Use of myrrh volatile oil in preparing medicine for treating colitis and myrrh volatile oil microemulsion

Abstract

The present invention provides the use of myrrh volatile oil in preparing a medicament for treating colitis. The present invention also provides a myrrh volatile oil microemulsion, which is an O / W type microemulsion prepared from myrrh volatile oil, an emulsifier, a co-emulsifier, and an auxiliary oil phase. The myrrh volatile oil of the present invention is used to treat colitis, particularly ulcerative colitis, and has a clear and controllable efficacy. After the myrrh volatile oil is prepared into a microemulsion, the drug's efficacy can be better exerted, providing a new medication option for clinical use.

Description

Technical Field

[0001] The invention relates to use of myrrh volatile oil in preparing medicine for treating colitis and a myrrh volatile oil microemulsion. Background Art

[0002] Myrrha is the dried resin of the hardwood tree (Commiphora myrrha Engl. or Commiphora molmol Engl.), a plant of the Oleaceae family. It has a yellow-brown or reddish-brown surface and is found in hard, lumpy forms. Myrrh is primarily distributed in tropical and subtropical regions, including Ethiopia, Somalia, and the Arabian Peninsula. Myrrh has the properties of promoting blood circulation and alleviating pain, and is commonly used to promote blood circulation and dissipate blood stasis. Modern pharmacological research has confirmed its anti-inflammatory, antioxidant, analgesic, anti-tumor, antibacterial, anti-malarial, lipid metabolism-regulating, anti-Alzheimer's, and wound healing activities. Myrrh has a complex chemical composition, primarily consisting of resins, gums, volatile oils, salts, and acids. The volatile oil is the characteristic component of myrrh and forms the basis for its pharmacological effects and aroma. Its complex composition exhibits diverse pharmacological activities, including anti-inflammatory, anti-tumor, antibacterial, hepatoprotective, neuroprotective, and analgesic properties. It also has a wide range of pharmacological effects, including lowering blood lipids, lowering blood sugar, combating Alzheimer's disease, and combating gastric ulcers. Because volatile oils are fat-soluble, conventional preparations are poorly absorbed and have low bioavailability. Currently, volatile oils are formulated into microemulsions, cyclodextrin inclusion complexes, liposomes, and other dosage forms to alter their solubility, enhance their stability, and improve their bioavailability. (Zhou Ying et al., Preparation and Anti-inflammatory Effects of Myrrh Volatile Oil Nanoemulsions, Animal Husbandry and Veterinary Medicine, Vol. 44, 2012)

[0003] Ulcerative colitis (UC) is a chronic inflammatory colonic disease primarily located in the large intestine, most commonly in the rectum and sigmoid colon. Lesions are diffuse and continuous, and can retrograde into the proximal colon. Clinical manifestations include persistent or recurrent diarrhea, bloody and mucus-purulent stools, accompanied by abdominal pain, tenesmus, and varying degrees of systemic symptoms. Its incidence is increasing worldwide. It is difficult to cure, prone to recurrence, and prone to cancer. It has been classified by the World Health Organization as a modern intractable disease, and specific treatment options are still lacking. Clinical treatment focuses on alleviating inflammatory lesions, controlling symptoms, reducing recurrences, and managing acute attacks. Traditional treatments, including glucocorticoids, aminosalicylic acid preparations, and immunosuppressants, are ineffective, and long-term use can result in severe adverse reactions. These treatments are limited in effectiveness and can lead to numerous complications and adverse consequences. For example, aminosalicylic acids can cause gastrointestinal and allergic reactions, while glucocorticoids can easily cause infections, osteoporosis, and hypertension. Immunomodulators can cause liver and kidney damage. Therefore, it is urgent to find a new treatment method that is effective, easily accepted by the public and inexpensive. Summary of the Invention

[0004] The present invention provides a new use of myrrh volatile oil, in particular, a use in preparing a medicine for treating colitis and a myrrh volatile oil microemulsion.

[0005] The present invention provides use of myrrh volatile oil in preparing a medicine for treating colitis.

[0006] Furthermore, the medicine is a medicine for treating ulcerative colitis.

[0007] The drug achieves its anti-ulcerative colitis effect by regulating p-JNK and p-ERK proteins in the MAPK signaling pathway.

[0008] The myrrh volatile oil contains furanoeudesma 1,3-diene (not less than 20% w / w), curzerene (not less than 17% w / w), lindestrene (not less than 9% w / w), atractylone (not less than 8.00% w / w), and beta-elemene (not less than 7.00% w / w).

[0009] The volatile oil is extracted by steam distillation, the soaking time is 0.5-2 hours, the amount of water is 4-12 times, and the extraction time is 6-10 hours.

[0010] The soaking time of the steam distillation extraction is 1.5 hours, the amount of water added is 8 times, and the extraction time is 8 hours.

[0011] The present invention also provides a myrrh volatile oil microemulsion, which is an O / W type microemulsion prepared from myrrh volatile oil, an emulsifier, a co-emulsifier, and an auxiliary oil phase; wherein the emulsifier is EL-40, RH-60, Tween-80, or Tween-20; the co-surfactant is PEG-400, glycerol, anhydrous ethanol, or 1,2-propylene glycol; and the auxiliary oil phase is isopropyl myristate IPM, oleic acid, or IPP; wherein the ratio Km of the emulsifier to the co-emulsifier is (4-1):1.

[0012] Wherein, the emulsifier is EL-40; the co-emulsifier is glycerol; the auxiliary oil phase is isopropyl myristate IPM; the Km is 2:1;

[0013] The weight ratio of myrrh volatile oil, emulsifier, co-emulsifier and oil phase is:

[0014] Myrrh essential oil 1-10 parts, EL-40 50-70 parts, glycerol 20-40 parts, IPM 1-10 parts.

[0015] The ratio of myrrh volatile oil to IPM is 1:9; the ratio of total emulsifier to oil phase is 9:1.

[0016] The average particle size of the microemulsion was 89.62±0.2487nm, the average PDI was 0.1126±0.0164, and the Zeta potential was -0.2135±0.27mV.

[0017] The present invention also provides a method for preparing the myrrh volatile oil microemulsion, which adopts a phase inversion emulsification method to prepare the microemulsion; the method comprises the following steps:

[0018] a. Mixing myrrh volatile oil with an auxiliary oil phase to prepare a mixed oil phase;

[0019] b. Mixing the emulsifier and the co-emulsifier to prepare a mixed emulsifier;

[0020] c. Evenly mix the mixed oil phase and the mixed emulsifier, dropwise add ultrapure water while stirring, to obtain an o / w emulsion; wherein the stirring temperature is 25°C-50°C; preferably, the stirring temperature is 25°C.

[0021] The present invention is by setting up mouse ulcerative colitis model, giving different concentrations of myrrh volatile oil treatment, and comparing its efficacy.Record the disease activity index (DAI) of each mouse of different groups, after putting to death, the colon length of each mouse is measured, HE staining, immunohistochemistry experiment, protein immunoblotting (WB, Western blot) experiment are carried out to colon tissue, verify the content of inflammatory factors TNF-α, IL-1β and the expression of p-JNK, p-ERK, determine the therapeutic effect of myrrh volatile oil on ulcerative colitis, and filter out the optimal dosage simultaneously.Considering the unstable quality of volatile oil, dissolution rate is not high, the time that plays a role in human body is short, and it is greasy so inconvenient when using, so change dosage form, make microemulsion with myrrh volatile oil as main drug, screen surfactant, cosurfactant, oil phase, Km value and preparation temperature, draw pseudo-ternary phase diagram, determine optimal preparation process in conjunction with conductivity and appearance character, carry out quality evaluation and methodological investigation to it. To confirm the efficacy of myrrh volatile oil microemulsions against ulcerative colitis, further pharmacodynamic validation was conducted and efficacy was compared between myrrh volatile oil groups. A mouse model of ulcerative colitis (UC) was established and treated with different concentrations of myrrh volatile oil and myrrh volatile oil microemulsions. The DAI values ​​of each group were recorded, colon length was measured, serum levels of inflammatory factors such as TNF-α and IL-1β were measured, and colon tissue was pathologically examined and immunohistochemically analyzed for p-JNK and p-ERK protein expression. Results showed that the myrrh volatile oil microemulsion group generally had superior efficacy compared to the corresponding volatile oil concentration group. Among the myrrh volatile oil microemulsion groups, the high-dose group had the greatest anti-UC effect.

