A pharmaceutical composition for treating chronic prostatitis and a preparation method thereof

Abstract

The present application provides the use of the volatile oil of Magnolia biondii in the preparation of a drug for chronic prostatitis. The present application also provides a pharmaceutical composition for treating chronic prostatitis, which comprises the volatile oil of Magnolia biondii and the volatile oil of Lavandula angustifolia; the weight ratio of the volatile oil of Magnolia biondii to the volatile oil of Lavandula angustifolia is 1.5-2.5:0.5-1.5. The present application also provides the preparation method and use of the extract. The present application further develops the medicinal function of the volatile oil of Magnolia biondii and makes a relevant study on the treatment of chronic prostatitis. The combination of the volatile oil of Lavandula angustifolia and the volatile oil of Magnolia biondii can better treat chronic prostatitis. The volatile oil of Lavandula angustifolia and the volatile oil of Magnolia biondii are prepared into a cream preparation, which is applied to the prostate of a rat with chronic prostatitis, and the drug effect is exerted through transdermal administration, thereby avoiding the side effects of the gastrointestinal tract and the prostate barrier, and achieving a synergistic effect of raw materials, which provides a new idea for the administration route for treating chronic prostatitis.

Description

TECHNICAL FIELD

[0001] The present application relates to a new use of the volatile oil of Magnolia denudata Desch, and a pharmaceutical composition for treating chronic prostatitis and a preparation method thereof. BACKGROUND

[0002] Prostatitis is a male urogenital disease. Type III prostatitis (chronic prostatitis / chronic prostatitis-pain syndrome, CP / CPPS) accounts for about 90-95% of all prostatitis and is the most common type of prostatitis. The pathogenesis of chronic prostatitis is complex, and although extensive research has been conducted on chronic prostatitis, it has not yet been fully elucidated. The pathogenic factors include infection, oxidative stress, abnormal urination, immune response disorder, endocrine disorder, abnormal nerve regulation, and mental and emotional factors. Currently, antibiotics and alpha-receptor blockers are commonly used in clinical practice, but these drugs have poor oral absorption and cannot reach the nerve endings. Therefore, in order to better treat chronic prostatitis, scholars are committed to the development of natural drugs for treating chronic prostatitis with less side effects.

[0003] The main problem in the treatment of prostatitis is the presence of the prostate barrier. The prostate barrier generally refers to some structural and functional characteristics present in the prostate tissue that protect the prostate from harmful substances while also affecting drug penetration and distribution. These include: 1. Blood-Prostate Barrier (BPB), which is a special barrier that limits the exchange of substances between blood and prostate tissue, making it difficult for certain drugs to penetrate into the prostate, thereby affecting drug concentration and efficacy in the prostate. 2. Prostate epithelial barrier: the prostate epithelial cells form a barrier through tight junctions, controlling the entry and exit of substances (Cui D, et al. Research on the function of prostate capillary endothelial cell barrier, [J]. Journal of the Third Military Medical University, 2016, 38(4): 385-389); 3. Prostate stromal barrier: the stromal components of the prostate, such as connective tissue and smooth muscle, may also have some limiting effect on substance exchange. 4. Prostate capsule: a thin fibromuscular tissue that surrounds the surface of the prostate parenchyma, called the prostate capsule, which, together with the prostate sheath, forms the external protective layer of the prostate. 5. Prostate anatomy: the location, adjacent relationship, and internal structure of the prostate, such as acini and ducts, also affect the distribution of substances within the prostate. For natural plants, it is even more difficult to reach the lesion site through the prostate barrier.

[0004] Lavandula angustifolia Mill. (Labiatae) Lavandula plant, evergreen shrubs, multi-year distribution in the global range. Lavender volatile oil is a kind of aromatic herbal extract, mainly containing linalool, linalyl acetate, lavender acetate and camphor and other components, often used in a variety of clinical conditions, such as anxiety, pain and inflammation. Lavender volatile oil has good anti-inflammatory activity. Magnoliae Flos volatile oil is Magnoliae Flos. Magnoliae Flos volatile oil active ingredients have eucalyptol, farnesol, alpha-pinene, beta-pinene, which has good anti-inflammatory, anti-allergic, antioxidant, antibacterial effect. However, there are many different structural types of components in volatile oil, not all are effective ingredients. At present, the related research on the effective components of volatile oil and its dose-effect relationship is less, so the lack of this research limits its application. There is no relevant literature report on the combination of lavender and Magnoliae Flos volatile oil for the treatment of prostatitis. SUMMARY

[0005] The technical scheme of the present application provides a new use of Magnoliae Flos volatile oil, specifically, the use in the preparation of a drug for chronic prostatitis. Another technical scheme of the present application provides a pharmaceutical composition for treating chronic prostatitis, a preparation method and use thereof, and a quality detection method of the pharmaceutical composition.

[0006] The present application provides the use of Magnoliae Flos volatile oil in the preparation of a drug for chronic prostatitis.

[0007] The drug is rectal administration or direct local injection into the prostate.

[0008] The present application provides a pharmaceutical composition for treating chronic prostatitis, which comprises Magnoliae Flos volatile oil and lavender volatile oil; the weight ratio is 1.5-2.5 parts of Magnoliae Flos volatile oil and 0.5-1.5 parts of lavender volatile oil.

[0009] The Magnoliae Flos volatile oil contains 1%-60% w / w of eucalyptol and 1%-30% w / w of farnesol; the lavender volatile oil contains 5%-55% w / w of linalool and 3%-40% w / w of linalyl acetate.

