Preparation method of vinegar-processed frankincense and myrrh for improving bioavailability and efficacy of frankincense and myrrh

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a vinegar-processed frankincense and myrrh preparation method capable of improving oral bioavailability and efficacy of frankincense and myrrh, which comprises the following steps: respectively removing impurities from frankincense and myrrh to obtain clean frankincense and clean myrrh, freezing and crushing once to obtain frankincense coarse powder and myrrh coarse powder, and mixing the frankincense coarse powder and the myrrh coarse powder to prepare a vinegar-processed frankincense and myrrh powder; baking the frankincense or myrrh coarse powder at 80-120 DEG C for 30-90 minutes, immediately spraying 2.5-30% of rice vinegar, or spraying 2.5-30% of rice vinegar, baking at 80-120 DEG C for 30-90 minutes, immediately putting into a colloid mill with water, circularly grinding, cooling the obtained suspension dispersion emulsion to room temperature, freeze-drying by using a vacuum freeze-drying machine, and crushing to obtain the frankincense or myrrh freeze-dried powder with small particle size. The vinegar processing method provided by the invention can improve oral bioavailability, efficacy and production efficiency of frankincense and myrrh, alleviate the problem of environmental pollution, and clarify the processing mechanism so as to better guide clinical reasonable medication.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a method for preparing vinegar-processed frankincense and myrrh for improving the bioavailability and efficacy of frankincense and myrrh, and vinegar-processed frankincense and myrrh prepared by the preparation method. Background Art

[0002] Frankincense is the resin exuded from the bark of Boswellia carterii Birdw. and Boswelliabhaw dajiana Birdw., both plants of the Oleaceae family. Myrrh is the dried resin of Commiphora myrrha Engl. or Commiphora molmol Engl., both plants of the Oleaceae family. Both are blood-activating and stasis-removing drugs, and have similar effects, both of which have the effects of activating blood circulation and relieving pain. The main active ingredients of frankincense are triterpene acids, and 11-carbonyl-β-boswellic acid (KBA) and acetyl-11-carbonyl-β-boswellic acid (AKBA) have very significant anti-inflammatory and anti-tumor effects. The active ingredients of myrrh, 2-methoxy-5-acetyl-4-furanozim-1(10)-en-6-one (FSA), 2-methoxy-8,12-epoxyazim-1(10),7,11-trien-6-one (MCS134) and myrrhone, have significant biological activities. However, the low oral bioavailability of frankincense and myrrh limits their clinical application and development.

[0003] The Chinese Pharmacopoeia (2020 edition) stipulates that vinegar-processed frankincense should weigh about 100g of frankincense, place it in a hot pot and stir-fry on low heat until the surface melts, then spray 5% vinegar, and stir-fry until dry. The preparation method of myrrh is the same as that of frankincense. However, the original medicinal material is in the form of large hard blocks. When it is processed by the existing vinegar roasting method, the heat is difficult to control and it is easy to carbonize overfire; the rice vinegar is unevenly distributed, and it is difficult to achieve uniform adhesion by steaming in advance or directly sprinkling vinegar and stir-frying; the large amount of white smoke produced by frying will pollute the environment; the volatile oils fried in the finished product cause the surface to be sticky, and adhesion to each other or sticking to the wall will affect the yield and inconvenience in operation, which is not conducive to standardized and eco-friendly production. For example, Hu Sulian et al. (Hu Sulian, Jiang Xiaohuang, Jiang Mengliang, et al. Study on the optimal frankincense processing technology using analgesia and stimulation experiments [J]. Chinese Patent Medicine, 2013, 7: 1515-1517) reported that the optimal frankincense baking process is 120℃ baking for 3h, drug placement thickness of 1cm, drug diameter of 1cm, and frankincense diameter of 1cm. However, this processing method has low crushing degree and long baking time. The baked frankincense is sticky, large in size, difficult to crush, and has low extraction efficiency. Niu Chao et al. (Niu Chao, Zhang Huimin. Orthogonal optimization and verification test of frankincense vinegar production process [J]. Modern Chinese Medicine, 2015, 17 (03): 247-249) conducted orthogonal optimization and verification test of frankincense vinegar production process and believed that the best process for frankincense vinegar production is to take 300g of frankincense, stir-fry it at 140℃ for 9 minutes, until the surface is slightly melted, spray 15mL of 5% rice vinegar, stir-fry for another 1 minute, until the vinegar liquid is absorbed and it is shiny, then quickly take it out of the pot and let it cool. However, this processing method is difficult to control the heat during frying, the rice vinegar is unevenly distributed, and the finished product is very sticky. Chinese patent application CN110478387A discloses a method for preparing vinegar myrrh, and specifically discloses that rice vinegar is poured into a container, then an appropriate amount of deionized water is added for dilution, and the mixture is stirred to obtain a rice vinegar dilution solution, wherein the ratio of deionized water to rice vinegar is 1-4:1, myrrh is added and simmered for 2-3 hours, wherein the weight ratio of myrrh to vinegar solution is 20-30:1; the mixture is poured into a constant temperature controlled frying device and fried at 150°C-155°C for 20-30 minutes. The processing process of the application bakes the myrrh powder at 100°C for 1 hour, and removes the irritating volatile oil components by heating, but the method does not examine the bioavailability and efficacy of the processed product.