[0022] The myrrh volatile oil of the present invention is used for treating colitis, especially for treating ulcerative colitis, and has clear and controllable medicinal effects. After the myrrh volatile oil is prepared into a microemulsion, the medicinal effect can be better exerted, thereby providing a new medication option for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of different soaking times on the yield of myrrh volatile oil;

[0024] Figure 2 The effect of different water addition ratios on the yield of myrrh volatile oil;

[0025] Figure 3 Effects of different extraction times on the yield of essential oil from Myrrh;

[0026] Figure 4 Pseudo-ternary area diagram of emulsifier formation ( n=3) (Note: A: RH-60, B: EL-40, C: Tween-80)

[0027] Figure 5 Pseudo-ternary area diagram formed by emulsifier ( n=3) (Note: A: anhydrous ethanol, B: 1,2-propylene glycol, C: glycerol, D: PEG-400);

[0028] Figure 6 Pseudo-ternary area plot of optimal auxiliary oil phase formation ( n=3) (Note: A: IPM, B: IPP, C: oleic acid);

[0029] Figure 7 Pseudo-ternary area chart formed by optimal KM n=3) (Note: A: 1:1, B: 2:1, C: 3:1, D: 4:1);

[0030] Figure 8 Pseudo-ternary area diagram of optimal temperature formation ( n=3) (Note: A: 25°C, B: 30°C, C: 40°C, D: 50°C);

[0031] Figure 9 Appearance and properties of MEO microemulsion (Note: A: appearance of MEO microemulsion, B: transmission electron microscopy image of MEO microemulsion);

[0032] Figure 10 Diffusion diagram of Sudan III and methylene blue in MEO microemulsion;

[0033] Figure 11 Particle size and zeta potential of MEO microemulsion (n=3);

[0034] Figure 12 Figure 3: Heating-cooling and freezing-thawing cycle experiments of MEO microemulsion (n=6) (Note: A: before heating-cooling cycle experiment, B: after heating-cooling cycle experiment, C: before freezing-thawing cycle experiment, D: after freezing-thawing cycle experiment);

[0035] Figure 13 Chromatograms of blank solvent, MEO microemulsion test sample, and reference sample;

[0036] Figure 14 Standard curves of standards (Note: A: standard curve of reference substance β-elemene, B: standard curve of reference substance curcumene);

[0037] Figure 15 DAI score and colon morphology of UC mice ( n=6) (Note: A: DAI score of mice in each group, B: weight change of mice in each group, C: colon morphology of mice in each group, D: difference in colon length of mice in each group compared with the Control group, ## p≤0.01; compared with the Model group, ## p≤0.01);

[0038] Figure 16 The levels of TNF-α and IL-1β in serum of UC mice ( n=6) (Note: A: TNF-α content in the serum of mice in each group, B: IL-1β content in the serum of mice in each group, compared with the Control group, ## p≤0.01; compared with the Model group, ** p≤0.01);

[0039] Figure 17 HE staining of the colon in each group of mice with ulcerative colitis treated with myrrh essential oil;

[0040] Figure 18 Expression of p-JNK, p-ERK and TNF-α in ulcerative colitis mice treated with myrrh essential oil (Note: A: Effect of MEO on the p-JNK, p-ERK and TNF-α protein levels in DSS-induced colon tissue, B: p-JNK protein level in colon tissue of mice in each group, C: p-ERK protein level in colon tissue of mice in each group, D: TNF-α protein level in colon tissue of mice in each group, compared with the Control group, ## p≤0.01; compared with the Model group, * p≤0.05, ** p≤0.01);

[0041] Figure 19Results of WB experiments on ulcerative colitis in mice treated with myrrh essential oil (Note: A: Changes in the levels of P-JNK, JNK, P-ERK, and ERK in DSS-induced colon tissue after MEO treatment; B: p-JNK / JNK protein levels in colon tissue of mice in each group; C: p-ERK / ERK protein levels in colon tissue of mice in each group compared with the control group. ## p≤0.01; compared with the Model group, * p≤0.05);

[0042] Figure 20 DAI score and colon morphology of UC mice ( n=6) (Note: A: DAI score of mice in each group, B: weight changes of mice in each group);

[0043] Figure 21 The levels of TNF-α and IL-1β in serum of UC mice ( n=6) (Note: A: TNF-α content in the serum of mice in each group, B: IL-1β content in the serum of mice in each group, compared with the Control group, ## p≤0.01; compared with the Model group, ** p≤0.01);

[0044] Figure 22 HE staining of colon in each group of mice with ulcerative colitis treated with myrrh essential oil and microemulsion. DETAILED DESCRIPTION

[0045] Example 1 Screening and Optimization of Extraction Process for Myrrh Volatile Oil of the Present Invention

[0046] 1 Effect of soaking time on volatile oil extraction rate

[0047] Weigh 50g of myrrh (identified by Professor Yan Yonggang of the School of Pharmacy of Shaanxi University of Chinese Medicine as the dry resin of the olive family plant Myrrha japonica) and place it in a 2000mL round-bottom flask. Soak for 0, 0.5, 1, 1.5, and 2 hours respectively, add 8 times the amount of water, and extract for 8 hours. Connect the condenser and place it in an electric heating mantle. Heat slowly until the liquid in the bottle is slightly boiling. Observe the volatile oil extractor and start timing when the first drop of liquid falls. Perform three parallel experiments and use a vernier caliper to measure the oil extraction amount in different time periods. After 8 hours, stop heating. After 1 hour, collect the volatile oil in the extractor and add anhydrous sodium sulfate to dry it. The final result is the volatile oil. Calculate the oil yield (%) of the volatile oil.

[0048] 2. Effect of adding water on the extraction rate of volatile oil

[0049] Weigh 50g of myrrh into a 2000mL round-bottom flask. Add 4, 6, 8, 10, and 12 times the volume of water, respectively. Soak for 1.5 hours, and extract for 8 hours. Connect a condenser and place it in an electric heating mantle. Slowly heat until the liquid in the flask is slightly boiling. Observe the volatile oil extractor and start timing when the first drop of liquid falls. Repeat the experiment three times in parallel, using a vernier caliper to measure the oil extraction at different time periods. After 8 hours, stop heating. After 1 hour, collect the volatile oil in the extractor and dry it over anhydrous sodium sulfate. The final result is the volatile oil. Calculate the volatile oil yield (%).

[0050] 3 Effect of extraction time on volatile oil extraction rate

[0051] Weigh 50g of myrrh into a 2000mL round-bottom flask and add 8 times the volume of water. Soak for 1.5 hours. Extract for 6, 7, 8, 9, and 10 hours, then connect a condenser and place in an electric heating mantle. Slowly heat to a boil and maintain a slight boil. Boiling is considered to begin when the first drop of liquid drips into the extractor, and the timer begins. Repeat the experiment three times. Use a vernier caliper to measure the oil extraction at different time periods. After the extraction time is complete, stop heating and let the flask stand for at least 1 hour. Collect the volatile oil and dry it overnight over anhydrous sodium sulfate to obtain an oily substance, which is the volatile oil. Calculate the oil yield (%).

[0052] 4 Experimental results

[0053] 4.1 Effect of soaking time on volatile oil extraction rate

[0054] Add 8 times the amount of water, extract for 8 hours, soak for 0, 0.5, 1, 1.5, 2 hours, and the changes in the amount of myrrh volatile oil extracted are as follows: Figure 1 As shown in the results, direct extraction without soaking the herbs resulted in a low extraction rate. When the soaking time gradually increased to 2 hours, the oil yield continued to increase. However, no significant difference in oil yield was found when the soaking time was 1.5 hours or 2 hours. After comprehensive consideration, the optimal soaking time was determined to be 1.5 hours.

[0055] 4.2 Effect of water addition on volatile oil extraction rate

[0056] Soaking for 1.5 hours, extraction time for 8 hours, adding 4, 6, 8, 10, 12 times the amount of water, the changes in the amount of myrrh volatile oil extracted are as follows Figure 2 As shown in the results, it can be seen that when the amount of water added is less than 6 times, the extraction rate is low and the volatile oil cannot be fully distilled out. When the amount of water added is 8 times, the oil yield continues to increase, but when the amount of water added exceeds 8 times, the extraction rate shows a downward trend. This may be because excessive boiling caused by excessive water addition causes loss of medicinal materials. After comprehensive consideration, it is finally determined that the amount of water added is 8 times the amount of medicine.

[0057] 4.3 Effect of extraction time on volatile oil extraction rate

[0058] Add 8 times the amount of water, soak for 1.5 hours, and extract for 6, 7, 8, 9, and 10 hours respectively. The changes in the amount of myrrh volatile oil extracted are as follows: Figure 3 As shown in the results, the yield of myrrh volatile oil increases with extraction time. However, after 8 hours, the yield gradually decreases. This decrease is likely due to the prolonged extraction time, which causes a small amount of water and volatile oil to evaporate, resulting in a decrease in oil yield. After comprehensive consideration, the final extraction time was determined to be 8 hours.

[0059] 5. Summary:

[0060] This section uses single-factor experiments to investigate the three influencing factors of the steam distillation extraction of myrrh volatile oil: water addition ratio, soaking time, and extraction time. Taking the extraction rate as the evaluation index, the investigation shows that the optimal soaking time for the extraction of myrrh volatile oil is 1.5h, the optimal water addition ratio is 8 times, and the optimal extraction time is 8h.

[0061] The specific preparation process is:

[0062] Weigh 50g of myrrh (identified by Professor Yan Yonggang of the School of Pharmacy at Shaanxi University of Chinese Medicine as dried resin from the olive family plant Myrrha sylvestris) into a 2000mL round-bottom flask and soak for 1.5 hours. Then, add 8 times the amount of water and extract for 8 hours. Connect the condenser and place it in an electric heating mantle. Slowly heat until the liquid in the flask is slightly boiling. Observe the volatile oil extractor and start timing when the first drop of liquid falls. Repeat the experiment three times in parallel, measuring the amount of oil extracted at different time periods with a vernier caliper. After 8 hours, stop heating. After 1 hour, collect the volatile oil in the extractor and dry it over anhydrous sodium sulfate to obtain myrrh volatile oil.