[0010] The weight ratio of Magnoliae Flos volatile oil to lavender volatile oil is:

[0011] 2 parts of Magnoliae Flos volatile oil and 1 part of lavender volatile oil.

[0012] The pharmaceutical composition is prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to the active ingredients as raw materials to prepare the commonly used pharmaceutical preparations; the preparation is rectal administration or direct local injection into the prostate.

[0013] The rectal administration preparation includes suppositories, enemas, foams, gels, creams.

[0014] The cream is an O / W type cream, which contains the following raw materials and bases by weight per 100g:

[0015] Liquid paraffin 6g, octadecanol 7.5g, white vaseline 10g, lily of the valley: lavender volatile oil (2:1) 2g, ethyl paraben 0.1g, sodium lauryl sulfate 1g, glycerol 5g.

[0016] The application provides a method for preparing the pharmaceutical composition for treating chronic prostatitis, and the emulsion comprises the following steps:

[0017] a. weighing each weight-proportioned raw material; wherein the oil phase is liquid paraffin and white vaseline, and the water phase is octadecanol, glycerol and water;

[0018] b. heating the oil phase and the water phase to 80℃ respectively, fully melting the oil phase, and then adding volatile oil;

[0019] c. continuously stirring the oil phase and the water phase respectively, so as to mix them uniformly; adding ethyl paraben and sodium lauryl sulfate into the water phase;

[0020] d. when the temperatures of the two phases are the same, slowly pouring the water phase into the oil phase, and continuously stirring in the same direction; firstly increasing the speed, and then gradually decreasing the speed until emulsification is completed; after stopping heating, continuously stirring until room temperature, and until the cream is formed.

[0021] The application provides the use of the pharmaceutical composition in the preparation of a medicine for chronic prostatitis.

[0022] The application provides a detection method of the pharmaceutical composition for treating chronic prostatitis, which adopts a GC-MS method for detection, and comprises the following steps:

[0023] a. sample pretreatment: precisely taking 100mL lily of the valley volatile oil and 100mL lavender volatile oil into two different 10mL brown volumetric flasks respectively, adding n-hexane to constant volume, mixing, adding anhydrous sodium sulfate to remove water, passing through a 0.22mm filter membrane, and then dropping into a sample bottle for standby;

[0024] b. the GC-MS condition is as follows:

[0025] The gas chromatography conditions of the volatile oil of lonicera japonica are as follows: Agilent HP-5ms (30m*250um*0.25um) capillary column, carrier gas He, no split, sample amount 3.0ul, flow rate 1ml / min; initial temperature 55 DEG C, 8 DEG C / min to 80 DEG C, 6 DEG C / min to 160 DEG C (holding time 2 min), 8 DEG C / min to 200 DEG C, 3 DEG C / min to 250 DEG C (holding time 2 min); mass spectrometry conditions are as follows: ionization source EI ion source temperature 230 DEG C, ionization energy 70 eV, transfer line temperature 280 DEG C, ion source temperature 230 DEG C, quadrupole temperature 150 DEG C, solvent delay time 3 min, scan mass scan range 28-555 amu;

[0026] The gas chromatography conditions of the volatile oil of lavender are as follows: Agilent HP-5ms (30m*250um*0.25um) capillary column, carrier gas He, no split, sample amount 1.0ul, flow rate 1ml / min; initial temperature 40 DEG C (holding time 1min), 5 DEG C / min to 90 DEG C (holding time 1min), 10 DEG C / min to 260 DEG C (holding time 6min); mass spectrometry conditions are as follows: ionization source EI ion source temperature 230 DEG C, ionization energy 70 eV, transfer line temperature 280 DEG C, ion source temperature 230 DEG C, quadrupole temperature 150 DEG C, solvent delay time 5 min, scan mass scan range 30-400 amu.

[0027] According to the TCM differentiation angle, the main pathogenesis of chronic prostatitis is qi stagnation and blood stasis, liver and kidney yin deficiency, and dampness-heat and stasis. The volatile oil of lonicera japonica and lavender is used in combination, lonicera japonica tastes pungent and is warm, and belongs to lung and stomach channels; lavender tastes pungent and is cool, and belongs to spleen and lung channels; and the volatile oil has aromatic and pungent and warm properties, can regulate the human body qi, and the two can be used together to achieve the effects of soothing liver and regulating qi, warming meridians, removing stasis and relieving pain, and can greatly reduce the pain of chronic prostatitis. Especially when lonicera japonica:lavender = 2:1, it has a significant synergistic effect.

[0028] The application further develops the medicinal function of the volatile oil of lonicera japonica, and carries out relevant research on the treatment of chronic prostatitis. The volatile oil of lavender and the volatile oil of lonicera japonica are combined, which can better treat chronic prostatitis. The volatile oil of lavender and the volatile oil of lonicera japonica are prepared into a cream preparation, which is used for external application on the prostate of chronic prostatitis rats, and plays a pharmaceutical effect through transdermal administration, avoids the gastrointestinal side effects and the prostate barrier, and realizes the synergistic effect of raw materials, thereby providing a new idea for the administration route of treating chronic prostatitis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The single factor test result graph is shown in the following figure.