[0004] In view of this, without destroying the integrity of the multi-component nature of traditional Chinese medicine, it is urgent to solve the problems of low crushing degree, long frying time, difficult to control the heat, uneven distribution of rice vinegar, large amount of white smoke polluting the environment, high viscosity of the finished product, unclear processing mechanism, etc. in the traditional vinegar-roasting method of frankincense and myrrh. Developing a new process for preparing frankincense and myrrh with vinegar that is simple in processing method and high in oral bioavailability is a problem to be solved in this field. Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to provide a method for preparing vinegar-processed frankincense that improves the oral bioavailability and efficacy of frankincense and myrrh, thereby increasing the dissolution and in vivo absorption of the main effective ingredients, significantly enhancing the efficacy of frankincense and myrrh, that is, significantly enhancing the anti-inflammatory and anti-ulcer effects of frankincense, and significantly enhancing the dissolution of the effective ingredients of myrrh and the treatment of cold coagulation and blood stasis. At the same time, the processing method of the present invention can improve production efficiency, alleviate environmental pollution problems, and clarify the processing mechanism, which is conducive to better guiding clinicians in medication.

[0006] Specifically, the present invention is realized through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing vinegar-processed frankincense and myrrh to improve the bioavailability and efficacy of frankincense and myrrh, the preparation method comprising the following steps:

[0008] (1) Purification: Weigh a certain amount of frankincense and myrrh respectively, remove impurities, and grind the obtained pure frankincense and pure myrrh once to obtain coarse frankincense powder and coarse myrrh powder;

[0009] (2) Preparation of Frankincense Vinegar: Myrrh is prepared by the method described in step (2) above, or the following method is used to prepare Myrrh Vinegar: The coarse frankincense powder prepared in step (1) is weighed and sieved, and then dried in an electric constant temperature forced air drying oven preheated at 100° C. to 150° C. for 30 to 90 minutes to obtain partially deoiled frankincense. After the prepared partially deoiled frankincense is taken out of the drying oven, it is immediately sprayed with 2.5% to 30% by weight of rice vinegar, and then immediately put into a colloid mill with water and circulated and ground to a fineness of 10 to 50 μm to obtain a suspension dispersion;

[0010] (3) Preparation of myrrh vinegar: The myrrh coarse powder prepared in step (1) is weighed and sieved, and then 2.5% to 30% by weight of rice vinegar is sprayed on and mixed evenly, and then dried in an electric constant temperature blast drying oven preheated at 80-120° C. for 30-90 min to obtain partially degreased myrrh, which is then immediately put into a colloid mill for circulation grinding to a certain fineness to obtain a myrrh suspension dispersion;

[0011] (4) Freeze-drying: The suspension dispersion prepared in step (2) and step (3) is cooled to room temperature and then freeze-dried, crushed and sieved, and the finished freeze-dried powder is stored.

[0012] As an optional manner, in the above preparation method, in step (2), a 20-mesh sieve is used, the preheating temperature is 120° C., and the baking time is 60 min.

[0013] As an optional manner, in the above preparation method, in step (2), the rice vinegar used is 5% by weight, and the colloid mill is set to 1-5 gears at 10-50 μm scale.

[0014] Preferably, in step (2), the rice vinegar used is 5% by weight, and the colloid mill is set to level 3 at 30 μm.

[0015] As an optional manner, in the above preparation method, in step (3), a 20-mesh sieve is used, the preheating temperature is 100° C., and the baking time is 60 min.

[0016] As an optional manner, in the above preparation method, in step (3), the rice vinegar used is 5% by weight, and the colloid mill is set to 1-5 gears at 10-50 μm scale.

[0017] Preferably, in step (3), the rice vinegar used is 5% by weight, and the colloid mill is set to level 3 at 30 μm.

[0018] As an optional manner, in the above preparation method, in step (4), freeze-drying is performed using a vacuum freeze dryer and the product is crushed to pass through a 60-80 mesh sieve.

[0019] In a second aspect, the present invention provides vinegar-processed frankincense and myrrh prepared by the preparation method described in the first aspect, wherein the vinegar-processed frankincense and myrrh have higher equilibrium solubility, smaller particle size and larger specific surface area than those prepared by traditional processing methods.

[0020] In a third aspect, the present invention provides vinegar-processed frankincense and myrrh prepared by the preparation method described in the first aspect, wherein the vinegar-processed frankincense and myrrh have higher bioavailability than those prepared by traditional processing methods.

[0021] In a fourth aspect, the present invention provides vinegar-processed frankincense prepared by the preparation method described in the first aspect, and the vinegar-processed frankincense has stronger anti-inflammatory and analgesic effects than the vinegar-processed frankincense prepared by the traditional processing method. The present invention also provides vinegar-processed myrrh prepared by the preparation method described in the first aspect, and the vinegar-processed myrrh has stronger therapeutic effect on cold stagnation and blood stasis than the vinegar-processed myrrh prepared by the traditional processing method.

