A magnolia flower crude extract, a preparation method and application thereof

Abstract

The present application relates to a kind of crude extract of magnolia flower and its preparation method and application, the preparation method of the crude extract of magnolia flower includes: fresh magnolia flower is sequentially dried, pulverized, screened and then obtains magnolia flower powder;The required quality of magnolia flower powder is weighed, under the conditions of extraction temperature 40-80 ℃, extraction pressure 10-30 Mpa, entrainer concentration 40%-80%, entrainer flow rate 0.5-2.5 ml / min, supercritical CO2 extraction is carried out for 1-3 hours to obtain the crude extract of magnolia flower;The crude extract of magnolia flower is rotary evaporated to dryness, and the crude extract of magnolia flower is obtained.The present application uses magnolia flower as raw material, uses supercritical CO2 extraction technology to prepare the crude extract of magnolia flower, uses excessive antibiotics to induce intestinal flora disorder of mouse, establishes weak mouse model of spleen deficiency, and by using different concentrations of the crude extract of magnolia flower to the mouse is treated by gavage, find that the crude extract of magnolia flower can balance its intestinal flora, adjust the weak state of mouse of spleen deficiency, and improve immunity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a crude Magnolia officinalis flower extract, a preparation method and application thereof. BACKGROUND

[0002] Intestinal flora is very important for the health of the body, and participates in various physiological metabolic activities of the host, and is an indispensable important part of normal physiological activities of the host. The intestinal flora plays an indispensable role in the occurrence of host health and diseases. When the structure of the normal intestinal flora changes, the "yin and yang balance" of the intestinal flora of the body will be destroyed. The relevant theory of traditional Chinese medicine states that the basic principle of treating diseases is to adjust yin and yang, and the so-called treatment of diseases is based on yin and yang. Once the yin and yang is out of balance, the human body is more susceptible to diseases caused by exogenous wind, cold, heat, dampness, dryness and heat. Those who get wind evil have symptoms such as spleen deficiency, blood deficiency, qi deficiency, lack of strength, loss of appetite, etc. In the development of modern traditional Chinese medicine, the pharmacological activity research of related traditional Chinese medicines is also more and more widely used in the treatment of such diseases. For example, Liuwei Dihuang Pill, Shenling Baizhu Powder and other traditional Chinese medicines containing Poria cocos and Chinese yam have the pharmacological activities of supporting healthy qi, tonifying the spleen and invigorating qi.

[0003] Magnolia officinalis resources include Magnolia officinalis, Magnolia officinalis var. biloba and Magnolia officinalis flowers. These are all state-protected traditional Chinese medicinal materials. Magnolia officinalis flowers are large and beautiful, with a sweet smell, a light taste, large leaves and dense shade. The flowers are white when they open. The main components include phenols, alkaloids, flavonoids, volatile oils and sugars. Related literature research shows that these components have certain antioxidant, antitumor, analgesic and anti-inflammatory effects. However, at present, the research mainly focuses on the physicochemical activity of single active substance such as phenols and volatile oils. In terms of pharmacology of traditional Chinese medicine, the action of traditional Chinese medicinal materials is mainly attributed to the interaction and coordination of complex active ingredients. Therefore, it has good commercial value and prospect to study more pharmacological activities of Magnolia officinalis flowers.

[0004] Supercritical CO2 extraction technology is a technology that uses the solubility of substances in supercritical fluid, which is greatly affected by pressure and temperature. In the supercritical state, the supercritical fluid is in contact with the substance to be separated, so that it selectively extracts components with different polarity, boiling point and molecular weight. The purpose of separation and purification can be achieved. The supercritical fluid extraction has the characteristics that when passing through the separator, CO2 and the extract rapidly become two phases (gas-liquid separation) due to the pressure drop, and immediately separate. There is no phase change process of the material, and the solvent does not need to be recovered. The operation is convenient, the extraction efficiency is high, the energy consumption is less, and the cost is saved. In addition, the supercritical fluid CO2 used in supercritical CO2 extraction is an inert gas, which is not easy to react and has good safety. It can prevent the oxidation and degradation of heat-sensitive substances. SUMMARY

[0005] The present application aims to provide a crude magnolia flower extract, a preparation method and application thereof.