[0030] Figure 2 Response surface experiment result graph;

[0031] Figure 3 Prostate index and kit result graph (wherein, (A) prostate index result graph, (B) TNF-alpha result graph, (C) IL-1beta result graph, (D) IL-6 result graph);

[0032] Figure 4 Prostate index and kit result graph of cream and volatile oil group (wherein, (A) cream prostate index result graph, (B) cream TNF-alpha result graph, (C) cream IL-1beta result graph, (D) cream IL-6 result graph);

[0033] Figure 5 HE staining result graph;

[0034] Figure 6 HE staining result graph of linaloe lavender volatile oil compatible cream;

[0035] Figure 7 Immunohistochemical result graph;

[0036] Figure 8 Immunohistochemical result graph (wherein, (A) COX2 result graph; (B) p-AKT result graph; (C) p-p65 result graph; (D) p-PI3K result graph);

[0037] Figure 9 Immunohistochemical result graph of linaloe lavender volatile oil compatible cream;

[0038] Figure 10 Immunohistochemical result of cream (wherein, (A) cream COX2 result graph, (B) cream p-AKT result graph, (C) cream p-p65 result graph, (D) cream p-PI3K result graph). DETAILED DESCRIPTION

[0039] The lavender volatile oil used in the application is purchased from Xinjiang Yiparkhan Spice Co., Ltd.; and the linaloe volatile oil is purchased from Ningbo Fragrance Wharf Biotechnology Co., Ltd.

[0040] Example 1 Preparation of the cream of the application

[0041] Liquid paraffin 6g, octadecanol 7.5g, white vaseline 10g, linaloe: lavender (2:1) volatile oil 2g, nipagin ethyl ester 0.1g, sodium dodecyl sulfate 1g, glycerol 5g, and water is added to 100g.

[0042] The specific preparation process is as follows:

[0043] 1) Take liquid paraffin and white vaseline, heat to 80℃ to melt, mix evenly to get oil phase; take octadecanol, glycerol and water, heat to 80℃, mix evenly to get water phase;

[0044] 2) Take the oil phase obtained in step 1), add volatile oil, mix evenly. Take the water phase obtained in step 1), then add nipagin ethyl and sodium lauryl sulfate, mix evenly. Finally, slowly add the water phase to the oil phase while stirring until it cools to room temperature.

[0045] Example 2 Screening test of excipient matrix of cream of the application

[0046] Cream is a semi-solid preparation formed by mixing and dispersing drugs in a matrix of emulsion type. Cream preparation can avoid liver first-pass effect and gastrointestinal irritation, and directly act on the target site to play a therapeutic role. The use of cream technology to prepare a cream of magnolia and lavender volatile oil can solve the poor experience of using volatile oil.

[0047] 1 Pre-formulation study

[0048] 1.1 W / O cream and O / W cream

[0049] In order to produce a cream of magnolia and lavender volatile oil, a series of pre-study work was carried out. Cream is a product divided into W / O and O / W types, of which W / O has a lower greasy feeling, is very easy to apply, and when the water on the skin surface evaporates, it brings a slow cooling effect; the latter can be mixed with water, has a lighter greasy feeling, is easy to apply and wash, and has a faster drug release speed and skin absorption speed. On the basis of these characteristics, some related literature reports show that, compared with W / O cream, O / W cream has stronger advantages in drug release speed, skin penetration and stability, etc. Therefore, the method of preparing O / W cream was adopted in this experiment.

[0050] 1.2 Matrix of O / W cream

[0051] In the preparation of O / W cream, the amount of water phase is usually greater than that of oil phase, and O / W emulsifier needs to be used. The ideal cream matrix should have stability, uniformity, water absorption, non-irritation, no effect on wound healing, easy to wash off and good drug release performance, etc.

[0052] 1.3 Water phase

[0053] The water phase often uses polyethylene glycol, cellulose derivatives and appropriate polyols, etc. Such as propylene glycol and glycerol can reduce the freezing point of the water phase.

[0054] 1.4 Oil phase

[0055] The oil phase is usually vaseline, liquid paraffin, solid paraffin, lanolin, etc., but the amount needs to be moderate to avoid affecting the absorption of the cream.

[0056] 1.5 Emulsifier

[0057] The emulsifier plays a crucial role in the preparation, stability maintenance, and efficacy of the cream. The cream can choose W / O or O / W emulsifier according to its type, and the selection of emulsifier is mainly based on HLB value. Common emulsifiers include Tween-80, Span-80, and sodium dodecyl sulfate, etc.

[0058] 1.6 Additives

[0059] Since the cream is prone to mold during storage, preservatives and other additives need to be added. Common preservatives include hydroxybenzoate, benzoic acid, sorbic acid, and benzalkonium bromide, among which hydroxybenzoate preservatives are commonly used in creams, usually at a concentration of 0.1%.

[0060] 2 Cream formulation screening

[0061] 2.1 Single factor experiment design

[0062] Using single factor experiment, combined with literature reports, this experiment will take A (Jinyi: lavender (2:1) volatile oil), B (liquid paraffin), C (octadecanol), D (white vaseline), E (glycerol), F (sodium dodecyl sulfate) as the observation factors, each factor takes 5 levels, the single factor experiment level design is shown in Table 1.

[0063] Table 1 Single factor experiment design

[0064]

[0065] 2.2 Investigation index and scoring standard

[0066] This test uses comprehensive scoring method as the index to screen the prescription, the total score is 100 points, and the appearance of the cream (30 points), physical stability (30 points), emulsion droplet size (20 points), and moisture retention (20 points) are investigated respectively.

[0067] Table 2 Investigation index and scoring standard of cream quality

[0068]

[0069] 2.2.1 Investigation of appearance

[0070] The appearance evaluation mainly assesses five indicators of the cream: gloss, uniformity, smoothness, viscosity, and spreadability. Each indicator is scored from 0 to 6 points, with each point representing a grade. The sum of the scores for each indicator is the appearance score of the cream.

