Application of different processed products of cortex phellodendri chinensis in preparation of antidepressant drugs

Abstract

The invention belongs to the technical field of traditional Chinese medicine and chemistry, and particularly relates to related preparation and analysis methods of different processed products of cortex phellodendri chinensis and application of the cortex phellodendri chinensis in preparation of antidepressant drugs. Research finds that main chemical components in different processed products of cortex phellodendri chinensis, including crude cortex phellodendri chinensis, cortex phellodendri chinensis processed with honey, cortex phellodendri chinensis processed with wine and cortex phellodendri chinensis processed with salt have different intimacy degrees with depression behavioral indexes, and the main chemical components jointly act to generate an anti-depression effect. The invention provides a theoretical basis and a new idea for making cortex phellodendri chinensis become an antidepressant drug.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine and chemistry, and particularly relates to preparation and analysis methods of different processed products of Phellodendron chinense and applications thereof in the preparation of antidepressant drugs. Background Art

[0002] At present, Western medicine has problems such as side effects and dependence in the treatment of depression, while Chinese medicine makes up for this defect with its safety and high efficiency, and Guan Huangbo is an indispensable part of this type of Chinese medicine. Although Guan Huangbo is mentioned in some Chinese medicine compound prescriptions for related diseases, there is a lack of in-depth research on the antidepressant effect of its different preparations and the correlation between chemical components and antidepressant effects. The present invention aims to fill this gap. Summary of the invention

[0003] To solve the above problems, the present invention provides preparation and analysis methods of different processed products of Phellodendron chinense and their application in the preparation of antidepressant drugs.

[0004] The present invention adopts the following technical solution:

[0005] Application of different processed products of Phellodendron chinense in the preparation of antidepressant drugs.

[0006] Furthermore, in the above-mentioned application, the different processed products of Guan Huangbo include raw Guan Huangbo, honey Huangbo, wine Huangbo and salt Huangbo.

[0007] Furthermore, in the above application, the different processed products of Phellodendron chinense are wine Phellodendron chinense or salt Phellodendron chinense.

[0008] Furthermore, in the above-mentioned application, the pretreatment method of different processed products of Phellodendron chinense includes the following steps: taking different processed products of Phellodendron chinense, drying them, grinding them into fine powder and sieving them, using ethanol solution as solvent, extracting them by ultrasonic extraction method, extracting them three times continuously under the same conditions, filtering them after the extraction is completed, combining the three filtrates, concentrating them under reduced pressure in a rotary evaporator, and evaporating them to extract.

[0009] Furthermore, in the above application, the volume concentration of the ethanol solution is 80%.

[0010] Furthermore, in the above application, the conditions for the ultrasonic extraction method are: ultrasonic power is 100W, temperature is controlled at 45°C, and the treatment is 30 minutes.

[0011] The present invention has the following beneficial effects:

[0012] 1. The present invention provides a method for analyzing the antidepressant correlation of a drug, and obtains some alkaloid components with strong antidepressant correlation.

[0013] 2. The present invention provides a possible theoretical basis and new ideas for Phellodendron chinense to become an antidepressant drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the total ion current graph of different processed products of Guan Huangbo in positive ion mode, among which A is raw Guan Huangbo, B is honey Huangbo, C is wine Huangbo, and D is salt Huangbo.

[0015] Figure 2 This is the PCA score chart of different processed products of Guanhuangbo (left), and the orthogonal partial least squares discriminant analysis and permutation test chart (right), among which, A and a are between the groups of raw Guanhuangbo, honeyed Huangbo, wine-made Huangbo and salted Huangbo, B and b are between the groups of raw Guanhuangbo and honeyed Huangbo, C and c are between the groups of raw Guanhuangbo and wine-made Huangbo, and D and d are between the groups of raw Guanhuangbo and salted Huangbo.

[0016] Figure 3 This is a cluster diagram of the chemical components of different processed products of Phellodendron chinense.