[0006] The present application realizes the above-mentioned purpose through the following technical solutions.

[0007] A crude magnolia flower extract, and a preparation method thereof, comprising the following steps:

[0008] Step one: drying, crushing and screening fresh magnolia flowers to obtain magnolia flower powder;

[0009] Step two: weighing the required amount of magnolia flower powder, and performing supercritical CO2 extraction under the following extraction conditions: extraction temperature of 40-80 DEG C, extraction pressure of 10-30 Mpa, entrainer concentration of 40%-80%, and entrainer flow rate of 0.5-2.5 ml / min, to obtain a crude magnolia flower extract after 1-3 h;

[0010] Step three: rotary evaporation of the crude magnolia flower extract to dryness, and leaving the solid part as the crude magnolia flower extract.

[0011] As a further optimization of the present application, in step one, the screening is 20 mesh screening.

[0012] As a further optimization of the present application, in step two, the entrainer is ethanol, and the mass ratio of the magnolia flower powder to anhydrous ethanol is 1:0.9-30.

[0013] As a further optimization of the present application, in step three, the rotary evaporation temperature is 60±3 DEG C.

[0014] A preparation method of a crude magnolia flower extract, which uses the above-mentioned preparation method to prepare the crude magnolia flower extract.

[0015] Application of a crude magnolia flower extract in the preparation of a medicine for treating or improving spleen deficiency and intestinal flora disorder, wherein the crude magnolia flower extract is used as the main component to prepare the medicine for treating or improving spleen deficiency and intestinal flora disorder.

[0016] As a further optimization of the present application, the medicinal dose of the crude magnolia flower extract is 100-300 mg / kg.

[0017] As a further optimization of the present application, the medicinal dose of the crude magnolia flower extract is 200 mg / kg.

[0018] As a further optimization of the present application, the intestinal flora disorder includes intestinal flora disorder caused by excessive use of antibiotics, in which Proteobacteria is the main dominant flora.

[0019] As a further optimization scheme of the present application, the antibiotic includes any one or more of ceftriaxone sodium and lincomycin hydrochloride.

[0020] The present application has the beneficial effects in that:

[0021] 1) The supercritical CO2 extraction technology adopted in the present application can better remove harmful biological substances in Magnolia officinalis flower raw materials and retain beneficial biological substance activity, thereby providing a more effective means for active substances of traditional Chinese medicines to exert sufficient drug efficacy.

[0022] 2) The present application discovers new medicinal value of Magnolia officinalis flower crude extract, which can be used to prepare drugs for treating or improving spleen deficiency and intestinal flora disorder, thereby opening up a new field for application of Magnolia officinalis flower crude extract.

[0023] 3) The present application uses fresh Magnolia officinalis flower as raw material, uses supercritical CO2 extraction technology to prepare Magnolia officinalis flower crude extract, uses excessive antibiotics to induce intestinal flora disorder of mice, establishes a spleen deficiency mouse model, and finds that Magnolia officinalis flower crude extract can balance intestinal flora of mice and adjust the physical state of mice, i.e., improve immunity and improve the state of spleen deficiency.

[0024] 4) The Magnolia officinalis flower crude extract adopted in the present application is derived from fresh Magnolia officinalis flower, which is abundant in resources and easy to obtain. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A Magnolia officinalis flower crude extract prepared by supercritical CO2 extraction technology, Figure 1 A Magnolia officinalis flower crude extract prepared by supercritical CO2 extraction technology, Figure 1 B Magnolia officinalis flower crude extract prepared by alcohol extraction method).

[0026] Figure 2 A mass concentration graph of immunoglobulin IgG, interleukin (IL)-4, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in serum of each group of mice (blank group, model group, low-concentration treatment group, medium-concentration treatment group and high-concentration treatment group) after 30 days of treatment.

[0027] Figure 3 An HE staining graph of spleen tissue of each group of mice (blank group, model group, low-concentration treatment group, medium-concentration treatment group and high-concentration treatment group) after 30 days of treatment.