[0063] Test Example 2 Quality Testing Method for Myrrh Volatile Oil of the Present Invention

[0064] The myrrh volatile oil contains furanoeudesma 1,3-diene (not less than 20% w / w), curzerene (not less than 17% w / w), lindestrene (not less than 9% w / w), atractylone (not less than 8.00%), and beta-elemene (not less than 7.00%).

[0065] The specific detection methods are:

[0066] 100 μL of the extracted myrrh essential oil was placed in a 10 mL brown volumetric flask and diluted to 10 mL with n-hexane. Anhydrous sodium sulfate was added and the supernatant was allowed to stand for 5 minutes. The supernatant was then filtered through a 0.22 μM organic filter membrane to obtain the prepared myrrh essential oil sample. The myrrh essential oil sample was analyzed using GC-MS. GC conditions included an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), high-purity helium carrier gas at a flow rate of 1 mL / min, a split ratio of 15:1, an injection volume of 1 μL, and an inlet temperature of 260°C. MS conditions included an EI ionization source with an ionization energy of 70 eV and a source temperature of 230°C. A temperature program was used: an initial temperature of 90°C, ramped to 175°C at a rate of 2°C / min, held for 5 minutes, and then ramped to 210°C at a rate of 1°C / min, held for 10 minutes. The results showed that the relative content of furanoeudesma 1,3-diene was 1.984%, the relative content of curzerene was 17.028%, the relative content of lindestrene was 9.223%, the relative content of atractylone was 8.894%, and the relative content of beta-Elemene was 7.715%.

[0067] Table 1 GC-MS identification results of myrrh essential oil

[0068]

[0069]

[0070] Test Example 3 Preparation and Quality Evaluation of Myrrh Volatile Oil Microemulsion Preparation of the Present Invention

[0071] Myrrh volatile oil is a fat-soluble component that is poorly soluble in water. It evaporates easily over time, making it inconvenient to use and having a short duration of action in the body. Therefore, myrrh volatile oil is formulated into a microemulsion. Microemulsions offer excellent stability, increase the solubility of fat-soluble components, and are easy to prepare. Therefore, experiments were conducted to investigate the effects of various factors on microemulsions and identify the optimal formulation and ratio.

[0072] 1 Preparation of Myrrh Essential Oil Microemulsion

[0073] Microemulsions were prepared using a phase inversion emulsification method, with a total formulation of 5 grams. The mixed oil phase consisted of myrrh volatile oil plus an auxiliary oil phase. Experiments were conducted using specific ratios of total emulsifier and mixed oil phase (9:1-1:9). Different types of emulsifiers, co-emulsifiers, temperatures, oil phases, and Km values ​​were examined. The conductivity and temperature were measured while adding water and stirring. With the continuous addition of water, it was found that the conductivity reached a peak at a certain value and gradually decreased with further water addition. The microemulsion was most stable at the peak conductivity, and the ratio of emulsifier to co-emulsifier at this point was the optimal formulation for that ratio. A pseudo-ternary phase diagram was constructed based on these formulations, and the optimal microemulsion process and ratio were determined by quantitatively calculating the microemulsion area.

[0074] First, the formulation was initially set at 5.0g, and the optimal emulsifier was screened. The preparation temperature was initially set at 25°C. Isopropyl myristate (IPM) was proposed as the auxiliary oil phase, and Tween-80, Tween-20, EL-40, and RH-60 were selected as emulsifiers. IPM and myrrh essential oil were mixed in a 9:1 ratio to form the mixed oil phase. Subsequently, the emulsifier was mixed in a 1:9 ratio (myrrh essential oil: 0.05g; IPM: 0.45g; emulsifier: 4.5g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1mL of ultrapure water was added. As ultrapure water was added, the conductivity initially increased and then decreased. When the conductivity peaked and then began to decline, this peak indicated the critical point of the microemulsion. The same procedure was followed, with a gradient of emulsifier ratios (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentages of each component at the critical point were recorded, and a pseudo-ternary phase diagram was plotted. The pseudo-ternary phase diagram revealed that EL-40, when used as an emulsifier, formed a microemulsion with the largest area, the smallest particle size, and the clearest and most transparent appearance. Therefore, EL-40 was selected as the optimal emulsifier for subsequent formulation screening.

[0075] Second, the formulation was initially set at 5.0g, EL-40 was chosen as the emulsifier, and the optimal co-emulsifier was screened. The preparation temperature was initially set at 25°C, IPM was chosen as the auxiliary oil phase, and the mixing ratio (KM value) of emulsifier to co-emulsifier was set at 2:1. Polyethylene glycol 400 (PEG-400), glycerol, anhydrous ethanol, and 1,2-propylene glycol were selected as co-emulsifiers. IPM and myrrh essential oil were mixed in a ratio of 9:1 to form the mixed oil phase. EL-40 and the co-emulsifier were mixed in a ratio of 2:1 to form the mixed emulsifier. Subsequently, the mixed oil phase and the mixed emulsifier were mixed in a ratio of 1:9 (myrrh essential oil: 0.05g; IPM: 0.45g; EL-40: 3g; co-emulsifier: 1.5g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1mL of ultrapure water was added. With the continuous addition of ultrapure water, the conductivity will show a trend of first increasing and then decreasing. When the conductivity reaches a peak and begins to show a downward trend, the peak point is the critical point of the microemulsion. The same operation method was used to conduct experiments with mixed emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reached the critical point was recorded, and a pseudo-ternary phase diagram was drawn. According to the results of the pseudo-ternary phase diagram, when glycerol was used as an emulsifier, the microemulsion formed had the largest area, the smallest particle size, and the clearest and most transparent appearance. Therefore, glycerol was selected as the optimal emulsifier for subsequent prescription screening.

[0076] Third, the formulation was initially set at 5.0g, EL-40 as the emulsifier, and glycerol as the co-emulsifier. The auxiliary oil phase was screened. The preparation temperature was initially set at 25°C, with a KM ratio of 2:1. Oleic acid, IPP, and IPM were selected as the auxiliary oil phases. The auxiliary oil phase was mixed with myrrh essential oil in a ratio of 9:1 to form the mixed oil phase. EL-40 and glycerol were mixed in a ratio of 2:1 to form the mixed emulsifier. Subsequently, the mixed oil phase was mixed with the mixed emulsifier in a ratio of 1:9 (myrrh essential oil: 0.05g; auxiliary oil phase: 0.45g; EL-40: 3g; glycerol: 1.5g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1mL of ultrapure water was added. As ultrapure water was added, the conductivity initially increased and then decreased. When the conductivity peaked and then began to decline, this peak indicated the critical point of the microemulsion. The same procedure was followed, with experiments conducted using a gradient ratio of mixed emulsifiers (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentages of each component at the critical point were recorded, and a pseudo-ternary phase diagram was plotted. The results of the pseudo-ternary phase diagram revealed that when IPM was used as the auxiliary oil phase, the microemulsion formed had the largest area, the smallest particle size, and the clearest appearance. Therefore, IPM was selected as the auxiliary oil phase for subsequent formulation screening.

[0077] Fourth, the formulation was initially set at 5.0g, EL-40 as the emulsifier, glycerol as the co-emulsifier, and IPM as the auxiliary oil phase. The optimal KM value was screened. The preparation temperature was initially set at 25°C, with KM values ​​of 1:1, 2:1, 3:1, and 4:1. Myrrh essential oil and IPM were mixed in ratios of 1:1, 2:1, 3:1, and 4:1 to form the mixed oil phase. EL-40 and glycerol were mixed in a 1:1 ratio to form the mixed emulsifier. The mixed oil phase and the mixed emulsifier were then mixed uniformly at a ratio of 1:9 (KM value 1:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 2.25 g, glycerol: 2.25 g; KM value 2:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3 g, glycerol: 1.5 g; KM value 3:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3.375 g, glycerol: 1.125 g; KM value 4:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3.6 g, glycerol: 0.9 g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1 mL of ultrapure water was added. With the continuous addition of ultrapure water, the conductivity will show a trend of first increasing and then decreasing. When the conductivity reaches a peak and begins to show a downward trend, the peak point at this time is the critical point of the microemulsion. The same operation method is used to conduct experiments with the mixed emulsifier ratio in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reaches the critical point is recorded, and a pseudo-ternary phase diagram is drawn. According to the results of the pseudo-ternary phase diagram, it is found that when the KM value is 2:1, the microemulsion area is the largest, the particle size is the smallest, and the microemulsion appearance is the clearest and most transparent. Therefore, a KM value of 2:1 is selected for subsequent prescription screening.