[0022] As an optional manner, in the second to fourth aspects above, the traditional preparation method of vinegar-made frankincense or myrrh includes the following steps: weigh frankincense or myrrh, fry over low heat in a hot pot until the surface is melted, spray 5% by weight of rice vinegar, and fry until dry.

[0023] More preferably, the traditional preparation method of vinegar-made frankincense comprises the following steps: breaking raw frankincense into small pieces, heating them in a pot over low heat, stir-frying until the surface is slightly melted into a spherical shape, spraying 5% rice vinegar by weight when smoke comes out of the pot, stir-frying while spraying to ensure uniform heating and full contact with the rice vinegar, and quickly taking out the frankincense when the surface has an oily luster, and collecting it for later use after the temperature drops to room temperature.

[0024] As an optional method, the traditional preparation method of vinegar-made myrrh includes the following steps: the bark and branches of the myrrh are removed, the myrrh is broken into small pieces, the pieces are placed in a pot and heated over low heat, and stir-fried until the surface is slightly melted into a spherical shape, when smoke comes out of the pot, 5% rice vinegar by weight is sprayed, and the spraying and stir-frying are carried out at the same time to ensure that the heat is evenly distributed and fully contacted with the rice vinegar, and the myrrh is quickly taken out when the surface has an oily luster, and the temperature is lowered to room temperature before being collected for later use.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention is based on the relevant processing methods recorded in ancient books of frankincense and myrrh and the theoretical achievements of modern research, and combines modern industrial machinery to realize controllable processing, and develops a novel vinegar-processed frankincense and myrrh preparation method for improving the oral bioavailability and efficacy of frankincense and myrrh. The processing method is to respectively remove impurities from frankincense and myrrh to obtain clean frankincense and clean myrrh, freeze-crush them once to obtain frankincense coarse powder and myrrh coarse powder, and immediately spray 5% rice vinegar after the frankincense coarse powder is baked at 120°C for 60 minutes, so that a large amount of white smoke will not be generated to pollute the environment and affect the health of workers. Then, immediately put it into a colloid mill with water for cyclic grinding, and after the obtained suspension dispersion milk is cooled to room temperature, it is freeze-dried by a vacuum freeze dryer, which is convenient for pulverization, and a frankincense freeze-dried powder with a small particle size is obtained, so that the equilibrium solubility of the main component triterpenoid compounds is high. The myrrh coarse powder is sprayed with 5% rice vinegar, baked at 100°C for 60 minutes, and immediately put into the colloid mill for cyclic grinding, and the obtained suspension dispersion milk is rotary evaporated, freeze-dried, and pulverized to obtain a finished freeze-dried powder. The method for preparing frankincense and myrrh vinegar of the present invention can improve the oral bioavailability and efficacy of frankincense and myrrh as well as the production efficiency, alleviate the problem of environmental pollution, and clarify the processing mechanism to better guide clinical rational drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The preparation process flow chart of the novel vinegar-processed frankincense and myrrh preparation method for improving the oral bioavailability and efficacy of frankincense of the present invention.

[0028] Figure 2 : Effect of baking time on equilibrium solubility of main components in frankincense.

[0029] Figure 3: Effect of rice vinegar dosage on equilibrium solubility of main components in frankincense.

[0030] Figure 4 : Effect of grinding degree on equilibrium solubility of main components in frankincense.

[0031] Figure 5 :The effect of baking time on the equilibrium solubility of the main components in myrrh. Among them: A: FSA; B: MCS134; C: myrrhone.

[0032] Figure 6 :The effect of vinegar dosage on the equilibrium solubility of the main components in myrrh. Among them: A: FSA; B: MCS134; C: myrrhone.

[0033] Figure 7 :The effect of grinding degree on the equilibrium solubility of the main components in myrrh. Among them: A: FSA; B: MCS134; C: myrrhone.

[0034] Figure 8 :Effects of each drug group on the colon length of DSS-induced ulcerative colitis mice. Compared with the control group: ## P<0.01; compared with the model group: * P<0.05.

[0035] Fig. 9 :Effects of each drug group on DSS-induced ulcerative colitis damage in mice. A. Control; B. Model; C. RX; D. CRX; E. XCRX; F. BPYCW, magnification is 100 times.

[0036] Fig.10 :Effects of each drug group on coagulation time of rats with cold coagulation and blood stasis syndrome. Data are expressed as mean ± SD, n = 6. Compared with the control group: ### P<0.001; compared with the model group: ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0037] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0040] Example 1: A method for preparing frankincense prepared with vinegar to improve oral bioavailability and efficacy of frankincense 1. Investigation of process parameters of new frankincense prepared with vinegar

[0041] (1) Baking time (A): Weigh 30 g of frankincense coarse powder and pass it through a 20-mesh sieve. Then, bake it in a preheated oven at 120° C. for 6 baking times of 0, 30, 45, 60, 75, and 90 min. Immediately spray 3 g of rice vinegar on it and then immediately put it into a colloid mill (first gear) for cycle grinding. The resulting suspension dispersion is appropriately concentrated, cooled to room temperature, and then freeze-dried. After being crushed, it is sieved through an 80-mesh sieve. The finished freeze-dried powder samples A0-A5 are stored. The equilibrium solubility of the samples is measured and the experiment is repeated 3 times.