[0028] Figure 4The staining chart of T lymphocyte differentiation factor CD3 and CD8 in the spleen of each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group, and high concentration treatment group) after 30 days of treatment.

[0029] Figure 5 The analysis chart of intestinal flora (classification at the level of phylum) of each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group) after 30 days of treatment.

[0030] Figure 6 The analysis chart of intestinal flora (classification at the level of genus) of each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group) after 30 days of treatment.

[0031] Figure 7 The principal component analysis chart in the beta diversity of intestinal flora of each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group) after 30 days of treatment. DETAILED DESCRIPTION

[0032] It is necessary to point out here that the following detailed description is only used to further illustrate the application and cannot be understood as limiting the scope of protection of the application. Those skilled in the art can make some non-essential improvements and adjustments to the application based on the above application content.

[0033] 1. Materials

[0034] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified. The reagents and other materials used are commercially available products unless otherwise specified.

[0035] (1) Magnolia flower, purchased from Anhui Tianhe Traditional Chinese Medicine Development Co., Ltd.

[0036] (2) SPF level BALB / C male mice, 6-7 weeks old, average body weight (22±2) g, purchased from Changzhou Cavens Experimental Animal Co., Ltd.

[0037] 2. Methods

[0038] 2.1 Preparation of Magnolia flower crude extract by supercritical CO2 extraction technology (SFE-CO2)

[0039] 2.1.1 Raw material pretreatment: Fresh Magnolia flowers were dried naturally or dried at a temperature below 50°C, crushed with a crusher at room temperature, and then sieved through a 20-mesh sieve to obtain Magnolia flower powder.

[0040] 2.1.2, Dynamic extraction: the required mass of Magnolia officinalis flower powder (obtained in step 2.1.1) is weighed and placed in a supercritical extraction kettle, and supercritical CO2 extraction is carried out under the following extraction conditions: extraction temperature of 40-80°C, extraction pressure of 10-30 MPa, concentration of entraining agent (ethanol) of 40%-80%, flow rate of entraining agent (ethanol) of 0.5-2.5 ml / min, and extraction time of 1-3 h to obtain Magnolia officinalis flower crude extract;

[0041] wherein the mass ratio of Magnolia officinalis flower powder to anhydrous ethanol is 1:0.9-30;

[0042] 2.1.3, Rotary evaporation treatment of Magnolia officinalis flower crude extract: the Magnolia officinalis flower crude extract obtained in step 2.1.2 is placed in a tomato-shaped bottle, and rotary evaporation is carried out at a temperature of 60°C until dryness, and the solid part is left to obtain Magnolia officinalis flower crude extract.

[0043] 2.2 Comparative analysis of Magnolia officinalis flower crude extract prepared by alcohol extraction and supercritical CO2 extraction technology

[0044] 2.2.1, Preparation of Magnolia officinalis flower crude extract by supercritical CO2 extraction technology: 10 g of dried and pulverized Magnolia officinalis flower powder is weighed and placed in an extraction kettle;

[0045] Under the following extraction conditions: extraction pressure of 20 MPa, extraction temperature of 60°C, concentration of entraining agent (ethanol) of 60%, flow rate of entraining agent (ethanol) of 1.5 ml / min, dynamic extraction for 2 h, collection of Magnolia officinalis flower crude extract, and rotary evaporation at a temperature of 60°C until dryness; wherein the amount of 60% ethanol is 180 mL, and 1.9584 g of Magnolia officinalis flower crude extract is obtained, i.e. the extraction rate is 19.584%;

[0046] Under the following extraction conditions: extraction pressure of 25 MPa, extraction temperature of 70°C, concentration of entraining agent (ethanol) of 60%, flow rate of entraining agent (ethanol) of 2.5 ml / min, dynamic extraction for 3 h, collection of Magnolia officinalis flower crude extract, and rotary evaporation at a temperature of 60°C until dryness; wherein the amount of 60% ethanol is 450 mL, and 2.3573 g of Magnolia officinalis flower crude extract is obtained, i.e. the extraction rate is 23.573%;

[0047] 2.2.2, Extraction of Magnolia officinalis flower crude extract by alcohol extraction: 10 g of dried and pulverized Magnolia officinalis flower powder is weighed, and the extraction conditions are as follows: extraction time of 2 h; extraction solvent of 60% ethanol; solid-liquid ratio of 1:20; extraction temperature of 60°C; after completion of extraction, Magnolia officinalis flower extract is obtained, and the extract is rotary evaporated at a temperature of 60°C until dryness; wherein the amount of 60% ethanol is 200 mL, and 1.8049 g of Magnolia officinalis flower crude extract is obtained, i.e. the extraction rate is 18.049%.