[0078] Fifth, the formulation was initially set at 5.0g, EL-40 as the emulsifier, glycerol as the co-emulsifier, IPM as the auxiliary oil phase, and a KM ratio of 2:1. The optimal preparation temperature was screened. 25°C, 30°C, 40°C, and 50°C were selected as the preparation temperatures. IPM and myrrh essential oil were mixed in a 9:1 ratio to form the mixed oil phase. EL-40 and glycerol were mixed in a 2:1 ratio to form the mixed emulsifier. Subsequently, the mixed oil phase and the mixed emulsifier were mixed in a 1:9 ratio (myrrh essential oil: 0.05g, auxiliary oil phase: 0.45g, EL-40: 3g, glycerol: 1.5g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1mL of ultrapure water was added. With the continuous addition of ultrapure water, the conductivity will show a trend of first increasing and then decreasing. When the conductivity reaches a peak and begins to show a downward trend, the peak point at this time is the critical point of the microemulsion. The same operation method is used to conduct experiments with the mixed emulsifier ratio in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reaches the critical point is recorded, and a pseudo-ternary phase diagram is drawn. According to the results of the pseudo-ternary phase diagram, it is found that at a temperature of 25°C, the microemulsion has the largest area, the smallest particle size, and the clearest appearance. Therefore, the preparation temperature of 25°C is selected.

[0079] 2 Investigation of emulsifiers

[0080] An emulsifier is a surfactant with both hydrophilic and lipophilic properties, enabling the uniform mixing of immiscible liquids. EL-40, RH-60, Tween-80, and Tween-20 were selected for emulsifier screening because their HLB values ​​range from 8 to 18. A higher HLB value indicates greater hydrophilicity. IPM was first used as the auxiliary oil phase and mixed with myrrh essential oil in a ratio of 9:1 to form the mixed oil phase.

[0081] 3 Investigation of co-emulsifiers

[0082] By adjusting the HLB value of the emulsifier, co-emulsifiers can change the polarity of the system, thereby improving the stability of fine emulsion polymerization. To explore the effect of co-emulsifiers on microemulsion formation, PEG-400, glycerol, anhydrous ethanol, and 1,2-propylene glycol were selected as candidate co-emulsifiers, and a series of experiments were conducted to screen the best co-emulsifier.

[0083] 4 Investigation of oil phase

[0084] The auxiliary oil phases include oleic acid, IPP and IPM, which are mixed with myrrh essential oil at a ratio of 9:1 to form a mixed oil phase.

[0085] Investigation of 5Km value

[0086] The Km value is the ratio of emulsifier to co-emulsifier. Studies have shown that different Km values ​​can lead to significant differences in the quality of microemulsions. Therefore, the Km values ​​were set at 4:1, 3:1, 2:1, and 1:1.

[0087] 6 Temperature Investigation

[0088] Temperature is an important factor affecting the performance of volatile oil drugs, so it is necessary to study the preparation of microemulsions at different temperatures. To this end, the temperature parameters were set at 25°C, 30°C, 40°C, and 50°C.

[0089] 7 Experimental results

[0090] 7.1 Selection of the best emulsifier

[0091] The initial formulation volume was set at 5.0 g, the preparation temperature at 25°C, and the auxiliary oil phase was isopropyl myristate (IPM). Tween-80, Tween-20, castor oil polyoxyethylene ether-40 (EL-40), and polyoxyethylene hydrogenated castor oil-60 (RH-60) were selected as emulsifiers. IPM and myrrh essential oil were mixed in a 9:1 ratio to form the mixed oil phase. Subsequently, the emulsifier was mixed in a 1:9 ratio (myrrh essential oil: 0.05 g; IPM: 0.45 g; emulsifier: 4.5 g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1 mL of ultrapure water was added. With the addition of ultrapure water, the conductivity initially increased and then decreased. When the conductivity peaked and then began to decline, this peak indicated the critical point of the microemulsion. The same operation method was used to conduct experiments with emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reached the critical point was recorded, and a pseudo-ternary phase diagram was drawn. The above experiments were conducted on four different emulsifiers: RH-60, EL-40, Tween-80, and Tween-20 to examine their emulsification abilities. In Origin 8.0 drawing software, the amount of water added and the corresponding conductivity value were recorded, and a pseudo-ternary phase diagram was drawn to obtain the microemulsion areas: S EL-40 :0.010091,S Tween-80 :0.009804,S RH-60 : 0.009804, Tween-20 was used as an emulsifier to make microemulsion, so it was eliminated. Figure 4 As shown in Table 2, when the emulsifier is EL-40, the microemulsion area is the largest, the appearance is clear and uniform, and the particle size is also within the range.

[0092] Table 2 Emulsifier screening results ( n=3)

[0093]

[0094]

[0095] 7.2 Selection of the best co-emulsifier

[0096] The formulation was initially set at 5.0g, EL-40 as the emulsifier, and 25°C as the preparation temperature. The auxiliary oil phase was IPM, with a proposed mixing ratio (KM value) of 2:1 between the emulsifier and the co-emulsifier. Polyethylene glycol 400 (PEG-400), glycerol, anhydrous ethanol, and 1,2-propylene glycol were selected as co-emulsifiers. IPM and myrrh essential oil were mixed in a 9:1 ratio to form the mixed oil phase. EL-40 and the co-emulsifier were mixed in a 2:1 ratio to form the mixed emulsifier. Subsequently, the mixed oil phase and the mixed emulsifier were mixed in a 1:9 ratio (myrrh essential oil: 0.05g; IPM: 0.45g; EL-40: 3g; co-emulsifier: 1.5g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1mL of ultrapure water was added. With the continuous addition of ultrapure water, the conductivity will show a trend of first increasing and then decreasing. When the conductivity has a peak point and begins to show a downward trend, the peak point at this time is the critical point of the microemulsion. The same operation method is used to conduct experiments with the ratio of mixed emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reaches the critical point is recorded, and a pseudo-ternary phase diagram is drawn. The above experiments were carried out on four different emulsifiers: propylene glycol, anhydrous ethanol, PEG-400 and 1,2-propylene glycol, to examine their emulsification ability. In the Origin 8.0 drawing software, the amount of water added and the corresponding conductivity value are recorded, and a pseudo-ternary phase diagram is drawn. The results can be found in Figure 5 and Table 3, the areas of the microemulsion regions are S 丙三醇 =0.011161>S 无水乙醇 =0.010091>S 1,2-丙二醇 =0.009804>S PEG400 =0.008921. The results showed that when glycerol was used as a co-emulsifier, the microemulsion formed the largest area, had a clear and uniform appearance, and met the required particle size and system stability. Therefore, glycerol was selected as the co-surfactant in this formulation.

[0097] Table 3 Screening results of co-emulsifiers ( n=3)

[0098]

[0099] 7.3 Screening of the best auxiliary oil phase

[0100] The formulation was initially set at 5.0 g, EL-40 as the emulsifier, and glycerol as the co-emulsifier. The preparation temperature was tentatively set at 25°C, with a KM ratio of 2:1. Oleic acid, IPP, and IPM were selected as auxiliary oil phases. The auxiliary oil phase was mixed with myrrh essential oil in a ratio of 9:1 to form the mixed oil phase. EL-40 and glycerol were mixed in a ratio of 2:1 to form the mixed emulsifier. Subsequently, the mixed oil phase was mixed with the mixed emulsifier in a ratio of 1:9 (myrrh essential oil: 0.05 g; auxiliary oil phase: 0.45 g; EL-40: 3 g; glycerol: 1.5 g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1 mL of ultrapure water was added. With the addition of ultrapure water, the conductivity initially increased and then decreased. When the conductivity peaked and then began to decline, this peak indicated the critical point of the microemulsion. The same operation method was used to conduct experiments with the ratio of mixed emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reached the critical point was recorded, and a pseudo-ternary phase diagram was drawn. The above experiments were conducted on three different auxiliary oil phases: IPM, IPP, and oleic acid. In Origin8.0 drawing software, the amount of water added and the corresponding conductivity value were recorded, and a pseudo-ternary phase diagram was drawn. The results are shown below. Figure 6 , as shown in Table 4. The microemulsion areas are: S IPM :0.011161,S IPP :0.010463,S 油酸 : 0.004467. It was found that when IPM was the oil phase, the microemulsion area was the largest, the appearance was clear and uniform, and the particle size was also within the range.

[0101] Table 4 Auxiliary oil phase screening results ( n=3)

[0102]

[0103] 7.4 Screening of the Optimal Km Value

[0104] The formulation was initially set at 5.0g, EL-40 as the emulsifier, glycerol as the co-emulsifier, and IPM as the auxiliary oil phase. The preparation temperature was tentatively set at 25°C, with KM values ​​of 1:1, 2:1, 3:1, and 4:1, respectively. Myrrh essential oil and IPM were mixed evenly in ratios of 1:1, 2:1, 3:1, and 4:1 to form the mixed oil phase. EL-40 and glycerol were mixed evenly in a 1:1 ratio to form the mixed emulsifier. The mixed oil phase and the mixed emulsifier were then mixed uniformly at a ratio of 1:9 (KM value 1:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 2.25 g, glycerol: 2.25 g; KM value 2:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3 g, glycerol: 1.5 g; KM value 3:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3.375 g, glycerol: 1.125 g; KM value 4:1: myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3.6 g, glycerol: 0.9 g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1 mL of ultrapure water was added. With the continuous addition of ultrapure water, the conductivity will show a trend of first increasing and then decreasing. When the conductivity has a peak point and begins to show a downward trend, the peak point at this time is the critical point of the microemulsion. The same operation method is used to conduct experiments with the ratio of mixed emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1), record the percentage of each component when each group reaches the critical point, and draw a pseudo-ternary phase diagram. The above experiments were carried out for the four KM values ​​of 4:1, 3:1, 2:1, and 1:1. In the Origin8.0 drawing software, the amount of water added and the corresponding conductivity value are recorded, and a pseudo-ternary phase diagram is drawn. The results are as follows. Figure 7 , as shown in Table 5. The microemulsion areas under different Km values ​​are S 2:1 =0.011161>S 1:1 =0.010764>S 3:1 =0.010463=S 4:1 =0.010463, and it was found that when Km=2:1, the microemulsion area was the largest, the appearance was clear and uniform, and the particle size was also within the range.