[0042] The results show that if Figure 2 As shown in the figure, the baking time directly affects the degreasing effect of frankincense. At 30 minutes, the surface of the medicinal material has obvious oiliness. At about 45 minutes, the medicinal material texture becomes soft and easy to grind. At 60 minutes, the equilibrium solubility of the main active ingredients KBA and AKBA is the highest. At 75 minutes, the equilibrium solubility decreases significantly, and then the change tends to be gentle. The index of the processed product baked for 60 minutes is 2-3 times that of the unbaked processed product.

[0043] (2) Amount of rice vinegar (B): Weigh 30 g of frankincense coarse powder and pass it through a 20-mesh sieve, then bake it in a preheated oven at 120° C. for 60 min, spray it with rice vinegar in five ratios (0%, 5%, 10%, 20%, and 30%), take it out, and immediately put it into a colloid mill (first gear) for cycle grinding. The resulting suspension dispersion is appropriately concentrated, cooled to room temperature, and then freeze-dried. After crushing, pass it through an 80-mesh sieve, and save the finished freeze-dried powder samples B1-B5. The equilibrium solubility of the samples is measured and the experiment is repeated 3 times.

[0044] The results show that if Figure 3 As shown, the amount of rice vinegar added has a significant effect on the change in equilibrium solubility. When 5% of rice vinegar is added, the equilibrium solubility of the main active ingredients is the highest, followed by a downward trend, and slowly decreases after 20% is added.

[0045] (3) Grinding degree (C): 30 g of frankincense powder was weighed and passed through a 20-mesh sieve. After drying in a preheated oven at 120°C for 60 min, 3 g of rice vinegar (Haitian brand white rice vinegar, total acid ≥ 3.50 mg / 100 mL) was immediately sprayed on the powder. The powder was then immediately put into a colloid mill and ground in a cycle with 6 fineness settings (0: no grinding, 1: 50 μm, 2: 40 μm, 3: 30 μm, 4: 20 μm, 5: 10 μm). The resulting suspension was appropriately concentrated, cooled to room temperature, and then freeze-dried. After being crushed, the powder was passed through an 80-mesh sieve. The finished freeze-dried powder samples C0-C5 were stored. The equilibrium solubility of the samples was measured and the experiment was repeated 3 times.

[0046] The results show that if Figure 4 As shown, the equilibrium solubility of the processed product that has not been ground by a colloid mill is the lowest, and the equilibrium solubility of the processed product obtained when set to "level 3" is the highest.

[0047] 2. Characterization of the properties of new vinegar frankincense

[0048] (1) Properties: The morphology of the powders of frankincense, vinegar frankincense, and new vinegar frankincense (prepared using the preferred process parameters in Part 1 above) after sieving was characterized using a scanning electron microscope. After 5000 times magnification, it can be seen that the surface of the frankincense powder is relatively smooth, the surface of the vinegar frankincense powder is rough, and the surface of the new vinegar frankincense is not only rough, but also has dense small pores.

[0049] The preparation method of vinegar frankincense is as follows: take raw frankincense and break it into small pieces, put it in a pot and heat it on low heat, stir-fry until the surface is slightly melted into a spherical shape, spray 5% rice vinegar by weight when smoke comes out of the pot, stir-fry while spraying to ensure uniform heating and full contact with the rice vinegar, and quickly take it out when the surface of the frankincense has an oily luster, wait for the temperature to drop to room temperature, and collect it for later use.

[0050] (2) Equilibrium solubility: 2 g each of frankincense (RX), vinegar frankincense (CRX), and new vinegar frankincense (XCRX) powder were prepared into equilibrium solubility solutions, and the equilibrium solubility was measured.

[0051] Table 1: Equilibrium solubility of frankincense, vinegar frankincense, and new vinegar frankincense

[0052]

[0053] (3) Particle size statistics: Electron microscope was used to scan and photograph randomly sampled frankincense, vinegar frankincense, and new vinegar frankincense powder samples for analysis and observation. Nano Measurer 1.2 software was used to mark and locate 100 powder particles in the SEM results, and the particle size was statistically analyzed as shown in Table 2. The obtained data was analyzed by distribution statistics using Origin 2021 software and Gaussian fitting was performed. The final particle size distribution showed a significant contrast in the sample particle size, with frankincense > vinegar frankincense > new vinegar frankincense.

[0054] Table 2: Particle size data of frankincense, vinegar frankincense, and new vinegar frankincense

[0055]

[0056] Embodiment 2: A new process for preparing myrrh vinegar

[0057] 1. Investigation of process parameters of new vinegar myrrh

[0058] (1) Baking time (A): Take 30g of coarse myrrh powder that has passed through a 20-mesh sieve, spray 1.5g of rice vinegar and stir evenly, bake in a preheated oven at 100°C for 6 baking times of 0, 30, 45, 60, 75, and 90min, and immediately put it into a colloid mill (level 2) for cyclic grinding. The resulting suspension dispersion is appropriately concentrated, cooled to room temperature, and then freeze-dried. After crushing, pass through an 80-mesh sieve, and save the finished freeze-dried powder samples A0-A5. The equilibrium solubility of the samples is determined and the experiment is conducted in parallel 3 times. The results show that the baking time affects the degreasing effect of myrrh. During the experiment, it was found that when myrrh was baked, there was obvious oiliness on the surface of the medicinal material after 15min, and the texture of the medicinal material became soft and easy to grind after about 30min. After baking for 60min, the equilibrium solubility of the main active ingredients FSA, MCS134 and myrrhone was the highest. Figure 5 .