[0017] Figure 4 The figure is the weight change of mice in each group at different time points.

[0018] Figure 5 This is a graph showing the changes in sucrose preference of each group of mice at different time points.

[0019] Figure 6 These are the movement trajectory diagrams of mice in each group in the open field test, among which A is the normal blank control group, B is the depression model group, C is the positive control group, D is the raw Phellodendron chinense group, E is the honey Phellodendron chinense group, F is the wine Phellodendron chinense group, and G is the salt Phellodendron chinense group.

[0020] Figure 7 Figure 3 is a graph of the immobility time of each group of mice in the forced swimming test (A) and the tail suspension test (B).

[0021] Figure 8 This is a correlation analysis chart between the main components of different processed products of Phellodendron chinense and depression. DETAILED DESCRIPTION

[0022] The technical scheme and technical effects of the present invention are described in detail below in conjunction with specific embodiments and drawings. Experimental methods without specifying specific conditions are usually based on conventional conditions, such as the conditions described in textbooks and experimental guides, or the conditions recommended by manufacturers, which are well known or easily known to ordinary technicians in the field. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0023] Example 1

[0024] The present invention adopts ultrasonic extraction method to prepare test solution and make animal model to conduct behavioral experiment, and then analyze the changes of chemical components in the prepared product to analyze the anti-depression correlation. The specific steps are as follows:

[0025] (I) Preparation and component analysis of Phellodendron chinense test solution

[0026] 1) Preparation of Phellodendron amurense test solution

[0027] 1.1) Take the raw material of Phellodendron chinense, remove impurities, spray with clean water, moisten thoroughly, cut into shreds, and dry to obtain the raw product of Phellodendron chinense.

[0028] 1.2) The preparation of honey Phellodendron amurense, wine Phellodendron amurense and salt Phellodendron amurense is based on the raw product of Guan Phellodendron amurense and is processed according to their specific processing techniques.

[0029] 1.3) Weigh 1.0000g of each processed product of Phellodendron chinense powder accurately, use 10mL of 80% ethanol solution as solvent, place in stoppered conical flasks, extract by ultrasonic extraction, ultrasonic power 100W, temperature 45℃, treatment for 30min, extract three times continuously under the same conditions, filter after extraction, combine three filtrates. Centrifuge at 12000r / min for 15min, take the supernatant and filter through 0.22μm filter membrane, store at low temperature, and obtain the test solution of different processed products of Phellodendron chinense for testing.

[0030] 2) The chemical compositions of different processed products of Guanhuangbai were analyzed by ultra-performance liquid chromatography-mass spectrometry. In order to further clarify the differences between the processed products and raw products, partial least squares discriminant analysis (OPLS-DA) modeling was used to analyze the difference components between the two groups.

[0031] 2.1) Chromatographic conditions:

[0032] A Thermo Hyperil Gold C18 column (100×2.1mm, 1.9μm) was used, with 0.1% formic acid solution (A) and acetonitrile (B) as the mobile phase gradient elution. The specific gradient settings were as follows: 0-1min, 2% B; 1-4min, 2%-20% B; 4-8min, 20%-55% B; 8-11min, 55%-85% B; 11-13min, 85% B; 13-13.1min, 85%-2% B; 13.1-15min, 2% B, flow rate 0.35mL / min. The injection volume was 3μL, and the column temperature was 40℃.

[0033] 2.2) Mass spectrometry conditions:

[0034] In order to qualitatively analyze the chemical components of different processed products of Phellodendron chinense, full MS-dd MS2 scanning was performed in positive and negative modes with a mass number acquisition range of m / z 80-1200. The MS resolution was 70000 FWHM, the spray voltage was 3.5 KV, the sheath gas flow rate was 40 mL / min, the auxiliary gas flow rate was 10 mL / min, and the capillary and auxiliary gas heater temperatures were 320 °C; the ddMS2 resolution was 70000 FWHM, the collision energy (NCE) was 20 / 40 / 60 V, and the separation window was 1.2 m / z.