[0105] Table 5 Km value screening results ( n=3)

[0106]

[0107] 7.5 Screening of optimal temperature

[0108] The initial formulation was set at 5.0 g, EL-40 as the emulsifier, glycerol as the co-emulsifier, IPM as the auxiliary oil phase, and a KM ratio of 2:1. Preparation temperatures were selected at 25°C, 30°C, 40°C, and 50°C. IPM and myrrh essential oil were mixed in a 9:1 ratio to form the mixed oil phase. EL-40 and glycerol were mixed in a 2:1 ratio to form the mixed emulsifier. Subsequently, the mixed oil phase and the mixed emulsifiers were mixed in a 1:9 ratio (myrrh essential oil: 0.05 g, auxiliary oil phase: 0.45 g, EL-40: 3 g, glycerol: 1.5 g). Ultrapure water was added dropwise with stirring, and the conductivity was measured after each 1 mL of ultrapure water was added. With the addition of ultrapure water, the conductivity initially increased and then decreased. When the conductivity peaked and then began to decline, this peak indicated the critical point of the microemulsion. The same operation method was used to conduct experiments with the ratio of mixed emulsifiers in a gradient ratio (2:8; 3:7; 4:6; 5:5; 6:4; 7:3; 8:2; 9:1). The percentage of each component when each group reached the critical point was recorded, and a pseudo-ternary phase diagram was drawn. The above experiment was conducted at four different temperatures: 25°C, 30°C, 40°C, and 50°C. In the Origin8.0 drawing software, the amount of water added and the corresponding conductivity value were recorded, and a pseudo-ternary phase diagram was drawn. The results are shown below. Figure 8 , as shown in Table 6. The microemulsion areas at different temperatures are: S 25℃ :0.011161、S 30℃ :.009804、S 50℃ :0.009227、S 40℃ =0.007767. The results showed that the microemulsion area was the largest at 25°C, the appearance was clear, and the particle size was within the range.

[0109] Table 6 Effect of temperature on the preparation of microemulsion ( n=3)

[0110]

[0111]

[0112] 8. Summary

[0113] Myrrh volatile oil is poorly soluble in water and has poor bioavailability. Therefore, the preparation of volatile oil into microemulsions is considered to address its water insolubility and the uneven quality of liquid preparations. Using microemulsions as carriers, other dosage forms can be prepared to enhance bioavailability and stability.

[0114] MEO microemulsions were prepared by water titration and phase inversion emulsification. Using the microemulsion area calculated from the pseudo-ternary phase diagram as an indicator, the types of emulsifier, oil phase, and co-emulsifier were screened. Temperature and Km values ​​were also examined. The optimal preparation process parameters were determined: EL-40 as the emulsifier, glycerol as the co-emulsifier, MEO:IPM ratio = 1:9, Km value = 2:1, and total emulsifier: mixed oil phase = 9:1. The resulting microemulsions were clear and uniform, with particle sizes meeting the standard.

[0115] Example 4 Quality Evaluation of Myrrh Volatile Oil Microemulsion Preparation System

[0116] This section conducted a preliminary evaluation of the quality of myrrh essential oil microemulsion by measuring the appearance, type, particle size, zeta potential, stability, and content of index components of the microemulsion.

[0117] 1 Appearance and properties of microemulsion

[0118] Take an appropriate amount of sealing film and fix it on the table. Place a carbon-coated support film copper mesh on the sealing film. Pipette about 10μL of MEO microemulsion and drop it on the carbon film. After 10 minutes, use filter paper to absorb the excess microemulsion. Next, stain with the prepared phosphotungstic acid stain (concentration of 20mg / mL, pH=7) and drop it on the carbon film. Keep it for 90 seconds, then use filter paper to absorb the excess phosphotungstic acid reagent. Let it dry and observe.

[0119] 2. Identification of microemulsion type by staining

[0120] Take appropriate amounts of dye Sudan III (oil-soluble) and methylene blue (water-soluble) and add them to MEO microemulsion respectively, and observe the diffusion rates of the two. If it is an O / W type microemulsion, the diffusion rate of methylene blue in MEO microemulsion is greater than that of Sudan III; if it is a W / O type microemulsion, the diffusion rate of methylene blue in MEO microemulsion is less than that of Sudan III.

[0121] 3 Determination of physicochemical parameters of microemulsion

[0122] 3.1 Determination of microemulsion particle size

[0123] Three batches of MEO microemulsion were prepared, and 1 mL of each was diluted and then used for particle size measurement.

[0124] 3.2 Determination of Zeta Potential of Microemulsion

[0125] Three batches of MEO microemulsion were prepared, and 1 mL of each was diluted and then used for zeta potential measurement.

[0126] 4 Study on the stability of myrrh essential oil microemulsion

[0127] 4.1 Physical stability of microemulsion

[0128] Prepare 3 batches of MEO microemulsion, take 5 mL of each batch, and spin at 10000 r·min -1 , centrifuge for 15 minutes, and observe whether the microemulsion is stratified.

[0129] 4.2 Thermodynamic stability experiments of microemulsions

[0130] 4.2.1 Heating-cooling cycle experiment

[0131] Prepare three batches of MEO microemulsions. Take 5 mL of each batch into a centrifuge tube and keep it at 40°C for 24 hours. Then immediately take it out and keep it at 4°C for 24 hours. Repeat this process 6 times and observe the properties of the microemulsion.

[0132] 4.2.2 Freeze-thaw cycle experiment

[0133] Prepare three batches of MEO microemulsions. Take the same volume of each into a centrifuge tube, keep it at -20°C for 24 hours, then immediately take it out and keep it at room temperature for 24 hours. Repeat this process 6 times and observe the properties of the microemulsion.

[0134] 4.2.3 Accelerated stability test

[0135] Prepare three batches of MEO microemulsions, transfer 5 mL of each batch into weighing bottles, seal them, and store them in an environment with a temperature of 28-32°C and a humidity of 60-70% for three months. Samples were taken at the end of each month and tested for changes in appearance, conductivity, and particle size.

[0136] 5GC-MS determination of the content of index components and methodology investigation

[0137] 5.1 GC-MS conditions

[0138] The GC-MS analysis conditions are shown in Tables 7 and 8.

[0139] Table 7 GC-MS injection conditions

[0140]

[0141] Table 8 Temperature program gradient

[0142]

[0143] 5.2 Preparation of sample solution

[0144] 5.2.1 Preparation of reference solution

[0145] 20 mg of β-elemene reference substance was diluted to a 2 mL brown volumetric flask with n-hexane to obtain a 10 mg / mL β-elemene standard stock solution.

[0146] Weigh 20 mg of curcumene reference substance, accurately pipette 3 mL of n-hexane, and dissolve it in a brown volumetric flask to obtain the curcumene standard solution (6.67 mg / mL).

[0147] 5.2.2 Preparation of test solution

[0148] Weigh 10 g of MEO microemulsion, dilute to 25 mL in a brown volumetric flask with n-hexane, break the emulsion, vortex, fully shake and extract, centrifuge, and filter the supernatant through a 0.22 μm microporous membrane to obtain the test solution.

[0149] 5.3 Methodological Review

[0150] 5.3.1 Specificity Investigation

[0151] Take blank solvent, test solution, β-elemene reference solution and curcumene reference solution and inject them respectively to observe whether there are other impurity peaks in the solvent or microemulsion that interfere with the chromatographic peaks of β-elemene and curcumene.

[0152] 5.3.2 Linear relationship investigation

[0153] Accurately pipette 500 μL of the β-elemene reference solution into a 1 mL brown volumetric flask, dilute it downwards to 8 concentrations using the half-and-half dilution method, and dilute to volume with n-hexane to prepare 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, and 0.0390625 mg·mL -1 The reference solution was used, and the concentration and peak area were used as the horizontal and vertical coordinates for linear regression calculation. The GC-MS injection conditions were as follows: HP-5MS capillary column (30m×0.25mm×0.25μm), high-purity helium carrier gas, flow rate 1mL / min, split ratio 15:1, injection volume 1μL, injection port temperature 260℃; ionization source EI, ionization energy 70eV, ion source temperature 230℃; programmed temperature was used, with an initial temperature of 90℃, increased to 175℃ at a rate of 2℃ / min, maintained for 5min, and then increased to 210℃ at a rate of 1℃ / min, maintained for 10min.