[0059] (2) Dosage of rice vinegar (B): Take 30g of coarse myrrh powder that has been passed through a 20-mesh sieve, spray it with rice vinegar in 6 proportions (0, 2.5%, 5%, 10%, 12.5%, 15%), bake in a preheated oven at 100°C for 60 minutes, and immediately put it into a colloid mill (2nd gear) for cycle grinding. The resulting suspension dispersion is appropriately concentrated, cooled to room temperature, and then freeze-dried. After crushing, pass through an 80-mesh sieve, and save the finished freeze-dried powder samples B0-B5. The equilibrium solubility of the samples is measured and the experiment is repeated 3 times. The results show that the amount of rice vinegar added has a significant effect on the change in equilibrium solubility. When the proportion of rice vinegar added is 5%, the equilibrium solubility of the main active ingredient is the highest, and then there is a downward trend. Figure 6 .

[0060] (3) Grinding degree (C): Take 30g of coarse myrrh powder that has passed through a 20-mesh sieve, spray 1.5g of rice vinegar and stir evenly, bake in a preheated oven at 100°C for 60 minutes, and immediately put it into a colloid mill for cyclic grinding with 6 fineness settings (0 gear without grinding, 1 gear, 2 gear, 3 gear, 4 gear, 5 gear). The resulting suspension dispersion is appropriately concentrated, cooled to room temperature, and then freeze-dried. After crushing, pass through an 80-mesh sieve, and save the finished freeze-dried powder samples C0-C5. The equilibrium solubility of the samples is determined and the experiment is repeated 3 times. The results show that the equilibrium solubility of the processed product that has not been ground by a colloid mill is the lowest, and the equilibrium solubility of the processed product obtained when set to "2 gear" is the highest, see Figure 7 .

[0061] 2. Characterization of the properties of new vinegar myrrh

[0062] (1) Properties: The powder samples of myrrh (MY), vinegar myrrh (CMY), and new vinegar myrrh (XCMY) were scanned and photographed by scanning electron microscopy (SEM), and the samples were analyzed and observed. After magnification of 1600 times, it can be seen that the surface of myrrh powder is relatively smooth, the surface of vinegar myrrh powder is relatively rough, and the surface of vinegar myrrh is rough and has obvious holes.

[0063] (2) Equilibrium solubility: 2 g of each of myrrh (MY), vinegar myrrh (CMY), and new vinegar myrrh (XCMY) powder were prepared into equilibrium solubility solutions, and the equilibrium solubility was measured.

[0064] Table 3: Equilibrium solubility of myrrh, vinegar myrrh, and new vinegar myrrh

[0065]

[0066] (3) Particle size: SEM was used to characterize the appearance of myrrh, vinegar myrrh, and new vinegar myrrh. NanoMeasurer 1.2 software was used to mark and locate 50 powder particles in the SEM results, and the particle size was counted. The obtained data was analyzed for distribution statistics and Gaussian fitting using Origin 2021 software, and the average particle size D was calculated. 50 , boundary particle size D 10 , D 90 The particle size comparison of myrrh, vinegar myrrh, and new vinegar myrrh samples under SEM magnification of 50 times is obvious, which is myrrh > vinegar myrrh > new vinegar myrrh. The relevant statistical values ​​of powder particle size are shown in Table 4. The results show that the particle size of myrrh powder is significantly reduced after processing.

[0067] Table 4: Particle size data of myrrh, vinegar myrrh, and new vinegar myrrh

[0068]

[0069] Example 3: Pharmacokinetic study of new vinegar frankincense in rats

[0070] 1. Chromatographic conditions:

[0071] UPLC H-Class ultra high performance liquid chromatograph; chromatographic column: ACQUITYUPLC BEH C18 column (100×2.1 mm, 1.7 μm); mobile phase: A: acetonitrile, B: water-formic acid (100:0.1, V / V); gradient elution program: 0-0.4 min, 30% B; 0.4-1.2 min, 30-15% B; 1.2-5.0 min, 15-10% B; 5.0-5 min, 10-5% B; 5.5-6.0 min, 5-30% B; detection wavelength: 250 nm; column temperature: 35°C; flow rate: 0.4 mL / min; injection volume: 2 μL.