[0035] 2.3) Compound Discoverer 3.3 software (Thermo Fisher Scientific Waltham, USA) UPLC-MS / MS raw data were used for database matching and chemical composition prediction to preliminarily identify the compounds. Xcalibur software (Thermo Fisher Scientific Waltham, USA) was then used to extract the characteristic peaks, molecular ion peaks, and fragment ion information of the compounds to be identified from the data, and the compounds were inferred based on the mass spectrometry fragmentation pathways of the compounds in combination with references. In addition, Compound Discoverer 3.3 and Mz Cloud software were used to calculate the peak area of ​​each substance according to the normalization method to obtain the relative content of each substance. The processed data matrix was imported into SIMCA-14.1 software for multivariate statistical analysis, and PLS-DA and OPLS-DA model analysis were established.

[0036] (II) Constructing animal depression models for behavioral experiments

[0037] 1) The mice were kept in an animal room at a temperature of (23±2)℃ and a humidity of 50%-60%, with a 12-h light-dark cycle every day, and were adapted for 1 week. During the adaptation period, the mice were allowed to take feed and clean drinking water on their own. All animal experiments were conducted in accordance with international animal ethics requirements.

[0038] 2) After the adaptation period, the experimental mice were divided into 7 groups (8 mice in each group) according to the random number table method, namely normal blank control group, depression model group, positive control group (fluoxetine, 2 mg / kg), raw Phellodendron chinense group, honey Phellodendron chinense group, wine Phellodendron chinense group, and salt Phellodendron chinense group.

[0039] 3) Solitary housing combined with chronic unpredictable stress (CUMS) was used to establish a depression model in experimental animals. The first day after the adaptation period of mice was taken as the first day of modeling. Except for the normal blank control group (n=8), the other 6 groups of mice (n=48) were housed in single cages to establish a depression model. During the modeling period, random selection was performed every day.

[0040] 4) The mice were stimulated with two of the unpredictable stress types (Table 1). The same type of stress could not appear consecutively, and the modeling period was 28 days. The specific stress treatment arrangements are as follows, Day 1: ①⑧; Day 2: ②⑨; Day3: ④⑥; Day 4: ③⑧; Day 5: ⑤⑩; Day 6: ②⑦; Day 7: ⑥⑨; Day 8: ①④; Day 9: ⑧③; Day Day 10: ①④; Day 11: ⑦⑤; Day 12: ⑥②; Day 13: ⑩⑧; Day 14: ⑨①; Day 15: ③⑦; Day 16: ⑤④; Day 17: ②⑩; Day 18: ⑧④; Day 19: ①⑥; Day 20: ⑨③; Day 21: ⑦⑤; Day 22: ⑩②; Day 23: ③①; Day 24: ④⑥; Day 25: ⑩⑧; Day Day 26: ②⑨; Day 27: ⑤⑦; Day 28: ①④.

[0041] Table 1 Types of unpredictable stress

[0042]

[0043] 5) Take a sufficient amount of the powder of different processed products of Phellodendron chinense after drying, grind it into fine powder, add 80% ethanol, and extract it by ultrasonic at 45°C for 30 minutes with an ultrasonic power of 100W. Repeat the extraction three times, filter, combine the three filtrates, and concentrate under reduced pressure in a rotary evaporator to evaporate to extract. The extracts of different processed products are fully dissolved in physiological saline (NaCl 0.9%) and prepared into 0.2g / mL for use. The positive drug fluoxetine is fully dissolved in physiological saline and set aside.