[0071] The specific evaluation process is as follows: First, six evaluators visually inspect the gloss of each formulation of the cream and score them, then calculate the average value. Next, similarly, the six evaluators visually inspect the color consistency of each formulation of the cream and score its uniformity, then calculate the average value. Next, the six evaluators visually inspect the cream for smoothness and absence of coarse particles and score it, then calculate the average value. Afterwards, the six evaluators apply a suitable amount of cream to the back of their hands, feel for any greasiness, score it, and then calculate the average value. Finally, the six evaluators apply a suitable amount of cream to the back of their hands, feel for how quickly and evenly it can be spread, score it, and finally calculate the average value as the spreadability score.

[0072] Table 3. Appearance Characteristics Evaluation Indicators and Scoring Standards for Creams

[0073]

[0074] 2.2.2 Physical Stability Assessment

[0075] The physical stability test of creams consists of centrifugation, heat resistance, and low-temperature resistance tests. Each indicator is scored from 0 to 10 points, and the sum of the scores for the three indicators yields the physical stability score.

[0076] The specific evaluation process is as follows: Centrifugation test: Take 1 mL of each prescription sample and place it in a 1.5 mL centrifuge tube. Centrifuge at 3000 rpm for 30 min. Observe the properties of the cream in each centrifuge tube to determine whether oil-water separation occurs. Score on a 1-point scale, with a maximum score of 10 points if no separation occurs. Heat resistance test: Take 15 g of sample from various locations and place it in a 25 mL beaker. Seal with plastic wrap and place in a constant temperature oven at 55 ± 2℃ for 6 hours. Afterward, remove the sample and allow it to cool to room temperature. Observe whether hardening, thinning, roughening, bubbling, or oil-water separation occurs. Each indicator is scored according to the degree of change; if there is no change, 2 points are given. A 1-point scale is used for downgrading the score. For the low-temperature resistance test, take 15g of each prescription sample and place it in a sealed bag. First, pre-freeze it at 4°C for 2 hours, then quickly place it at -20°C. After 24 hours, take it out and let it stand at room temperature to observe whether there is cracking, hardening, thinning, roughening, or oil-water separation. Each indicator is scored out of 2 points. The score is determined according to the degree of change. No change is scored out of 2 points, and large changes are scored out of 0 points. Each level is downgraded by 1 point.

[0077] Table 4. Indicators and Scoring Standards for Physical Stability Evaluation of Creams

[0078]

[0079] 2.2.3 Examination of droplet size and uniformity

[0080] A suitable amount of cream was evenly spread on a glass slide, then covered with a coverslip, and the size and distribution of the droplets were observed under a 100x microscope. Based on the observation results, the scoring criteria were divided into three levels: good, medium, and poor, with 8-10 points being good, 4-7 points being medium, and 0-3 points being poor. Evaluation of droplet size and uniformity showed that high-scoring creams were characterized by smaller particle size and more uniform particle size distribution.

[0081] Table 5. Evaluation Indicators and Scoring Standards for Droplet Size and Uniformity of Creams

[0082]

[0083] 2.2.4 Moisturizing effect assessment

[0084] Apply 2g of the cream onto a glass slide, weigh it precisely, and place it in a 37°C incubator for 8 hours. Afterward, remove it and weigh it precisely in triplicate, and calculate the average value of the cream's water loss rate.

[0085] Moisturizing performance scoring method: Score = 20 - water loss rate × 20. The lower the score, the better the moisturizing performance of the cream.

[0086] 2.3 Analysis of Single-Factor Experiment Results

[0087] Depend on Figure 1 It can be seen that the quality of the Magnolia biondii:Lavender (2:1) volatile oil cream is affected by the amounts of Magnolia biondii:Lavender (2:1) volatile oil, liquid paraffin, stearyl alcohol, white petrolatum, glycerin, and sodium lauryl sulfate. Except for the trend of gradually decreasing amounts of Magnolia biondii:Lavender (2:1) volatile oil, the amounts of other ingredients generally show a relationship of first increasing and then decreasing. Based on the results of the single-factor experiments, the optimal formulation ratio was found to be 2% Magnolia biondii:Lavender (2:1) volatile oil, 6% liquid paraffin, 7.5% stearyl alcohol, 10% white petrolatum, 5% glycerin, and 1% sodium lauryl sulfate, which resulted in the highest cream score. Based on the magnitude of the influence of each factor, response surface methodology was designed using sodium lauryl sulfate, stearyl alcohol, and glycerin as the three factors.

[0088] Table 6 Scoring of Single-Factor Experiments

[0089]

[0090] 2.4 Response Surface Design

[0091] The optimal level of each single factor was determined by single factor experiment results. Box-Behnken Design experiment was designed by using Design-Expert 8.0.6 software. Sodium dodecyl sulfate, octadecanol and glycerol were selected for response surface experiment. The response surface experiment factors and levels are shown in the following table. All experiments were performed in triplicate.

[0092] Table 7 Response surface experiment factors and levels

[0093]

[0094] 2.5 Response surface optimization experiment results

[0095] 2.5.1 Response surface experiment results

[0096] Design-Expert 8.0.6 software was used for response surface experiment design, as shown in the following table.