[0048] As Figure 1The morphological chart of the Magnolia officinalis flower crude extract prepared by the two methods is shown in the figure Figure 1 A is the Magnolia officinalis flower crude extract prepared by the supercritical CO2 extraction technology, Figure 1 B is the Magnolia officinalis flower crude extract prepared by the alcohol extraction method);

[0049] The experimental results are as follows: the extraction rate of the Magnolia officinalis flower crude extract prepared by the supercritical CO2 extraction technology is 19.584% and 23.573%, the extraction rate of the Magnolia officinalis flower crude extract prepared by the alcohol extraction method is 18.049%, therefore, the extraction rate of the Magnolia officinalis flower crude extract prepared by the supercritical CO2 extraction technology is improved, and compared with the alcohol extraction method, the supercritical CO2 extraction technology (extraction condition one) needs to use less amount of ethanol; and the quality of the Magnolia officinalis flower crude extract prepared by the two methods is simply evaluated by the sensory evaluation, the Magnolia officinalis flower crude extract prepared by the alcohol extraction method is dark yellow solid, has special fragrance but mixed with sour taste, while the Magnolia officinalis flower crude extract prepared by the supercritical CO2 extraction technology has special fragrance and no sour taste.

[0050] 2.3 Antioxidant activity test of the Magnolia officinalis flower crude extract

[0051] 10 g of the Magnolia officinalis flower powder screened by the 20-mesh sieve in step 2.1.1 and the Magnolia officinalis flower crude extract prepared by the alcohol extraction method and the supercritical CO2 extraction technology are taken for antioxidant activity determination, the antioxidant activity is determined by DPPH free radical scavenging method and hydroxyl free radical (OH - ) scavenging method respectively, and the test data is shown in Table 1:

[0052] Table 1: DPPH and OH- free radical scavenging results

[0053]

[0054] The experimental results are as follows: in the supercritical CO2 extraction conditions I and condition II, the half-inhibition rate (IC50) of the crude extract of Magnolia officinalis flower on DPPH free radicals is 0.102 mg / ml and 0.095 mg / ml, respectively, and the half-inhibition rate (IC50) on hydroxyl free radicals is 1.541 mg / ml and 1.476 mg / ml, respectively; the half-inhibition rate (IC50) of the crude extract of Magnolia officinalis flower prepared by alcohol extraction and the Magnolia officinalis flower powder (the Magnolia officinalis flower powder of step 2.1.1 sieved through a 20-mesh sieve) on DPPH free radicals is 0.124 mg / ml and 0.586 mg / ml, respectively, and the half-inhibition rate (IC50) on hydroxyl free radicals is 1.866 mg / ml and 8.125 mg / ml, respectively; thus, it can be seen that, in order to achieve the inhibition rate IC50 of DPPH free radicals and OH- free radicals, the amount of the crude extract of Magnolia officinalis flower prepared by supercritical CO2 extraction is less than that of the crude extract of Magnolia officinalis flower prepared by alcohol extraction and the Magnolia officinalis flower powder (the Magnolia officinalis flower powder of step 2.1.1 sieved through a 20-mesh sieve), which means that the crude extract of Magnolia officinalis flower prepared by supercritical CO2 extraction has stronger antioxidant capacity, and thus it can be concluded that the supercritical CO2 extraction method has higher extraction rate and obvious advantages in the preservation of active ingredients of Magnolia officinalis flower.