[0044] 6) The dosage was referred to the "Pharmacological Experimental Methodology", and the weight of mice was converted to 20g / 70kg for adults. The maximum dosage of Phellodendron chinense bark extract for adults is 3.12g / d, which is converted to a medium dose of 400mg / kg / d. The Phellodendron chinense experimental groups (raw product group, honey Phellodendron chinense group, wine Phellodendron chinense group and salt Phellodendron chinense group) were gavaged at 400mg / kg / d. The normal blank control group and the model group (depression) were given an equal amount of normal saline, and the positive control group was given fluoxetine 2mg / kg / d. The mice were gavaged at 9:00 am every day. The dosing cycle was 14d.

[0045] 3. Model verification

[0046] 1) Body weight monitoring: Taking 7 days as the node, weigh and record the body weight of the experimental mice at 7:00 in the morning on the last day of the adaptation period (day 0 of modeling), day 1 of modeling, day 14 of modeling, day 21 of modeling, day 28 of modeling, day 7 of drug administration, and day 14 of drug administration.

[0047] 2) Sucrose consumption experiment: The sucrose preference of mice was measured on the last day of the adaptation period, i.e., Day 0 of modeling (Day 0), Day 1 of modeling (Day 1), Day 14 of modeling (Day 14), Day 21 of modeling (Day 21), Day 28 of modeling (Day 28), Day 7 of drug administration (Day 35), and Day 14 of drug administration (Day 42). A water bottle containing 1% sucrose solution and a water bottle containing regular drinking water were placed in the cage of each mouse at the same time. After 6 hours, the two water bottles were replaced to avoid unilateral preference of mice. After another 6 hours, all food and water bottles in the cage were removed, and the mice were deprived of food and water for 12 hours. On the second day, the sucrose consumption experiment was formally carried out. A bottle containing 1% sucrose solution and a bottle containing regular drinking water were placed in each cage at the same time. The bottles were replaced after 6 hours, and the experiment was ended after another 6 hours. The liquid remaining in the two bottles was recorded, and the consumption volume of the two liquids for each mouse was calculated. The sucrose preference SP (%) of the mouse was calculated according to the following formula. The formula is as follows:

[0048]

[0049] Where: V1—volume of sucrose solution consumed, mL; V2—volume of regular water consumed, mL.

[0050] 3) Behavioral experiments: After the dosing period, the 7 groups of mice in the antidepressant experiment were subjected to open field test (OFT), forced swimming test (FST), and tail suspension test (TST) in turn. Because this experiment involves a large number of mice and cannot be completed at the same time, in order to ensure the quality of the experiment, only one behavioral experiment was conducted a day. All animal behavioral experiments must be conducted in a place without external interference to avoid environmental impact on the accuracy and reliability of the experimental test results. After each mouse experiment, wipe the feces, urine, etc. left by the test mice in the experimental device with a paper towel, and use 75% alcohol to spray the device to remove odors to prevent excrement from affecting the test results of subsequent mice. After the experimental device is clean and odorless, the next animal will be experimented.

[0051] (IV) Correlation analysis

[0052] Origin 2021 software was used to perform Correlation Plot analysis on the antidepressant effect indicators and main chemical components of different processed products of Phellodendron chinense. The relative content of the main chemical components in different processed products of Phellodendron chinense was set as the independent variable (X); the behavioral indicators, pathological scores, and neurobiochemical indicators in the antidepressant experiment were all dependent variables (Y). Data statistics were performed separately to obtain the Pearson correlation coefficient, which was visualized using a bubble chart.

[0053] Figure 1This is the total ion chromatogram of Phellodendron chinense in different positive ion modes. Under the optimized UPLC-MS / MS conditions, the main components in the Phellodendron chinense sample were well separated and detected within 12 minutes. The collected results were compared with the self-built library components and other literature, and multiple components of different processed Phellodendron chinense were identified. In the positive ion mode, a total of 26 components were identified through comparative analysis of retention time, molecular weight, MS / MS and related references, including 22 alkaloids, 1 organic acid, 2 triterpenoids and 1 unknown compound. 21, 22, 24 and 23 compounds were identified in raw Phellodendron chinense, honey Phellodendron chinense, wine Phellodendron chinense and salt Phellodendron chinense, respectively.

