucommia leaf tea extract provided by the present invention has a synergistic effect and enhances the antidepressant effect.

[0076] (2) Open field test

[0077] The open field test is an experimental method commonly used in behavioral neuroscience, mainly used to evaluate the anxiety behavior, exploratory behavior and motor activity of mice. Mice were placed in an open field device of 50cm*50cm*40cm, and the time they stayed in the central area was examined. A longer stay time is usually interpreted as a lower level of anxiety, while a longer stay time in the edge area may indicate a higher level of anxiety. The mice in the above treatment groups were placed in the open field device respectively, and the time when the mice entered the central area within 5 minutes was recorded. The results are shown in Table 3 below.

[0078] Table 3 Mouse open field test

[0079]

[0080] As shown in Table 3, the time mice in group M stayed in the central area was significantly reduced, indicating that the model was successfully established. Compared with groups M, D1, D3, and D4, the time mice in group S1 stayed in the central area was significantly increased, indicating that Eucommia tea extract had a certain anti-anxiety effect. Compared with groups C, H, L, and L, group S1 also had a significant increase, which again showed that Eucommia leaf tea extract could resist anxiety and improve depression.

[0081] (3) Tail suspension test

[0082] Tail suspension test (TST) is a classic method that can quickly evaluate antidepressant drugs. In this animal model, the tail of the experimental animal is fixed, so that its head is suspended downward. The animal struggles in this environment, trying to get rid of the predicament. After trying hard and still unable to get rid of it, it becomes intermittent and immobile, showing a state of "behavioral despair". The mice were suspended and fixed for 6 minutes, and the tail suspension immobility time of the mice in the last 4 minutes was recorded. The statistical results are shown in Table 4 below.

[0083] Table 4 Tail suspension test

[0084]

[0085] As shown in Table 4, compared with group B, the immobility time of the tail suspension in group M was significantly increased, indicating that the model was successfully established. Compared with groups M, D1, D3, and D4, the immobility time of the tail suspension in group S1 was significantly reduced, indicating that the extract of Eucommia ulmoides tea has a certain antidepressant effect. Compared with groups C, H, L, and L, the extract of Eucommia ulmoides tea in group S1 was also significantly reduced, which again showed that the extract of Eucommia ulmoides tea has an improving effect on depression.

[0086] (4) Investigation of inflammatory factors

[0087] The hippocampal tissue of mice in each treatment group was taken, homogenized and ground, and the supernatant was taken after centrifugation. IL-6 (interleukin-6), IL-1β (interleukin-1β), and TNF-α (tumor necrosis factor-α) were determined using Elisa kits. Blood was taken from the eyeballs of mice, and the supernatant was taken after centrifugation. IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α) were determined using Elisa kits. The results are shown in Table 5.

[0088] Table 5 Inflammatory factor detection results

[0089]

[0090] As shown in Table 5, compared with group B, the levels of IL-6 and TNF-α in the hippocampus and serum of group M were significantly reduced. Compared with groups M, D1, D3, and D4, the levels of IL-6 and TNF-α in serum and hippocampus of group S1 were significantly reduced, indicating that the extract of Eucommia leaf tea has anti-inflammatory effects. Compared with groups C, H, L, and L, the levels of IL-6 and TNF-α in serum and hippocampus of group S1 were also significantly reduced, indicating that the extract of Eucommia leaf tea also has anti-inflammatory effects.

[0091] (5) Investigation of the Hypothalamic-Pituitary-Adrenal (HPA) Axis

[0092] Mouse hippocampal tissue was obtained, homogenized and ground, and the supernatant was obtained after centrifugation. The content of ACTH (adrenocorticotropic hormone) was determined using an Elisa kit. Blood was collected from mouse eyeballs, and the supernatant was obtained after centrifugation. The content of ACTH (adrenocorticotropic hormone) and CORT (corticosterone) was determined using an Elisa kit. The results are shown in Table 6.

[0093] Table 6 HPA axis investigation

[0094]

[0095]

[0096] As shown in Table 6, compared with group B, the levels of adrenocorticotropic hormone in the hippocampus of group M, and the levels of adrenocorticotropic hormone and corticosterone in serum were significantly increased. Compared with groups M, D1, D3, and D4, the levels of adrenocorticotropic hormone in the hippocampus of group S1, the levels of corticosterone in serum, and the levels of adrenocorticotropic hormone were significantly reduced, and group S1 was also significantly reduced compared with groups C, H, L, and L. The levels of adrenocorticotropic hormone in the hippocampus of group S1, the levels of corticosterone in serum, and the levels of adrenocorticotropic hormone were significantly reduced, indicating that the extract of Eucommia ulmoides tea extracted by this method has an inhibitory effect on the excitation of the HPA axis, and has a synergistic effect with respect to the functional substances in the extract.