[0154] Accurately pipette 100 μL of the curcumene reference solution into a 1 mL brown volumetric flask and prepare according to the method in Table 5-10 to obtain concentrations of 4.44, 2.96, 1.98, 1.32, and 0.88 mg mL -1The reference solution was used, and the concentration and peak area were used as the horizontal and vertical coordinates for linear regression calculation. The GC-MS injection conditions were as follows: HP-5MS capillary column (30m×0.25mm×0.25μm), high-purity helium carrier gas, flow rate 1mL / min, split ratio 15:1, injection volume 1μL, injection port temperature 260℃; ionization source EI, ionization energy 70eV, ion source temperature 230℃; programmed temperature was used, with an initial temperature of 90℃, increased to 175℃ at a rate of 2℃ / min, maintained for 5min, and then increased to 210℃ at a rate of 1℃ / min, maintained for 10min.

[0155] Table 9 Configuration of different concentrations of curcumene reference substance

[0156]

[0157] 5.3.3 Precision experiment

[0158] Chromatographic conditions were as follows: an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), high-purity helium carrier gas at a flow rate of 1 mL / min, a split ratio of 15:1, an injection volume of 1 μL, and an inlet temperature of 260°C. An EI ionization source with an ionization energy of 70 eV and a source temperature of 230°C was used. A temperature program was used: initial temperature of 90°C, increasing at a rate of 2°C / min to 175°C, holding for 5 minutes, and then increasing at a rate of 1°C / min to 210°C, holding for 10 minutes. Six consecutive injections of the MEO microemulsion test solution were made, and the RSD values ​​were calculated based on the peak areas.

[0159] 5.3.4 Stability test

[0160] In this experiment, MEO microemulsion test solutions were injected at 0, 1, 2, 4, 8, 10, 12, and 24 hours. Chromatographic injection conditions were as follows: an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), high-purity helium carrier gas at a flow rate of 1 mL / min, a split ratio of 15:1, an injection volume of 1 μL, and an inlet temperature of 260°C. An EI ionization source with an ionization energy of 70 eV and a source temperature of 230°C was used. A temperature program was used: an initial temperature of 90°C, which was increased at a rate of 2°C / min to 175°C, held for 5 minutes, and then increased at a rate of 1°C / min to 210°C, held for 10 minutes. RSDs were calculated based on peak areas.

[0161] 5.3.5 Repeatability Experiment

[0162] Six equal aliquots of the MEO microemulsion solution were taken to prepare the test solution. Chromatographic injection conditions were as follows: an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), high-purity helium carrier gas at a flow rate of 1 mL / min, a split ratio of 15:1, an injection volume of 1 μL, and an inlet temperature of 260°C. An EI ionization source with an ionization energy of 70 eV and a source temperature of 230°C was used. A temperature program was used: an initial temperature of 90°C, which was increased at a rate of 2°C / min to 175°C, held for 5 minutes, and then increased at a rate of 1°C / min to 210°C, held for 10 minutes. RSDs were calculated based on peak areas.

[0163] 5.3.6 Sample recovery experiment

[0164] Accurately weigh 1.56 g of microemulsion, 6 portions, were added to known 100% β-elemene and curcumene reference substance solutions, and the chromatographic injection conditions were as follows: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), high-purity helium as carrier gas, flow rate 1 mL / min, split ratio 15:1, injection volume 1 μL, injection port temperature 260°C; ionization source EI, ionization energy 70 eV, ion source temperature 230°C; programmed temperature, initial temperature 90°C, raised to 175°C at a rate of 2°C / min, held for 5 min, then raised to 210°C at a rate of 1°C / min, held for 10 min, and the average recovery of the sample was calculated.

[0165] 5.4 Sample content determination

[0166] Weigh 1.0 g of the microemulsion and prepare the test solution. Chromatographic injection conditions are as follows: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), carrier gas: high-purity helium, flow rate: 1 mL / min, split ratio: 15:1, injection volume: 1 μL, inlet temperature: 260°C; ionization source: EI, ionization energy: 70 eV, ionization source temperature: 230°C; temperature program: initial temperature: 90°C, increase to 175°C at a rate of 2°C / min, hold for 5 min, then increase to 210°C at a rate of 1°C / min, hold for 10 min. The contents of β-elemene and curcumene in the sample were calculated based on the peak areas.

[0167] 6 Experimental results

[0168] 6.1 Appearance and Properties of Microemulsion

[0169] The appearance of MEO microemulsion is clear, uniform, transparent and has good fluidity. The MEO microemulsion droplets under transmission electron microscope are round with clear edges and the particle size meets the requirements. Figure 9 .

[0170] 6.2 Identification of MEO Microemulsion Types

[0171] After Sudan III and methylene blue were added to MEO microemulsion, the methylene blue diffused faster, so it was judged that MEO microemulsion was O / W type. Figure 10 shown.

[0172] 6.3 Determination of Particle Size and Zeta Potential of MEO Microemulsion

[0173] The average particle size of MEO microemulsion was 89.62±0.2487nm, the average PDI was 0.1126±0.0164 (n=3), and the Zeta potential of MEO microemulsion was -0.2135±0.27mV (n=3). Figure 11 .

[0174] 6.4 Study on the Stability of MEO Microemulsion

[0175] 6.4.1 Physical stability of microemulsions

[0176] After high-speed centrifugation, the MEO microemulsion did not separate into layers and had a clear appearance, which indicated that the centrifugal stability was good.

[0177] 6.4.2 Thermodynamic Stability of Microemulsions

[0178] After the MEO microemulsion was subjected to heating-cooling and freezing-thawing cycle experiments, it was found that the appearance of the microemulsion remained clear and transparent without stratification. Figure 12 As shown, it shows that MEO microemulsion has good thermodynamic stability.

[0179] 6.4.3 Accelerated Stability of Microemulsions

[0180] During the 3 months of storage, samples were taken at the end of each month to test the conductivity, particle size and appearance of the MEO microemulsion. The results are shown in Table 10.

[0181] Table 10 Accelerated stability results

[0182]

[0183] 6.5 GC-MS determination of index components and methodology

[0184] 6.5.1 Methodological Review

[0185] 6.5.1.1 Specificity Inspection

[0186] See the results Figure 13 After comparison, it was found that the peak at around 16.5 min in the MEO microemulsion test sample was β-elemene, and the peak at around 21.7 min was curcumene. There were no other impurity peaks nearby, and neither of them was detected in the blank solvent. This shows that β-elemene and curcumene can be selected as reference substances in the specificity experiment.

[0187] 6.5.1.2 Linear Relationship Investigation

[0188] β-elemene reference was injected at the following concentrations: 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625 mg mL -1 The curcumene reference substance was injected at the following concentrations: 4.44, 2.96, 1.98, 1.32, and 0.88 mg mL -1 , the data were obtained by measuring the corresponding chromatographic peak area. Then, linear regression analysis was performed using concentration and peak area as the horizontal and vertical coordinates, and the linear regression equation of β-elemene was obtained: y = 8.94657 × 10 12 ±1.53271×10 15 +(-4.37171×10 7 ±3.39992×10 7 -8.94657×10 12 ±1.53271×10 15 ) / (1+(x / 100784.27313±2.14843×10 7 )^0.80648±0.05659); R2=0.99977 and the linear regression equation of curcumene: y=8.47238×10 11 ±4.62057×10 14 +(3.50099×10 7 ±4.22917×10 8 -8.47238×10 11 ±4.62057×10 14 ) / (1+(x / 18416.91104±1.28824×10 7 )^0.79077±0.88583), R2=0.9982, standard curve see Figure 14 The results showed that within the selected concentration gradient range, the reference substances β-elemene and curcumene showed a good linear relationship.

[0189] 6.5.1.3 Precision test

[0190] The results are shown in Table 11. The data show that the RSD values ​​of the peak areas of β-elemene and curcumene in MEO microemulsion are 1.05% and 1.51%, respectively, indicating that the instrument has good precision.

[0191] Table 11 Precision test of standard products

[0192]

[0193] 6.5.1.4 Stability test

[0194] Table 12 shows the stability test data. The results show that under the chromatographic conditions, the relative standard deviations of the peak areas of β-elemene and curcumene in the microemulsion were 0.96% and 0.80%, respectively, which indicates that the test sample remained stable within 24 hours.

[0195] Table 12 MEO microemulsion stability test results

[0196]

[0197] 6.5.1.5 Repeatability Experiment

[0198] The repeatability test data shown in Table 13 show that the RSD values ​​of the peak areas of β-elemene and curcumene in the microemulsion were 2.26% and 1.46%, respectively, which indicates that the method has good repeatability.

[0199] Table 13 MEO microemulsion repeatability test results

[0200]

[0201] 6.5.1.6 Sample recovery test

[0202] The results are shown in Table 14 and Table 15. The average recoveries of β-elemene and curcumene in MEO microemulsion were 99.1% and 101.39%, respectively, and the RSD values ​​were 1.45% and 1.81%, respectively.

[0203] Table 14 β-elemene recovery experimental results in MEO microemulsion

[0204]

[0205] Table 15 Experimental results of recovery of curcumene in MEO microemulsion

[0206]

[0207] 6.5.1.7 Sample content determination

[0208] The average contents of β-elemene and curcumene in the samples were calculated to be 0.7127 mg·g -1 , 1.7124mg·g -1 , RSD values ​​were 3.06% and 3.29% respectively. The results are shown in Table 16.