[0055] 3. Effect verification experiment of the crude extract of Magnolia officinalis flower

[0056] 3.1 Modeling of mouse animal experiment

[0057] The mice are divided into 5 groups (6 mice in each group), namely, a blank group (Control), a model group (Model), a low-concentration treatment group (100 mg / kg), a medium-concentration treatment group (200 mg / kg), and a high-concentration treatment group (300 mg / kg); the modeling cycle of the mice is 5 days, and the model group and the treatment groups are intragastrically administered with 0.2 mL of ceftriaxone sodium (400 mg·mL-1) and 0.1 mL of lincomycin hydrochloride (300 mg·mL-1) per mouse per time, twice a day for the first 5 days, and the blank group is replaced with normal saline.

[0058] 3.2 Drug treatment of mice

[0059] The treatment cycle of the mice is 30 days, the blank group and the model group are fed with normal saline, and the low-concentration treatment group, the medium-concentration treatment group, and the high-concentration treatment group are intragastrically administered with the crude extract of Magnolia officinalis flower at a mass concentration of 100 mg / kg, 200 mg / kg, and 300 mg / kg, respectively.

[0060] 3.3 Detection of related indexes

[0061] 3.3.1 Detection of the mass concentrations of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, and immunoglobulin IgG in the blood samples of mice

[0062] After the completion of drug treatment in step 3.2, blood was collected from the eyeballs of mice in each group (blank group, model group, low-concentration treatment group, medium-concentration treatment group, and high-concentration treatment group). All blood samples were allowed to stand at room temperature for 60 min and then centrifuged at 3000 r / min in a low-temperature centrifuge for 15 min. The supernatant pale yellow serum was then collected, aliquoted into small test tubes, and the concentrations of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, and immunoglobulin IgG in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.

[0063] Immunoglobulin G (IgG) is the main component of serum immunoglobulins, possessing antiviral, virus-neutralizing, antibacterial, and immunomodulatory functions. Interleukin (IL)-4 is a cytokine whose biological effects mainly include stimulating the proliferation of activated B cells and T cells. Interferon (IFN)-γ is a pleiotropic cytokine with antiviral, antitumor, and immunomodulatory properties. Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine mainly produced by macrophages and monocytes, and participates in normal inflammatory and immune responses. Among these, IgG, (IL)-4, and (IF)-γ are positively correlated with the strength of immunity, while (TNF)-α is negatively correlated with the strength of immunity.

[0064] Experimental results are as follows Figure 2 As shown: by Figure 2 A, Figure 2 B. Figure 2 As shown in C, the number of relevant proteins and cytokines in each indicator showed a significant difference between the model group and the control group, indicating that there were differences in the immune status of mice in the model group and the treatment groups (low concentration treatment group (100mg / kg), medium concentration treatment group (200mg / kg), and high concentration treatment group (300mg / kg). Mice generally had low immunity after modeling and before drug treatment. After drug treatment, the concentrations of each indicator increased in the latter three treatment groups, especially in the medium concentration treatment group (200mg / kg) and the high concentration treatment group (300mg / kg), where the increases were more significant and approached those of the control group. This indicates that the medium concentration treatment group (200mg / kg) and the high concentration treatment group (300mg / kg) had better efficacy, and the difference in efficacy between the two groups was not significant.

[0065] And by Figure 2DIt can be known that, similar to the previous three indicators (immunoglobulin IgG, interleukin (IL)-4, interferon (IFN)-γ), the number of related proteins and cytokines of the model group and the blank group has a greater significance, and (TNF)-α and immunity are negatively correlated, so it can also be explained that the immune status of the model group and each treatment group (low concentration treatment group (100 mg / kg), medium concentration treatment group (200 mg / kg), high concentration treatment group (300 mg / kg)) is different, and the immune status of the mice is generally low before the drug treatment after the modeling, and the decrease of the mass concentration of the corresponding index after the drug treatment of the mice in the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg) also indicates that the curative effects of the two groups are better.

[0066] 3.3.2, Detection of changes in the structure of the mouse spleen and staining of T lymphocyte factors CD3 and CD8 in the mouse spleen

[0067] Each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group, high concentration treatment group) after the end of drug treatment in step 3.2 was dissected, and fresh mouse spleen was taken, soaked in 4% paraformaldehyde solution overnight, and the next day, the mouse spleen tissue was paraffin-embedded, sectioned, deparaffinized, stained, and mounted. Experimental operation. Finally, the changes in the structure of the mouse spleen and the staining of CD3 and CD8 cytokines in each group of mice were observed under an optical microscope.