[0097] In summary, the Eucommia leaf extract obtained by the preparation method provided by the present invention has the effects of improving depression, anti-inflammation, and inhibiting HPA axis excitement. It can be used as a pharmaceutical composition, or added with conventional additives used to prepare drugs for improving depression, anti-inflammation, and inhibiting HPA axis excitement. It can be prepared into a variety of dosage forms, including but not limited to tablets, capsules, pills, mixed solutions, injections, etc., and has significant practical value.

[0098] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a Eucommia leaf medicinal composition for improving depression, characterized in that: The following steps are involved: (1) The immobilized complex enzyme is prepared by a sodium alginate immobilization method, wherein the enzymes include cellulase and pectinase; (2) preparing acetic acid bacteria seed solution; (3) freezing the eucommia leaf tea with liquid nitrogen and then low-temperature crushing it, sieving it to obtain eucommia leaf tea powder, and enzymolyzing the eucommia leaf tea powder with the immobilized complex enzyme to obtain an eucommia leaf enzymolysis solution; (4) adding honey having β-glucosidase activity to the Eucommia ulmoides leaf hydrolyzate for secondary hydrolysis, adding 60° C. water after the hydrolysis, and filtering to obtain a secondary hydrolyzate and a filter residue; (5) subjecting the filter residue to ultrasonic ethanol secondary extraction, and filtering to obtain a secondary extract after the extraction is completed; (6) diluting the secondary extract with water to an ethanol concentration of 5%, inoculating the acetic acid bacteria seed solution with a volume ratio of 2% to 4% to perform acetic acid fermentation to obtain acetic acid fermentation solution; (7) The secondary enzymatic hydrolyzate and the acetic acid fermentation broth are combined and concentrated to obtain an extract which is the Eucommia ulmoides leaf medicinal composition.

2. The preparation method according to claim 1, characterized in that: The preparation method of the immobilized complex enzyme is as follows: taking a sodium alginate solution with a mass fraction of 4% at a volume ratio of 2:1:1 、 A 25 mg / mL complex enzyme solution and a 5% volume fraction of glutaraldehyde are mixed evenly, and dripped dropwise into a 1% CaCl2 solution. After forming small balls, the solution is replaced with a 1% CaCl2 solution for immersion, and stored in a refrigerator at 4°C for 2 hours. After filtering, the filter residue is immersed in a 0.5% volume fraction of glutaraldehyde for 4 hours, and then the filter residue is washed with distilled water for several times. After drying, the immobilized complex enzyme is obtained; wherein the enzyme activity ratio of cellulase to pectinase in the complex enzyme solution is 1:

1.

3. The preparation method according to claim 1, characterized in that: The concentration of the acetic acid bacteria seed solution is OD600=0.4-0.

6.

4. The preparation method according to claim 1, characterized in that: The sieving is through an 80-mesh sieve.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis treatment in step (3) is specifically as follows: taking the eucommia leaf tea powder and the immobilized complex enzyme at a mass ratio of 1:0.0345, adding disodium hydrogen phosphate-citric acid buffer at a material-liquid ratio of 1:20, adjusting the pH to 4.5, and performing enzymatic hydrolysis at 55° C. for 4.5 hours.

6. The preparation method according to claim 5, characterized in that: In the step (4), the amount of honey added is 1 / 10 of the weight of the eucommia leaf tea powder, the β-glucosidase activity in the honey is 40.9 mU / g; the pH of the secondary enzymolysis is 4.2, the enzymolysis temperature is 60°C, and the enzymolysis time is 2.5 h; the ratio of the added water to the eucommia leaf tea powder is 40:1 mL / g.

7. The preparation method according to claim 1, characterized in that: The ultrasonic ethanol secondary extraction has an ultrasonic time of 50 minutes, an ultrasonic temperature of 60° C., an ethanol concentration of 65%, and a solid-liquid ratio of 1:35 g / mL.

8. The preparation method according to claim 1, characterized in that: The acetic acid fermentation is as follows: the fermentation temperature is 30° C., the stirring speed is 400 r / min, and the fermentation time is 72 h.

9. The Eucommia ulmoides leaf medicinal composition prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the Eucommia leaf medicinal composition according to claim 9 in any of the following items, characterized in that: Contains the efficient use of Eucommia leaves and improved treatment of depression.

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