[0209] Table 16 β-elemene and curcumene content in MEO microemulsion

[0210]

[0211] 7. Summary

[0212] This section evaluated the quality of the microemulsion and characterized it using transmission electron microscopy. The results showed that the microemulsion had a rounded appearance and a relatively uniform particle size distribution. The diffusion rate of methylene blue was significantly faster than that of Sudan III, indicating that it was an O / W type microemulsion. The average particle size of the microemulsion was 89.62±0.2487nm, the average PDI was 0.1126±0.0164, and the zeta potential was -0.2135±0.27mV. Microemulsion stability testing revealed no significant changes in appearance across high-speed centrifugation, heating-cooling cycles, or freeze-thaw cycles. In accelerated stability testing, the MEO microemulsion remained clear and uniform in monthly tests for three consecutive months, with no significant changes in particle size or conductivity. The content determination experiment of β-elemene and curcumene in MEO microemulsion showed that β-elemene and curcumene had good linearity within a certain concentration range. The linear regression equation of β-elemene was y=8.94657×10 12 ±1.53271×10 15 +(-4.37171×10 7 ±3.39992×10 7 -8.94657×10 12 ±1.53271×10 15 ) / (1+(x / 100784.27313±2.14843×10 7 )^0.80648±0.05659); R2=0.99977 and the linear regression equation of curcumene: y=8.47238×10 11 ±4.62057×10 14 +(3.50099×10 7 ±4.22917×10 8 -8.47238×10 11 ±4.62057×10 14 ) / (1+(x / 18416.91104±1.28824×10 7 )^0.79077±0.88583), R2=0.9982, y=374632x+2×108, R 2=0.9992. In the methodological investigation, the specificity of the two index components, β-elemene and curcumene, in the MEO microemulsion was strong, with no interference from other peaks. The RSDs for precision were 1.05% and 1.51%, respectively; the RSDs for repeatability were 2.26% and 1.46%, respectively; and the RSDs for stability within 24 hours were 0.96% and 0.80%, respectively. The average recoveries were 99.1% and 101.39%, respectively, with RSDs of 1.45% and 1.81%, respectively. These results demonstrate that the prepared microemulsion exhibits high stability, and the preparation process is feasible and reliable, providing a solid foundation for the development of new microemulsion formulations of myrrh essential oil.

[0213] The beneficial effects of the present invention are demonstrated by the following pharmacodynamic tests.

[0214] Experimental Example 1 Pharmacodynamic Study on the Treatment of Ulcerative Colitis with Myrrh Essential Oil

[0215] 1 Animal grouping and establishment of UC model

[0216] Sixty SPF-grade BALB / c male mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., China, weighing (20±2) g, with animal license SCXK (Sichuan) 2020-030. The mice were adapted for one week in a constant pressure, temperature of 22±2°C, and relative humidity of 55±2% with good ventilation. This experiment was approved by the Ethics Committee of Shaanxi University of Chinese Medicine (approval number SUCMDC20220530003). BALB / c mice were randomly divided into 6 groups of 10 mice each, namely the Control group, Model group, Positive group, MEO low-dose group, MEO medium-dose group, and MEO high-dose group. Except for the Control group, the drinking water of the other groups was replaced with 3% DSS solution for modeling, and drug administration began on the second day of modeling. The control group was gavaged with rapeseed oil at 0.01 ml / g body weight, while the MEO low-dose (concentration of 3.75 mg / kg), MEO medium-dose (concentration of 7.5 mg / kg), and MEO high-dose (concentration of 15 mg / kg) groups were gavaged with myrrh volatile oil using rapeseed oil as a carrier at 0.01 ml / g body weight, and DAI scores were performed.

[0217] 2. Biological sample collection and processing

[0218] After the last dose, mice were fasted for 24 hours but not water. They were anesthetized with isoflurane, and their eyeballs were removed for blood collection. The serum was centrifuged (4°C, 4000 rpm) for 10 minutes, aliquoted, and stored at -20°C. Mice were euthanized, and their colons were dissected and their natural lengths recorded. One portion of the colon was fixed with paraformaldehyde, and the other portion was snap-frozen in liquid nitrogen and then transferred to -80°C for storage.

[0219] Determination of inflammatory factors in serum of 3UC mice

[0220] ELISA was used to determine the levels of TNF-α and IL-1β in mouse serum. Frozen serum was removed and assayed for the proinflammatory factors TNF-α and IL-1β according to the kit instructions. OD values ​​were measured at a wavelength of 450 nm using a microplate reader. Standard curves were drawn and the levels of each indicator were calculated.

[0221] 4. Hematoxylin and eosin (HE) staining

[0222] Colon tissues of mice in each group were collected, dehydrated with gradient ethanol, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and counterstained with eosin, dehydrated until transparent, and mounted.

[0223] 5. Immunohistochemical detection of protein expression of p-JNK, p-ERK and TNF-α in colon

[0224] The colon tissues of each group of mice fixed with 4% paraformaldehyde were taken and routine washing, dehydration, wax immersion, embedding, and sectioning were performed. After dewaxing with xylene, gradient hydration with ethanol was performed. Non-immune, normal sheep serum was added to block nonspecific antigens. After washing with PBS, primary antibody was added and incubated at 4°C overnight. The next day, it was placed at room temperature for 40 minutes and washed again with PBS. HRP-labeled secondary antibody (rabbit antibody) was added and incubated at 37°C for 1 hour, and then washed with PBS. After DAB staining, hematoxylin counterstaining was performed, and gradient ethanol dehydration was performed. After xylene transparentization, the slides were mounted.

[0225] 6. Western blot analysis experiments

[0226] Colon tissue from three mice in each group was weighed, placed in a mixture of RIPA lysis buffer and protease inhibitors, and then broken in a high-speed homogenizer. The cells were centrifuged at 12,000 rpm for 5 minutes at 4°C. Serum protein concentration was then determined using a BCA kit. After electrophoresis using sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, the proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. Primary antibodies were added to p-JNK (Mitaka, 1:1000), JNK (Mitaka, 1:3000), p-ERK (Sevier, 1:400), and ERK (abcam, 1:1000), with GAPDH (Hua'an Biotechnology, 1:5000) as an internal control. The cells were incubated overnight at 4°C. The membranes were washed three times with TBST at room temperature. A horseradish peroxidase (HRP)-labeled secondary antibody (Jackson, 1:5000) was added and incubated at room temperature for 30 min. The membrane was washed three times with TBST and developed with ELC colorimetric reagent. Proteins were visualized using an imager and the relative expression levels of the target proteins were calculated.

[0227] 7 Statistical analysis

[0228] The data were statistically analyzed using GraphPad Prism 9.0.0 software, and the differences between the groups were compared using one-way analysis of variance (ANOVA). Statistical significance was considered when p < 0.05.

[0229] 8 Experimental results

[0230] 8.1 DAI score and colon morphology of UC mice

[0231] The weight of mice in the control group gradually increased. The feces of mice in the model and MEO groups were loose or even thin on the third day after modeling, and they lost weight severely. On the fourth day, blood in the stool appeared. However, the condition of mice in the MEO group improved after the fourth day, with a slow increase in weight and normal stool characteristics. The colon length of mice in the model group was short, and the mucosa was congested and swollen, while the colon of mice in the MEO group was longer, indicating that MEO can effectively alleviate ulcerative colitis in mice. DAI scores and weight changes of mice in each group, colon pictures and colon length differences are shown in the figure. Figure 15 .

[0232] 8.2 Results of determination of inflammatory factors in UC mouse serum

[0233] ELISA kits were used to detect the levels of TNF-α and IL-1β in mouse serum. Figure 16 Compared with the Control group, the levels of TNF-α and IL-1β in the Model group were significantly increased (p<0.01). Compared with the Model group, the levels of TNF-α and IL-1β in the Positive group and MEO group decreased to varying degrees (p<0.01). The above data indicate that MEO can reduce the expression of inflammatory factors in UC mice.

[0234] 8.3 HE staining results

[0235] The results of colon tissue staining of mice in different groups are as follows Figure 17 As shown in the figure, the colonic tissue structure of mice in the control group was normal; the overall intestinal tissue structure of mice in the model group was significantly abnormal, with a large number of inflammatory cells infiltrating the tissue; the high-dose MEO group showed normal structure, a small amount of inflammatory cell infiltration, and no necrosis. This indicates that the high-dose myrrh volatile oil group has a more effective effect in inhibiting ulcerative colitis.

[0236] 8.4 Immunohistochemistry Results

[0237] Immunohistochemistry results are shown in Figure 18 A large number of positive cells were stained in the colon tissue of the Model group; the area of ​​positive cell staining in the colon tissue of the MEO group was reduced to varying degrees. This suggests that myrrh essential oil can significantly reduce the expression of p-JNK, p-ERK, and TNF-α in the colon of diseased mice.

[0238] 8.5 Western blotting results

[0239] The experimental results showed that the expression levels of p-JNK, JNK, p-ERK and ERK in the Model group mice were significantly increased (p < 0.01). The expression levels of p-JNK, JNK, p-ERK and ERK in the MEO group decreased (p < 0.05), and the effect was close to that of the positive drug group. This shows that MEO can effectively inhibit the activation of the MAPK signaling pathway in the colon after DSS induction. Figure 19 shown.