[0097] Table 8 Response surface design results table

[0098]

[0099] 2.5.2 Establishment of regression model and significance analysis

[0100] According to the results in the above table, the amounts of sodium dodecyl sulfate (A), octadecanol (B) and glycerol (C) were polynomial fitted, and a multiple quadratic regression equation was established:

[0101] Quality score of Magnolia xueca compatibility volatile oil cream =

[0102] Variance analysis of response surface regression model was performed using software, and the variance analysis table is shown in the following table:

[0103] Table 9 Regression model variance analysis

[0104]

[0105] From the above table, it can be seen that the model p -value<0.0001, Lack of Fit p =0.9997>0.05, indicating that the regression model has statistical significance and can describe the relationship between the three factors and the quality score of the cream. The model determination coefficient R 2 =0.9939, the adjusted R 2 =0.9860, also proving the effectiveness of the model. Therefore, the regression equation can be used to predict the changes of the cream score with the three factors.

[0106] By the interaction analysis of the dosage of sodium dodecyl sulfate, octadecanol and glycerol, it is found that the three factors have different effects on the quality of the cream. According to the F value, the largest influencing factor is sodium dodecyl sulfate, followed by glycerol, and the last is octadecanol. The response surface test result graph is shown in Figure 2 .

[0107] 3CONCLUSION

[0108] The study selects the best component dosage by screening the prescription of the Magnolia liliflora lavender volatile oil cream. Then, the three factors that have greater influence on the quality are determined through single factor experiment and response surface experiment, and the preparation process of the cream is optimized. Finally, the best prescription is obtained: liquid paraffin 6g, octadecanol 7.5g, white vaseline 10g, Magnolia liliflora: lavender (2:1) volatile oil 2g, nipagin ethyl ester 0.1g, sodium dodecyl sulfate 1g, glycerol 5g, and water 100g. That is, the Magnolia liliflora lavender volatile oil cream preparation is obtained.

[0109] Example 3 Preparation of the drug suppository of the present application

[0110] 1) Weigh 5.0g of polyethylene glycol 6000, 3.0g of polyethylene glycol 4000 and appropriate amount of water, and place them in an evaporating dish. Heat and melt in a water bath, stir evenly, and prepare 10.0g of water-soluble matrix.

[0111] 2) Precisely weigh 0.15g of Magnolia liliflora: lavender (2:1) volatile oil, and place it in an evaporating dish. Add 0.015g of polysorbate-80 and appropriate amount of potassium sorbate, and then add 1.35g of the prepared molten polyethylene glycol mixed matrix. Heat and melt at 60-65℃ in a water bath, stir evenly, and then quickly fill into a suppository mold that has been coated with a lubricant. Solidify and demold, and then use water-absorbing paper to absorb the water on the surface of the suppository. Thus, the suppository is obtained.

[0112] Example 4 Preparation of the emulsion for injection of the present application

[0113] Preparation method:

[0114] 1) Weigh 100g of Magnolia liliflora: lavender (2:1) volatile oil and 100g of glyceryl tricaprylatemonocaprate for injection, mix them, heat in a water bath to 78℃, add 12g of egg yolk phospholipid for injection, shear to dissolve, and stir evenly to obtain an oil phase.

[0115] 2) Measure 750ml of water for injection, add 22.5g of glycerol, stir to dissolve, and heat to 78℃ to obtain an aqueous phase.

[0116] 3) The oil phase and the water phase are mixed at a temperature of 78℃, emulsified with a shearing emulsifier for 8 minutes (rotation speed 20000 r / min), to obtain a primary emulsion; the primary emulsion is further emulsified with a high-pressure homogenizer (pressure 15000 psi), and the injection water is quantified to 1000 mL, the pH value is adjusted to 8.50 with a sodium hydroxide solution, filtered with a microporous filter membrane, and then filled into plastic or glass bottles, filled with nitrogen, sealed, sterilized with a rotary high-pressure steam sterilization pot at 121℃ for 15 minutes, to obtain an injection emulsion.

[0117] Example 4 Quality detection method of the drug of the present application

[0118] 1 Identification of volatile oil components of lily and lavender

[0119] 1.1 Preparation of lily and lavender volatile oil samples

[0120] 100 mL of lily volatile oil and 100 mL of lavender volatile oil are precisely measured into two different 10 mL brown volumetric flasks, and then diluted with n-hexane, mixed, and then dehydrated by adding anhydrous sodium sulfate, filtered through a 0.22 mm filter membrane, and then dropped into a sample bottle for standby.

[0121] 1.2 GC-MS experimental conditions

[0122] The gas chromatography-mass spectrometry (GC-MS) conditions of the lily volatile oil are as follows: Agilent HP-5ms (30 m x 250 µm x 0.25 µm) capillary column, carrier gas He, no split, sample injection amount 3.0 µL, flow rate 1 mL / min. The initial temperature is 55℃, increased by 8℃ / min to 80℃, increased by 6℃ / min to 160℃ (holding time 2 min), increased by 8℃ / min to 200℃, increased by 3℃ / min to 250℃ (holding time 2 min). The mass spectrometry conditions are as follows: ionization source EI ion source temperature 230℃, ionization energy 70 eV, transfer line temperature 280℃, ion source temperature 230℃, quadrupole rod temperature 150℃, solvent delay time 3 min, scanning mass scanning range 28-555 amu.

[0123] The gas chromatography-mass spectrometry (GC-MS) conditions of the lavender volatile oil are as follows: Agilent HP-5ms (30 m x 250 µm x 0.25 µm) capillary column, carrier gas He, no split, sample injection amount 1.0 µL, flow rate 1 mL / min. The initial temperature is 40℃ (holding time 1 min), increased by 5℃ / min to 90℃ (holding time 1 min), increased by 10℃ / min to 260℃ (holding time 6 min). The mass spectrometry conditions are as follows: ionization source EI ion source temperature 230℃, ionization energy 70 eV, transfer line temperature 280℃, ion source temperature 230℃, quadrupole rod temperature 150℃, solvent delay time 5 min, scanning mass scanning range 30-400 amu.