[0054] Figure 2 This is the PCA score chart, orthogonal partial least squares discriminant analysis and permutation test of different processed products of Guan Huangbo. Figure 3 This is a cluster diagram of the chemical components of different processed products of Guan Huangbai. The principal component analysis (PCA) was used to analyze the differences between the raw Guan Huangbai, honey Huangbai, wine Huangbai and salt Huangbai. The results are as follows Figure 2 As shown in (A), the PCA score diagram realizes the difference and effective distinction between the groups of samples. Figure 3 It can be seen that the clustering within the group is good, and all samples are within the 95% confidence interval, indicating that the chemical composition of the processed Guan Huangbai and the raw product are significantly different. In order to further clarify the differences between the processed products and the raw products, the partial least squares discriminant analysis (OPLS-DA) modeling was used to analyze the difference components between the two groups. Figure 2 (BD). Both groups were randomly permuted 200 times for model permutation test. Figure 2 (ad), R2Y represents the interpretability of the model, and Q2 represents the predictability of the model, so as to evaluate the reliability of the model and prevent the overfitting of the model. The R2Y and Q2 of raw and honey cork were 99% and 97% respectively; the R2Y and Q2 of raw and wine cork were 95% and 99% respectively; the R2Y and Q2 of raw and salt cork were 98% and 96% respectively. The model fitting parameter is close to 1, indicating that the model established in this study has good reliability and can well explain the differences between the two groups of samples.

[0055] Figure 4It is the weight change of mice at different time points. Except for the normal group, the mice in other groups gained weight slowly after chronic unpredictable stress. This shows that the depression model established by chronic unpredictable stress combined with the solitary foster home significantly reduced the weight gain rate of mice, and the depression model was successfully established. After 14 days of administration, compared with the depression model group, the weight of the positive control group (fluoxetine) and the Guanhuangbo treatment group both recovered significantly, and the weight gain rate increased. These results mean that different preparations of Guanhuangbo can alleviate the slow weight gain caused by depression. Among them, the weight of mice treated with wine Huangbo and salt Huangbo recovered better and was similar to that of the positive control group.

[0056] Figure 5 is the sucrose preference of mice at different time points. When mice are in a depressed state, their sucrose preference will be significantly reduced. Figure 5 As shown in the figure, there was no significant difference in the sucrose preference of mice in each group on day 0 (p>0.05). After chronic unpredictable stress, except for the normal group, the sucrose preference of mice in the other groups decreased significantly (p<0.001), indicating that chronic unpredictable stress combined with the depression model established by solitary care had a significant effect on the anhedonia of mice, and the depression model was successfully established. After 14 days of administration, the sucrose preference of fluoxetine and Guan Huangbo groups increased compared with the model group (p<0.001). The raw group and honey Huangbo group were significantly lower than the normal group (p<0.05, p<0.001). There was no significant difference between wine Huangbo and salt Huangbo and the normal group (p>0.05). The results showed that different preparations of Guan Huangbo can effectively improve the anhedonia caused by depression in mice, and wine Huangbo and salt Huangbo can restore depressed mice to normal.

[0057] Figure 6 is the movement trajectory of the mouse in the open field test. In OFT, the behavior and movement trajectory of the mouse are recorded by the camera. Figure 6 As shown. Detailed data are shown in Table 2. The total distance of mice in the depression model group was less than that in the normal group (p<0.001). After administration of positive drugs fluoxetine and Guan Huangbo, the total distance of mice's movement increased, which was close to that of the normal group (p>0.05). This shows that depression can reduce the autonomous activity of mice, and positive drugs fluoxetine and different preparations of Guan Huangbo can significantly improve the movement behavior of mice. Chronic unpredictable stress can reduce the time mice stay in the central area and the time they comb. Compared with the depression model group, the time mice stay in the central area and the time they comb in the fluoxetine and Guan Huangbo groups increased significantly (p<0.05, p<0.01, p<0.001). The results show that different preparations of Guan Huangbo can effectively alleviate the degree of depression in mice. Among them, the effects of wine Huangbo and salt Huangbo are close to or better than fluoxetine, which means that they may have a good antidepressant effect.