[0240] 4 Summary

[0241] In this chapter, a mouse UC model was established using DSS. The body weight and fecal status of the mice were observed, and the condition of the mice in each myrrh essential oil dose group improved compared to the model group. Combined with the results of HE staining, it can be concluded that MEO can significantly improve colonic inflammation in UC mice. Immunohistochemistry was used to preliminarily verify proteins in the MAPK signaling pathway, and the expression of p-JNK, p-ERK, and TNF-α proteins in the colon was significantly reduced after MEO treatment. Further verification was performed using Western-blot experiments, and the results showed that the expression of p-JNK, JNK, p-ERK, and ERK in the MEO group was decreased (p < 0.05). This suggests that myrrh essential oil has a significant inhibitory effect on ulcerative colitis and may inhibit the MAPK signaling pathway by regulating proteins such as p-JNK and p-ERK.

[0242] Experimental Example 2 Pharmacodynamic Study on Myrrh Volatile Oil Microemulsion in the Treatment of Ulcerative Colitis

[0243] 1 Animal grouping and establishment of UC model

[0244] Ninety SPF-grade BALB / c male mice, weighing (20 ± 2) g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., China, under animal license SCXK (Sichuan) 2020-030. The mice were housed in a constant pressure environment with a temperature of 22 ± 2°C and a relative humidity of 55 ± 2% under good ventilation for one week. This experiment was approved by the Ethics Committee of Shaanxi University of Chinese Medicine (approval number SUCMDC20220530003). BALB / c mice were randomly assigned to nine groups of 10 mice each: control, model, positive, low-dose MEO, medium-dose MEO, high-dose MEO, low-dose myrrh oil microemulsion (MM), medium-dose MM, and high-dose MM. Except for the control group, the drinking water of all other groups was replaced with 3% DSS solution for modeling. Drug administration began on the second day of modeling. The control group was gavaged with rapeseed oil at 0.01 ml / g body weight, the MEO low-dose and MM low-dose groups (concentration was 3.75 mg / kg), MEO medium-dose and MM medium-dose groups (concentration was 7.5 mg / kg), MEO high-dose and MM high-dose groups (concentration was 15 mg / kg) were gavaged with 0.01 ml / g body weight, and DAI scores were performed.

[0245] 2. Biological sample collection and processing

[0246] After the last dose, mice were fasted for 24 hours but not water. They were anesthetized with isoflurane, and their eyeballs were removed for blood collection. The serum was centrifuged (4°C, 4000 rpm) for 10 minutes, aliquoted, and stored at -20°C. Mice were euthanized, and their colons were dissected and their natural lengths recorded. One portion of the colon was fixed with paraformaldehyde, and the other portion was snap-frozen in liquid nitrogen and then transferred to -80°C for storage.

[0247] Determination of inflammatory factors in serum of 3UC mice

[0248] ELISA was used to determine the levels of TNF-α and IL-1β in mouse serum. Frozen serum was removed and assayed for the proinflammatory factors TNF-α and IL-1β according to the kit instructions. OD values ​​were measured at a wavelength of 450 nm using a microplate reader. Standard curves were drawn and the levels of each indicator were calculated.

[0249] 4. Hematoxylin and eosin (HE) staining

[0250] Colon tissues of mice in each group were collected, dehydrated with gradient ethanol, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and counterstained with eosin, dehydrated until transparent, and mounted.

[0251] 5 Statistical analysis

[0252] The data were statistically analyzed using GraphPad Prism 9.0 software, and the differences between the groups were compared using one-way analysis of variance (ANOVA). A statistically significant difference was considered when p < 0.05.

[0253] 3 Experimental results

[0254] 3.1 DAI score and colon morphology of UC mice

[0255] The weight of mice in the Control group gradually increased, and the feces of mice in the Model, MEO and MM groups were loose on the second to third days after modeling. Some mice had thinner feces, lost weight, and had bloody stools on the fourth day. However, mice in the MEO and MM groups began to improve on the fifth day, gradually gained weight, and their fecal characteristics returned to normal. In terms of colon length, the colon length of mice in the Model group was short, and the mucosa was congested and swollen, while the colon of mice in the MEO and MM groups was longer, indicating that myrrh volatile oil and myrrh volatile oil microemulsion can effectively relieve ulcerative colitis in mice. DAI scores and weight changes of mice in each group, colon pictures and colon length difference are shown in the figure. Figure 20 .

[0256] 3.2 Results of determination of inflammatory factors in serum of UC mice

[0257] ELISA kits were used to detect the levels of TNF-α and IL-1β in mouse serum. The higher the levels of TNF-α and IL-1β in serum, the more severe the inflammation. Figure 21 As shown in the results, compared with the Control group, the TNF-α and IL-1β levels in the Model group were significantly increased (p<0.01). Compared with the Model group, the TNF-α and IL-1β levels in the Positive, MEO, and MM groups decreased to varying degrees (p<0.01). Comparison between the MEO and MM groups showed that the MM group had a better effect in inhibiting the release of TNF-α and IL-1β factors. The results showed that myrrh volatile oil and myrrh volatile oil microemulsion can effectively reduce the expression of inflammatory factors in UC mice, and the high-dose myrrh volatile oil microemulsion group had the best effect.

[0258] 3.3 HE staining results

[0259] The results of HE staining of the colon tissues of mice in each group are shown in Figure 22 The intestinal tissue structure of mice in the control group was normal;

[0260] The overall intestinal structure of mice in the Model group was significantly abnormal, with a large number of inflammatory cells infiltrating the tissue. In the MEO group, the structure was basically normal at both the medium and high doses, with only a small amount of inflammatory cells infiltrating. The area of ​​necrosis was small at the medium dose, and no necrosis was observed at the high dose. In the MM group, the structure was normal at both the medium and high doses, with minimal inflammatory cell infiltration and no necrosis. This suggests that myrrh volatile oil and myrrh volatile oil microemulsion can improve the condition of ulcerative colitis, and the high-dose myrrh volatile oil microemulsion group is more effective in inhibiting ulcerative colitis.

[0261] 4 Summary

[0262] In this chapter, a mouse ulcerative colitis model was established using DSS, the DAI scores of the mice were observed and recorded, and the UC mice were treated with myrrh volatile oil and myrrh volatile oil microemulsion of the same concentration by gavage. The pharmacodynamics of myrrh volatile oil and myrrh volatile oil microemulsion were evaluated based on the disease activity index of the mice, the levels of TNF-α and IL-1β in serum, HE staining results and immunohistochemistry results, and the effects of myrrh volatile oil and myrrh volatile oil microemulsion in treating UC were compared.

[0263] ELISA assays revealed that both MEO and MM reduced the expression of inflammatory factors TNF-α and IL-1β at different doses, but the extent of reduction varied. Comparison of the data revealed that the MM group had a superior overall therapeutic effect to the MEO group, with the highest dose in the MM group demonstrating the greatest improvement. Combined with HE staining, it was clear that both MEO and MM significantly improved colonic inflammation in mice, with the MM group exhibiting superior efficacy in treating UC. These results suggest that myrrh volatile oil microemulsions are effective, even surpassing volatile oils. Appropriate microemulsion formulations can effectively improve UC and therefore represent a promising new treatment option.

Claims

1. Use of myrrh volatile oil in preparing medicine for treating colitis.

2. The use according to claim 1, characterized in that: The medicine is a medicine for treating ulcerative colitis.

3. The use according to claim 2, characterized in that: The drug achieves the effect of resisting ulcerative colitis by regulating p-JNK and p-ERK proteins on the MAPK signaling pathway.

4. The use according to any one of claims 1 to 3, characterized in that: The myrrh volatile oil contains furanoeudesma 1,3-diene (not less than 20% w / w), curzerene (not less than 17% w / w), lindestrene (not less than 9% w / w), atractylone (not less than 8.00% w / w), and beta-elemene (not less than 7.00% w / w).

5. The use according to claim 4, characterized in that: The volatile oil is extracted by steam distillation, the soaking time is 0.5-2 hours, the amount of water is 4-12 times, and the extraction time is 6-10 hours.

6. The use according to claim 5, characterized in that: The soaking time of the steam distillation extraction is 1.5 hours, the amount of water added is 8 times, and the extraction time is 8 hours.

7. A myrrh volatile oil microemulsion, characterized by: The O / W microemulsion is prepared from myrrh volatile oil, an emulsifier, an auxiliary emulsifier, and an auxiliary oil phase; wherein the emulsifier is EL-40; the auxiliary emulsifier is glycerol; the auxiliary oil phase is isopropyl myristate IPM; and the Km is 2:

1. The myrrh volatile oil:IPM=1:9; total emulsifier:oil phase=9:1; The average particle size of the microemulsion was 89.62 ± 0.2487 nm, the average PDI was 0.1126 ± 0.0164, and the Zeta potential was -0.2135 ± 0.27 mV.

8. A method for preparing the myrrh volatile oil microemulsion according to claim 7, characterized in that: The microemulsion is prepared by a phase inversion emulsification method, which includes the following steps: a. Mixing myrrh volatile oil with an auxiliary oil phase to prepare a mixed oil phase; b. Mixing the emulsifier and the co-emulsifier to prepare a mixed emulsifier; c. Evenly mix the mixed oil phase and the mixed emulsifier, dropwise add ultrapure water while stirring, to obtain an o / w emulsion; wherein the stirring temperature is 25°C.

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