[0068] The experimental results of the changes in the structure of the mouse spleen are shown in Figure 3 As can be seen from the optical microscope, the staining of the mouse spleen tissue sections in each group can be seen, the mouse spleen cells in the blank group (Control) are arranged closely and orderly, and the cell nucleus is clear; the number of mouse spleen cells in the model group (Model) is significantly reduced, the cell size is different, and the arrangement is sparse and disordered. For the three different concentration drug treatment groups (low concentration treatment group (100 mg / kg), medium concentration treatment group (200 mg / kg), high concentration treatment group (300 mg / kg)), compared with the model group, after 30 days of treatment, the morphology of the spleen cells in each drug treatment group has changed in a good direction, especially in the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg), the arrangement of the spleen cells becomes close, and the cell nucleus is also clear. It can be deduced that the treatment effect of the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg) is better.

[0069] CD3 and CD8 are T lymphocyte differentiation factors, and low CD3 and CD8 are usually due to the body weakness and low resistance of the human body. CD3 is dyed red, and CD8 is dyed green during the experiment.

[0070] The experimental results of T lymphocyte factor CD3 and CD8 staining in the mouse spleen are shown in Figure 4 It can be seen that the CD3 and CD8 distribution in the spleen of normal mice in the blank group (Control) is wide and the number is large, which indicates that there are enough immune cells in the body of the mice and the immune system in the body is normal. For the model group, it can be seen that the number of CD3 and CD8 is relatively small, which indicates that there are differences in the immune state of the model group (Model) and each treatment group (low concentration treatment group (100 mg / kg), medium concentration treatment group (200 mg / kg), high concentration treatment group (300 mg / kg)), and the immune function of the mice decreased after the experiment modeling and before drug treatment, that is, it is easier to be infected by some diseases.

[0071] Compared with the model group, the number of CD3 and CD8 of the mice in the low concentration treatment group (100 mg / kg), the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg) after drug treatment increased, which indicates that the physical activity of the mice is improved after drug treatment. Among them, the number of CD3 and CD8 in the spleen of the mice in the low concentration treatment group (100 mg / kg) increased slightly, that is, the improvement of the body activity is not much, which may be caused by insufficient drug dosage. The number of CD3 and CD8 in the spleen of the mice in the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg) increased obviously, and the number of increase of the two was close, which indicates that the drug effect of the medium concentration treatment group (200 mg / kg) and the high concentration treatment group (300 mg / kg) is better and close, which also indicates that the thick shell flower crude extract has the effect of regulating the body weakness and low activity of the mice.

[0072] 3.3.3. High-throughput sequencing of 16S rRNA gene of intestinal flora of mice

[0073] The mice in each group (blank group, model group, low concentration treatment group, medium concentration treatment group) after drug treatment in step 3.2 were dissected, and the cecal contents of the mice in each group were collected aseptically in a super-clean bench and stored in a-80℃ refrigerator for use. Then the DNA of the cecal contents of the mice was extracted by a kit, and high-throughput sequencing of 16S rRNA gene of intestinal flora was performed.

[0074] The experimental results are shown in Figures 5-7 Figure 5 ​For the classification at the level of phylum, it can be seen that in the normal blank group (Control), the proportions of Firmicutes and Bacteroidota are relatively high, accounting for 39.57% and 48.98% respectively, and these two phyla belong to the dominant phyla of intestinal flora, mainly composed of some beneficial flora; compared with the blank group, it can be seen that in the model group (Model), the proportions of Firmicutes and Bacteroidota are reduced, while the proportion of Proteobacteria is relatively large and as high as 62.47%, which indicates that the diversity and richness of the flora in the model group have changed, and although Proteobacteria is the dominant phylum, it is mainly composed of some harmful flora; compared with the model group, the proportion of Proteobacteria in the two treatment groups is reduced, and the proportions of Firmicutes and Bacteroidota are also increased accordingly, and more notably, the proportions of phylum and flora richness in the medium concentration treatment group (200 mg / kg) are closer to those in the blank group than in the low concentration treatment group (100 mg / kg), which indicates that the medium concentration treatment group (200 mg / kg) has a better treatment effect.