[0072] 2. Collection and processing of plasma samples:

[0073] Twelve healthy male SD rats were fasted for 12 hours but not water, and then randomly divided into three groups (n=4): A (frankincense group), B (vinegar frankincense group), and C (new vinegar frankincense group). The drug was administered orally at a dose of 2.7 g / kg. Blood was collected from the rats' eye sockets at 0h, 15min, 30min, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 6h, 8h, 12h, and 24h, and heparin sodium was added for anticoagulation. The plasma was allowed to stand for 30min, then centrifuged at 13000r / min for 10min, and the supernatant was taken and placed in a -80℃ refrigerator for later use. 200 μL of plasma was collected from the rat orbit, placed in a 1.5 mL centrifuge tube, and 20 μL of glycyrrhetinic acid internal standard solution was added and vortexed for 30 seconds; 600 μL of ethyl acetate was added and vortexed for 5 minutes for extraction, centrifuged at 13000 r / min for 10 minutes, the supernatant was drawn and dried under a nitrogen flow in a 40 ° C water bath, and the residue was re-dissolved with 50 μL of acetonitrile-water (70:30), vortexed for 5 minutes, centrifuged at 13000 r / min for 10 minutes, and the supernatant was drawn for injection to obtain the internal standard, KBA and AKBA peak areas, and the drug-time curves of KBA and AKBA were drawn using Origin.2021 software. The data were analyzed and fitted using DAS2.0 software to obtain the main pharmacokinetic parameters of KBA and AKBA components in rats after oral administration of frankincense, vinegar frankincense and new vinegar frankincense suspension, as shown in Tables 5 and 6, respectively.

[0074] Table 5: In vivo KBA pharmacokinetic parameters after oral administration of frankincense, vinegar frankincense, and new vinegar frankincense

[0075]

[0076]

[0077] Table 6: Pharmacokinetic parameters of AKBA in vivo after oral administration of frankincense, vinegar frankincense, and new vinegar frankincense

[0078]

[0079] The experimental results showed that the metabolism of KBA in rats conforms to the two-compartment open model. max , AUC 0-t and AUC 0-∞ The results showed that the new vinegar frankincense > vinegar frankincense > frankincense, indicating that frankincense processing can increase the absorption of KBA and delay the elimination of KBA, and the new process has a more obvious promoting effect than the vinegar roasting method. The AUC of vinegar frankincense 0-t and AUC 0-∞ It is about 3 times that of frankincense, while the AUC of new vinegar frankincense is 0-t and AUC 0-∞ Both are about 6 times that of frankincense.

[0080] The experimental results showed that the metabolism of AKBA in rats conformed to the one-compartment open model. max , AUC 0-t and AUC 0-∞ The results showed that the new vinegar frankincense > vinegar frankincense > frankincense, indicating that frankincense processing can increase the absorption of AKBA, and the promoting effect of the new process is significantly better than that of vinegar roasting. The AUC of vinegar frankincense AKBA in rats 0-t and AUC 0-∞ Both are more than twice that of frankincense, while the AUC of new vinegar frankincense is 0-t and AUC 0-∞ It can be up to about 7 times that of frankincense.

[0081] Example 4: Study on the anti-inflammatory and analgesic effects of new vinegar frankincense on mice

[0082] 1. Therapeutic effect of new vinegar frankincense on DSS-induced ulcerative colitis in mice

[0083] (1) Modeling method:

[0084] The mouse UC model was established by the dextran sulfate sodium induction method. All mice were first adaptively fed and their body weights were monitored. At the beginning of the modeling, drinking water was replaced with DSS solution. During this period, each mouse was given 5 mL of 2% dextran sulfate sodium (DSS) solution every day, and mouse food was supplied normally. On the 8th day of modeling, the remaining DSS solution was replaced with drinking water in time. The ulcerative colitis model was considered to be successfully established when the mice showed a decrease in body weight, an increase in the number of bowel movements, thinner feces, blood and (or) mucus in the stool.

[0085] (2) Grouping and administration:

[0086] Except for the blank group (A), the successfully modeled animals were randomly divided into the model group (B), RX frankincense group (C), CRX vinegar frankincense group (D), XCRX new vinegar frankincense group (E) and positive drug BPYCW Bupi Yichang pill group (F), with 6 mice in each group. All mice were given medication on the first day of the experiment, and the blank group and model group were gavaged with 5% sodium carboxymethyl cellulose solution instead of drugs every day for 14 consecutive days. Modeling started on the fifth day of medication and lasted for one week.

[0087] (3) Detection:

[0088] The model was established on the fifth day of administration for one week, and the DAI level of mice was observed every day. After 14 days of administration, the mice were anesthetized and dissected, and blood was collected from the abdominal aorta and processed and the serum was preserved. The colon tissue was gently peeled off and rinsed with saline, the colon mucosal injury index was observed and the colon length was measured. The diseased colon segment was taken to make HE-stained sections and observe the histological changes of the colon. The levels of cytokines TNF-α, IL-1β, and IL-6 in the serum of mice were determined using ELISA kits, and the experimental data were sorted and processed.

[0089] 1) Observation of Disease Activity Index (DAI):

[0090] From the beginning of modeling to the end of the experiment, the mice were weighed and their feces were observed daily to calculate the daily DAI score of the mice. The scoring method is shown in Table 5 and the calculation method is as follows:

[0091] DAI = (weight change score + stool shape score + stool bleeding score) / 3

[0092] The changes in the disease activity index of mice showed that the health status of the blank group mice did not change significantly within two weeks; the model group mice began to show disease reactions from the fifth day of modeling, manifested as hair loss and weight loss, diarrhea and even bloody stools in the last few days, indicating that the modeling was successful; the mice in the four treatment groups also showed the same disease manifestations from the 5th to the 11th day, but as the number of treatment days increased, the degree of change in disease activity was different. Compared with the model group, the index of the frankincense group was lower and the treatment inflection point appeared on the 13th day; the vinegar frankincense group, the new vinegar frankincense group and the Bupi Yichang Pill group basically showed an inflection point on the 12th day, and the condition improved. The treatment effect was generally Bupi Yichang Pill group> new vinegar frankincense group> vinegar frankincense group> frankincense group.