[0124] 1.3 Data processing

[0125] The data used were processed by data analysis software to screen the content of the components of the volatile oil of Magnolia denudata Desch and Lavandula angustifolia Mill. The NIST standard library was used for retrieval, and the components were screened according to the matching degree and related literature.

[0126] 1.4 Data analysis results

[0127] The analysis identified that the volatile oil of Magnolia denudata Desch contains 21 effective components, and the results are shown in Table 10 below. The analysis identified that the volatile oil of Lavandula angustifolia Mill contains 14 effective components, and the results are shown in Table 11.

[0128] Table 10 Analysis table of volatile oil components of Magnolia denudata Desch

[0129]

[0130] Table 11 Analysis table of volatile oil components of Lavandula angustifolia Mill

[0131]

[0132] The beneficial effects of the present application are demonstrated by the following pharmacodynamic test.

[0133] Test Example 1 Effect of Magnolia denudata Desch and Lavandula angustifolia Mill volatile oil cream preparation on the treatment of chronic prostatitis

[0134] 1 Experimental method

[0135] 1.1 Experimental animals

[0136] 100 male Sprague-Dawley SPF level rats (180 ~ 220 g), certificate number: SCXK (Shaanxi) 2018-001, purchased from the Experimental Animal Center of Xi'an Jiaotong University Medical School, and the rats were raised in the animal room of the Traditional Chinese Medicine Pharmacology Laboratory of Shaanxi University of Chinese Medicine (room temperature 23 ~ 26 ℃, relative humidity 60 % ~ 24 %). During the adaptive feeding period, the rats could freely eat and drink water, and the duration was one week.

[0137] 1.2 Establishment of rat chronic prostatitis model and sampling

[0138] Carrageenan is a chemical extracted from plants, which is widely used to induce chronic prostatitis animal model in rats. Its main role is to increase the synthesis of local prostaglandins and interact with vasoactive peptides and kinins, thereby triggering edema and establishing chronic prostatitis. 100 rats were randomly divided into blank group, model group, celecoxib group, flos magnoliae volatile oil group, lavender volatile oil group, flos magnoliae: lavender (1:1) volatile oil combination group, flos magnoliae: lavender (2:1) volatile oil combination group, flos magnoliae: lavender (3:1) volatile oil combination group, flos magnoliae: lavender (1:2) volatile oil combination group, lavender (2:1) volatile oil cream combination group, each group of 10 rats, for the experiment (in which, the ratio is the mass ratio of flos magnoliae volatile oil and lavender volatile oil). 100 μL of 3% w / v carrageenan was injected into the left and right lateral lobes of the prostate to prepare a stable and individual response consistent rat chronic prostatitis model, and sutured after successful injection. All rats were injected with 0.2 mL of penicillin sodium, for three consecutive days. One week after modeling, the treatment of chronic prostatitis rats was carried out. The celecoxib group was given celecoxib capsules (18 mg·kg -1 ) by gavage; all dosing groups: including volatile oil groups and cream groups were given consistent dosages, i.e. 50 mg·kg -1 of volatile oil and volatile oil cream, for four weeks. After treatment, 2% pentobarbital sodium (40 mg·kg -1 ) was injected for anesthesia, and whole blood was taken from all rats, and after standing and centrifugation, the serum was aliquoted and stored at -20 ℃ for later use; all rats were euthanized, and the prostate tissue was weighed after washing with normal saline for the determination of the prostate index. Part of the prostate tissue was fixed and treated with 4% paraformaldehyde and stored at room temperature; part of the tissue was quickly frozen using liquid nitrogen and transferred to a -80 ℃ refrigerator for subsequent protein detection.

[0139] 1.3 Detection of TNF-α, IL-6 and IL-1β content in rat serum

[0140] The contents of TNF-α, IL-6 and IL-1β in the serum of rats in each group were detected by using ELISA scientific kit and following the method in the kit instruction. Then, the contents of inflammatory factors in the serum of each group were detected by using an enzyme label instrument and a standard curve was drawn.

[0141] 1.4 HE staining to observe the morphological changes of rat prostate tissue cells

[0142] The prostate tissue fixed in 4% paraformaldehyde embedded in paraffin was taken, the tissue was cut into 5 µm thick sections and stained with hematoxylin-eosin (HE), and pathological examination was performed under a 200-fold microscope.

[0143] 1.5 Immunohistochemistry

[0144] After sectioning the prostate tissue, it was soaked twice, followed by hydration using a gradient of ethanol (100%, 95%, 85%, 75%) and double-distilled water. The sections were then subjected to high-pressure antigen retrieval in 0.01 M sodium citrate buffer for 20-30 min, with the addition of 3% H2O2 to block endogenous peroxidase, followed by incubation in a humidified chamber for 10 min. Next, non-immune, normal sheep serum was added to block non-specific antigens, and the sections were incubated in a humidified chamber at 37 °C for 30 min. Primary antibody was then added and the sections were incubated overnight at 4 °C. The samples were then removed and washed three times for 3 minutes each at room temperature. HRP-labeled secondary antibody was added, and the sections were incubated at 37 °C for 60 min. Finally, DAB staining was performed, followed by rinsing with tap water and counterstaining with hematoxylin. Differentiation was then performed using 0.1% hydrochloric acid ethanol, and the staining degree was observed under a microscope. Finally, dehydration, clearing, and mounting were performed.