[0058] Table 2 Effects of different processed products of Phellodendron chinense on the open field test (OFT) of mice (x±s, n=8)

[0059]

[0060] Figure 7 is the immobility time of mice in the forced swimming test (A) and tail suspension test (B). Figure 7 As shown in (A), the immobility time of mice in the depression model group in FST was significantly longer than that of mice in the normal group (p<0.001); after treatment with positive drugs fluoxetine and Guan Huangbo, the immobility time of mice was shorter than that of the model group (p<0.001). In addition, the immobility time of mice in the wine Huangbo and salt Huangbo groups was shorter, similar to that of the normal group (p>0.05). The results showed that different preparations of Guan Huangbo reduced the despair behavior of mice in FST and had the potential to relieve depression. At the same time, wine Huangbo and salt Huangbo could better restore the health of mice. In TST, Figure 7 As shown in (B), the immobility time of mice in the depression model group in TST was significantly longer than that of mice in the normal group (p<0.001); the immobility time of mice treated with positive drugs fluoxetine and Guan Huangbai was shorter than that of the model group (p<0.001); the immobility time of Jiu Huangbai and Yan Huangbai was shorter, similar to that of the normal group (p>0.05). The results showed that different preparations of Guan Huangbai reduced the despair behavior of mice in TST, and had the potential to relieve depression, and Jiu Huangbai and Yan Huangbai had better therapeutic effects.

[0061] Figure 8 This is a correlation analysis chart of the main components of different processed products of Phellodendron chinense and depression. Figure 8 ) The close correlation between the main components of different processed products of Phellodendron chinense and the antidepressant effect of mice was analyzed. In the depressive behavioral test, each main component had different correlation coefficients with the behavioral tests in OFT, FST, and TST. Among them, 8 components were strongly correlated with the depressive behavioral indicators, and the correlation was in the order of daurine>magnolia pine>berberine>oblongine>tetrahydrojatrorrhizine>codonine>γ-truncatine>Phellodendron lactone. Some other components were strongly correlated with some behavioral indicators, such as N-methylhordenine, palmatine, N-methyltetrahydroafricanine, N-methyltetrahydropalmatine, protopine, chlorogenic acid, berberine, and N-methylrutine. The results showed that the main components of different processed products of Phellodendron chinense had different degrees of closeness with the depressive behavioral indicators, and they worked together to produce antidepressant effects. Overall, the strongly correlated components had a greater impact on the antidepressant effect, and it was speculated that they might be the potential active components of the antidepressant effect.

Claims

1. Application of different processed products of Phellodendron chinense in the preparation of antidepressant drugs.

2. The use according to claim 1, characterized in that: The different processed products of Guan Huangbo include raw Guan Huangbo, honey Huangbo, wine Huangbo and salt Huangbo.

3. The use according to claim 2, characterized in that: The different processed products of Phellodendron chinense are wine Phellodendron chinense or salt Phellodendron chinense.

4. The use according to claim 1, characterized in that: The pretreatment method of different processed products of Phellodendron chinense comprises the following steps: taking different processed products of Phellodendron chinense, drying them, grinding them into fine powder and sieving them, using ethanol solution as solvent, extracting them by ultrasonic extraction method, extracting them three times in succession under the same conditions, filtering them after the extraction is completed, combining the three filtrates, concentrating them under reduced pressure in a rotary evaporator, and evaporating them to extract.

5. The use according to claim 4, characterized in that: The volume concentration of the ethanol solution is 80%.

6. The use according to claim 4, characterized in that: The ultrasonic extraction method is carried out under the following conditions: ultrasonic power is 100W, temperature is controlled at 45°C, and treatment is carried out for 30 minutes.

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