[0093] 2) Observation of colon mucosal damage index (CMDI):

[0094] Two weeks later, 10% chloral hydrate was injected into the mouse abdominal cavity, and the anesthetized mouse was fixed on the operating table. The colon tissue was peeled off and rinsed with normal saline. The degree of colon mucosal damage in the mouse was observed with the naked eye and scored. The colon tissue of the mouse was dissected and observed, and it was found that the intestinal mucosa of the model group mice was edematous or even ulcerated to varying degrees. The specific colon mucosal damage index and colon histological score are shown in Table 7. From the perspective of the colon mucosal damage index, the model group had a significant difference compared with the blank group, indicating that the model was successful. Compared with the model group, the frankincense group and the vinegar frankincense group had certain differences in each treatment group, indicating that there was a certain therapeutic effect. The new vinegar frankincense group and the Bupi Yichang pill group were significantly different, indicating that the new vinegar frankincense had a good therapeutic effect on the colon mucosal lesions of UC mice.

[0095] Table 7: CMDI scores of ulcerative colitis mice (mean ± SD, n = 6)

[0096]

[0097]

[0098] Note: Compared with the control group: ### P<0.001; compared with the model group: * P<0.05, ** P<0.01.

[0099] 3) Colon length measurement

[0100] The gently peeled colon tissue was soaked in a culture dish filled with physiological saline, and tweezers were used to help it stretch naturally. Do not pull it hard. The length of the colon of each mouse was measured and recorded. Figure 8 .

[0101] 4) Colon histological observation and scoring (HS)

[0102] Take a section of colon tissue with the most obvious lesions in mice, immerse it in 10% neutral formalin for fixation, and perform routine paraffin embedding, sectioning, and HE staining. Observe the pathological changes of colon tissue under a light microscope and score. Fig. 9 Among them, the microscopic changes related to ulcerative colitis include abnormal crypt structure, abnormal epithelial morphological changes, and morphological changes of inflammatory infiltration.

[0103] The model group had significant differences compared with the blank group, indicating that the model was successfully established. Compared with the model group, the treatment groups were lower in the frankincense group and the vinegar frankincense group but not significant. The new vinegar frankincense group had significant differences, and the Bupi Yichang pill group had more significant differences. Overall, the influence of different processed products on the histological lesions of the mouse colon was new vinegar frankincense> vinegar frankincense> frankincense.

[0104] Table 8: Colon histological scoring table of ulcerative colitis mice

[0105]

[0106] Note: Compared with the control group: ## P<0.01; compared with the model group: * P<0.05, ** P<0.01.

[0107] 5) Detection of cytokines TNF-α, IL-1β, and IL-6 in serum

[0108] Blood was collected from the abdominal aorta of mice and stored in 1.5 mL EP tubes. After standing for 1 hour, the blood was centrifuged at 4°C for 10 minutes (3500 r / min -1 ), the supernatant was drawn into a clean EP tube, and the serum was stored at -40℃ for later use. The levels of TNF-α, IL-1β, and IL-6 in the serum of each group of mice were measured according to the instructions of the ELISA kit. The results showed that compared with the blank group, the TNF-α level in the model group was significantly increased, and the levels were significantly reduced after treatment in the drug-treated group. The treatment effect was Bupi Yichang Pills> New Vinegar Frankincense> Vinegar Frankincense> Frankincense; compared with the blank group, the IL-1β level in the model group was significantly increased, and its expression level in mice administered by oral administration was significantly reduced, and the treatment effect was New Vinegar Frankincense> Bupi Yichang Pills> Vinegar Frankincense> Frankincense; the IL-6 level in the model group was significantly higher than that in the blank group, indicating that the inflammatory model was successfully replicated, and its level was downregulated after oral administration of frankincense, while Vinegar Frankincense, New Vinegar Frankincense and Bupi Yichang Pills could significantly reduce the IL-6 level, and the effect was Bupi Yichang Pills> New Vinegar Frankincense> Vinegar Frankincense.

[0109] 2. Study on the analgesic effect of acetic acid-induced writhing reaction in mice

[0110] (1) Experimental groups and treatments

[0111] Kunming mice weighing 20 g ± 2 g were randomly divided into 5 groups, with 6 mice in each group. The blank group, positive drug group, frankincense group, vinegar frankincense group, and new vinegar frankincense group were intragastrically administered, and acetic acid solution was intraperitoneally injected, and the number of twisting behaviors of the mice was observed and recorded.

[0112] (2) Criteria for determining mouse writhing behavior

[0113] The abdomen of the mouse is obviously sunken inward, accompanied by the extension of the hind limbs or the lifting of the buttocks. The inhibition rate of the test drug on the writhing reaction of the mouse is calculated according to the following formula to evaluate the analgesic effect of frankincense, vinegar frankincense and new vinegar frankincense.