[0145] 1.6 Statistical Methods

[0146] Data were analyzed using GraphPad Prism 9.0.1 software. Independent samples t-tests were used for comparisons between two groups. Data are expressed as mean ± standard deviation (x ± SD). p < 0.05 indicates statistical significance.

[0147] 2 Results and Analysis

[0148] 2.1 Comparison of prostate index among different groups of rats

[0149] The effects of the drug administration group on chronic prostatitis in rats were preliminarily evaluated using the prostate index. The results are as follows: Figure 3 As shown in Figure A, the bar chart shows that, compared with the control group, the prostate index of rats in the model group was significantly increased ( p < 0.01), compared with the model group, the prostate index of the celecoxib capsule group, magnolia flower volatile oil group, lavender volatile oil group, magnolia flower:lavender (1:1) volatile oil combination group, magnolia flower:lavender (2:1) volatile oil combination group, magnolia flower:lavender (3:1) volatile oil combination group, and magnolia flower:lavender (1:2) volatile oil combination group all decreased, and there were differences in all of them. p < 0.01).

[0150] Compared with other treatment groups, the prostate index of the Magnolia biondii:Lavender (2:1) volatile oil combination group was lower. p < 0.05 and p < 0.01).

[0151] Depend on Figure 4As shown in Figure A, compared with the group using a 2:1 ratio of Magnolia biondii to Lavender (volatile oil) volatile oil in a cream formulation, the prostate index of the group was lower, and there was a difference. p < 0.05).

[0152] 2.2 Serum levels of TNF-α, IL-6, and IL-1β in rats

[0153] Figure 3 (BD) Results showed that, compared with the control group, the serum levels of inflammatory factors TNF-α, IL-6, and IL-1β in the model group were significantly increased, and the differences were statistically significant. p < 0.01). Compared with the model group, the serum levels of TNF-α, IL-6, and IL-1β were decreased in the celecoxib capsule group, magnolia flower volatile oil group, lavender volatile oil group, magnolia flower:lavender (1:1) volatile oil combination group, magnolia flower:lavender (2:1) volatile oil combination group, magnolia flower:lavender (3:1) volatile oil combination group, and magnolia flower:lavender (1:2) volatile oil combination group. p < 0.05 and p < 0.01).

[0154] Compared with other treatment groups, the serum levels of TNF-α, IL-6, and IL-1β were all decreased in the group treated with a volatile oil ratio of magnolia flower and lavender (2:1). p < 0.05 and p < 0.01).

[0155] Depend on Figure 4 As shown in (BD), compared with the group treated with a 2:1 ratio of Magnolia biondii to Lavender volatile oil in a cream, the serum levels of TNF-α, IL-6, and IL-1β were all decreased in the Magnolia biondii: Lavender volatile oil combination group, and there were significant differences. p < 0.05 and p < 0.01).

[0156] Prostate index and reagent kit data are shown in Tables 12, 13 and 14.

[0157] Table 12 Prostate Index and Kit Data Sheet

[0158]

[0159] Note: Compared with the blank group, ## p <0.01; compared with the model group, * p <0.05, ** p <0.01

[0160] Table 132: Prostate index and kit data comparison table of the compatible group and other administration groups

[0161]

[0162] Note: Compared with the Magnolia: Lavender (2: 1) volatile oil compatible group, * p <0.05, ** p <0.01

[0163] Table 14: Prostate index and kit data table of the cream and volatile oil groups

[0164]

[0165] Note: Compared with the Magnolia: Lavender (2: 1) volatile oil compatible group, * p <0.05, ** p <0.01

[0166] 2.3 HE staining results

[0167] The HE staining results are as follows Figure 5 The blank group had normal prostate tissue structure, and the tissue acinus was full of colloid, and no obvious loss was observed. The model group had abnormal structure, and a large number of inflammatory cells were observed in the interstitial tissue, and the inflammatory cells invaded the epithelial cells, resulting in the necrosis of part of the epithelial cells. The Magnolia volatile oil group and the Lavender volatile oil group were slightly improved. The Celecoxib capsule group, the Magnolia: Lavender (1: 1) volatile oil compatible group, the Magnolia: Lavender (2: 1) volatile oil compatible group, the Magnolia: Lavender (3: 1) volatile oil compatible group, and the Magnolia: Lavender (1: 2) volatile oil compatible group were improved to different degrees.

[0168] Figure 6 The results showed that the Magnolia: Lavender (2: 1) volatile oil compatible cream group was improved compared with the Magnolia: Lavender (2: 1) volatile oil compatible group. Among them, the overall structure of the prostate tissue of the Magnolia: Lavender (2: 1) volatile oil compatible cream group was basically normal, and no obvious inflammatory cell infiltration was observed.

[0169] 2.4 Immunohistochemical results

[0170] The study used immunohistochemical experiments to measure the expression levels of COX2, p-AKT, p-p65 and p-PI3K in the rat prostate tissue. According to Figure 7 and Figure 8 The results showed that compared with the blank group, the expression levels of COX2, p-AKT, p-p65 and p-PI3K in the model group were significantly increased (P < 0.05) p< 0.05). Compared with the model group, the expression levels of COX2, p-AKT, p-p65 and p-PI3K in the celecoxib capsule group, the magnolia volatile oil group, the lavender volatile oil group, the magnolia: lavender (1:1) volatile oil combination group, the magnolia: lavender (2:1) volatile oil combination group, the magnolia: lavender (3:1) volatile oil combination group, and the magnolia: lavender (1:2) volatile oil combination group were reduced to different degrees p < 0.05 and p < 0.01).