[0075] Figure 6For the classification at the genus level, similar to the analysis of phylum, in the classification at the genus level, it can be seen that in the blank group (Control), benign genera such as Muribaculaceae-unclassified, Akkermansia, Ligilactobacillus, etc. accounted for 33.0%, 21.23%, and 6.11%, respectively, while harmful genera such as Desulfovibrio accounted for a relatively small proportion of 4.33%. Compared with the blank group, the model group (Model) had fewer of these benign genera, and the proportion of inferior genera such as Brevundimonas, Vibrio, and Desulfovibrio was relatively high in the model group. Desulfovibrio in the model group accounted for 10.19%, which was 2.35 times that of the blank group. From the two treatment groups (low concentration treatment group (100 mg / kg), medium concentration treatment group (200 mg / kg)), it can be seen that some benign genera increased significantly, and harmful genera such as Desulfovibrio decreased significantly. The medium concentration treatment group (200 mg / kg) was very similar to the blank group in the classification at the genus level, which also indicated that the treatment groups (low concentration treatment group (100 mg / kg), medium concentration treatment group (200 mg / kg)) had the effect of restoring normal flora structure, and the medium concentration treatment group (200 mg / kg) had a better effect of Magnolia flower crude extract.

[0076] Figure 7 For the principal component analysis chart of the intestinal flora Beta diversity of each group of mice (blank group, model group, low concentration treatment group, medium concentration treatment group), the closer the distance of the sample species composition in the PCA chart, the more similar they are. The similarity within biological replicates and whether the difference between groups is significant can be visually observed through PCA analysis.

[0077] From Figure 7 It can be seen that the distance between the model group and the low concentration treatment group (100 mg / kg) and the normal blank group is far, which indicates that there is a large difference in the microbial composition structure of the three groups. Compared with the blank group and the medium concentration treatment group (200 mg / kg), the confidence ellipse area of the two is relatively close and has an intersection, which indicates that the microbial composition structure of the intestinal flora of the two groups is very similar. This result also indicates that the treatment effect of the medium concentration treatment group (200 mg / kg) is better.

[0078] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. The application of a crude extract of Magnolia officinalis flower in the preparation of drugs for treating or improving spleen deficiency and intestinal flora imbalance, characterized in that, The preparation method of the crude extract of Magnolia officinalis flower includes the following steps: Step 1: Dry, crush, and sieve the fresh Magnolia officinalis flowers to obtain Magnolia officinalis pollen; Step 2: Weigh the required amount of Magnolia officinalis pollen and perform supercritical CO2 extraction for 1-3 hours under the following conditions: extraction temperature 40-80℃, extraction pressure 10-30 MPa, entrainer concentration 40%-80%, and entrainer flow rate 0.5-2.5 ml / min to obtain crude Magnolia officinalis extract; the entrainer is ethanol. Step 3: Rotary evaporate the crude extract of Magnolia officinalis flower to dryness, leaving the solid part as the crude extract of Magnolia officinalis flower; Drugs for treating or improving spleen deficiency and weakness and intestinal flora imbalance were prepared using the crude extract of Magnolia officinalis flower as the main component. The gut microbiota dysbiosis includes the overuse of antibiotics leading to the dominance of Proteobacteria as the dominant bacterial group in the gut. The antibiotics include any one or more of ceftriaxone sodium and lincomycin hydrochloride.

2. The application according to claim 1, characterized in that, In step one, the screening is performed using a 20-mesh sieve.

3. The application according to claim 1, characterized in that, In step three, the rotary evaporation temperature is 60±3℃.

4. The application according to claim 1, characterized in that: The medicinal dosage of the crude extract of Magnolia officinalis flower is 100–300 mg / kg.

5. The application according to claim 4, characterized in that: The medicinal dosage of the crude extract of Magnolia officinalis flower is 200 mg / kg.

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