[0171] The expression levels of COX2, p-AKT, p-p65 and p-PI3K in the prostate of the magnolia: lavender (2:1) volatile oil combination group were lower than those in other drug groups p < 0.05 and p < 0.01).

[0172] Figure 9 and Figure 10 The results showed that the magnolia: lavender (2:1) volatile oil combination cream group reduced the expression of COX2, p-AKT, p-p65 and p-PI3K in the prostate tissue compared with the magnolia: lavender (2:1) volatile oil combination group p < 0.05 and p <0.01). It showed that the magnolia: lavender (2:1) volatile oil combination cream could better reduce the expression of COX2, p-AKT, p-p65 and p-PI3K in the prostate tissue of chronic prostatitis rats.

[0173] The immunohistochemical data is shown in Tables 15, 16 and 17.

[0174] Table 15 Immunohistochemical result data table

[0175]

[0176] Note: compared with the blank group, ## p <0.01; compared with the model group, * p <0.05, ** p <0.01

[0177] Table 16 Comparison of immunohistochemical data between the 2:1 combination group and other drug groups

[0178]

[0179] Note: compared with the magnolia: lavender (2:1) volatile oil combination group, * p <0.05,** p <0.01

[0180] Table 17 Data table of cream immunohistochemical results

[0181]

[0182] Note: Compared with the combination of Magnolia and Lavender (2:1) volatile oil, * p <0.05, ** p <0.01

[0183] 3 Summary and discussion

[0184] A rat model of chronic prostatitis was established by using 3% w / v carrageenan. The results showed that compared with the model group, the prostate index of the lavender volatile oil group, the Magnolia volatile oil group, the combination of Magnolia and Lavender (1:1) volatile oil group, the combination of Magnolia and Lavender (2:1) volatile oil group, the combination of Magnolia and Lavender (3:1) volatile oil group, the combination of Magnolia and Lavender (1:2) volatile oil group, and the combination of Magnolia and Lavender (2:1) volatile oil cream group were smaller, and the secretion of inflammatory factors TNF-α, IL-1β and IL-6 in the serum of rats was also reduced. At the same time, through the HE staining results, it was found that the inflammatory cell infiltration and the number of mast cells in the prostate tissue of the rats in the drug groups were reduced. The key proteins COX2, p-AKT, p-p65 and p-PI3K in the prostate tissue of rats were tested by immunohistochemical experiment, and the results showed that the drug groups could reduce the expression of COX2, p-AKT, p-p65 and p-PI3K in the prostate tissue of rats with chronic prostatitis. The experimental results showed that the combination of Magnolia and Lavender (2:1) volatile oil cream group had the best results and achieved therapeutic effect, which confirmed the efficacy of the combination of Magnolia and Lavender volatile oil cream group.

Claims

1. A pharmaceutical composition for treating chronic prostatitis, characterized by comprising: It is prepared from the magnolia volatile oil and the lavender volatile oil; the weight ratio of the magnolia volatile oil and the lavender volatile oil is: 1.5-2.5 parts of the magnolia volatile oil and 0.5-1.5 parts of the lavender volatile oil; ​ The magnolia volatile oil contains 1%-60% w / w of eucalyptol and 1%-30% w / w of farnesol; the lavender volatile oil contains 5%-55% w / w of linalool and 3%-40% w / w of linalyl acetate.

2. The pharmaceutical composition for treating chronic prostatitis according to claim 1, wherein: The weight ratio of the magnolia volatile oil and the lavender volatile oil is: 2 parts of the magnolia volatile oil and 1 part of the lavender volatile oil.

3. The pharmaceutical composition for treating chronic prostatitis according to claim 1 or 2, characterized by: It is prepared from the active ingredients, and the pharmaceutically acceptable adjuvant or auxiliary ingredients.

4. The pharmaceutical composition for treating chronic prostatitis according to claim 3, wherein: The preparation is rectal administration, intraprostatic direct local injection.

5. The pharmaceutical composition for treating chronic prostatitis according to claim 4, wherein: The rectal administration preparation includes: suppositories, enemas, foams, gels, creams.

6. The pharmaceutical composition for treating chronic prostatitis according to claim 5, wherein: The cream is an O / W cream, which contains the following ingredients and bases in the weight of per 100g: Liquid paraffin 6g, octadecanol 7.5g, white vaseline 10g, magnolia: lavender volatile oil (2:1) 2g, ethyl paraben 0.1g, sodium lauryl sulfate 1g, glycerol 5g.

7. A method of preparing a pharmaceutical composition for treating chronic prostatitis according to claim 6, characterized by: The emulsion includes the following steps: a. weigh the ingredients according to the weight ratio; The oil phase is liquid paraffin and white vaseline, and the water phase is octadecanol, glycerol and water; b. heat the oil phase and the water phase to 80℃ respectively, melt the oil phase completely, and then add the volatile oil; c. continuously stir the oil phase and the water phase respectively to mix them evenly; add ethyl paraben and sodium lauryl sulfate to the water phase; d. when the temperature of the two phases is the same, slowly pour the water phase into the oil phase, and continue to stir in the same direction; first increase the speed, then gradually slow down, until the emulsification is completed; stop heating, continue stirring until room temperature, until the cream is formed.

8. The use of the pharmaceutical composition of any one of claims 1-6 in the preparation of a drug for chronic prostatitis.

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