[0114] Inhibition rate (%) = (mean number of writhing in the blank group - mean number of writhing in the drug-treated group) ÷ mean number of writhing in the blank group × 100%

[0115] (3) Experimental results

[0116] The effects of different preparations on the analgesic effect of mice showed that mice began to have writhing reactions about 5 minutes after the injection of acetic acid solution. The mice in the blank group writhe frequently, while the number of writhings of the mice in the positive drug aspirin group was significantly reduced within 15 minutes, and the inhibition rate could reach 76.06%. The analgesic effects of frankincense, vinegar frankincense, and new vinegar frankincense were also very significant, and the number of writhings was significantly reduced compared with the blank group, and the new vinegar frankincense was significantly lower than that of vinegar frankincense. The inhibition ability was new vinegar frankincense>vinegar frankincense>frankincense.

[0117] Table 9: The number of writhing times and pain inhibition rate of mice in different drug groups

[0118]

[0119] Note: Compared with the control group: *** P<0.001; compared with the CRX group: ### P<0.001.

[0120] Example 5: Study on the therapeutic effect of new vinegar myrrh on rats with cold coagulation and blood stasis

[0121] 1. Modeling method

[0122] Adrenaline combined with ice water bath method was used to replicate the cold coagulation and blood stasis rat model. SD rats were placed in 0-4℃ ice water for 5 minutes, and the rats were taken out after their whole body became stiff, and the back hair was wiped dry. This was done once a day for 6 consecutive days. On the seventh day, adrenaline hydrochloride was subcutaneously injected (0.8 mg / kg, 2 times, 4 hours apart). Two hours after the first administration of adrenaline hydrochloride, the rats were placed in an ice water bath for 5 minutes.

[0123] 2. Grouping and Dosing

[0124] Thirty-six SD male rats were randomly divided into a blank group (Control), a model group (Model), a myrrh group (MY), a vinegar myrrh group (CMY), a new vinegar myrrh group (XCMY) and a compound Danshen dripping pill group (FFDSDW), with 6 rats in each group. After one week of adaptive feeding, the blank group and the model group were gavaged with an equivalent dose of 0.5% sodium carboxymethylcellulose solution every day, and each drug group was given a corresponding dose of drug (raw drug 1.8g / kg) once a day for 7 consecutive days. On the morning of the 8th day, blood was collected from the retro-orbital venous plexus of the rats 1 hour after gavage of each group.

[0125] 3. Detection

[0126] During the experiment, the experimental animals were observed and recorded at the same time every morning. Eight days after administration, the blood coagulation time of rats was measured by the capillary glass tube method, and blood was collected from the retroorbital venous plexus and the serum was processed and stored. The rats were anesthetized for dissection, the abdominal aorta tissue was gently peeled off, HE-stained sections were made, and the histological changes of the abdominal aorta were observed. The levels of coagulation indicators TXB2 and 6-keto-PGF1α in rat serum, and the levels of oxidative stress indicators SOD and MDA were determined by ELISA kits. The experimental data were sorted and processed.

[0127] (1) Coagulation time

[0128] The capillary glass tube method was used to measure the changes in rat blood coagulation time. On the morning of the 8th day, 1 hour after each group of rats were gavaged, a capillary was inserted into the rat's eye socket vein. Timing began when the blood flowed into the capillary. The capillary was pulled out after the blood filled the capillary and placed on a dry and smooth table. At regular intervals, 0.5 cm of the capillaries at both ends were broken and slowly pulled apart to observe whether there was coagulation at the break. When obvious coagulation was found, the time was recorded and the average value of the capillaries at both ends was taken as the coagulation time of the rat. Fig.10 Compared with the blank group, the coagulation time of the model group rats decreased significantly, indicating that the model was successfully established. Compared with the model group, the coagulation time of each drug-treated group was prolonged. There was no statistically significant difference between the myrrh group and the model group, but from the numerical intuitive analysis, myrrh has a therapeutic effect. There was a significant difference in treatment between the vinegar myrrh group and the new vinegar myrrh group, and the difference in the compound Danshen dripping pill group was more significant. Overall, the degree of influence of different preparations on the coagulation time of rats was new vinegar myrrh> vinegar myrrh> myrrh.

[0129] (2) Determination of coagulation indexes TXB2 and 6-keto-PGF1α

[0130] The collected blood samples were placed for 1 hour and then centrifuged at 4000r / min for 10min. The serum was transferred to EP tubes, and the content of thromboxane B2 (TXB2) and 6-keto-PGF1α in each group was determined by ELISA, and the TXB2 / 6-keto-PGF1α ratio was calculated.

[0131] The results showed that compared with the normal group, the TXB2 content of the model group rats was significantly increased, and the 6-keto-PGF1α content was significantly decreased. Compared with the model group, the TXB2 content of the drug administration group showed an increasing trend, with significant differences. Compared with the model group, the 6-keto-PGF1α content of the drug administration group showed a decreasing trend, with significant differences. Compared with the myrrh group, the TXB2 content of the new vinegar myrrh group was significantly reduced, and the 6-keto-PGF1α content was increased, indicating that the new process of myrrh vinegar can effectively enhance the effect of treating rats with cold coagulation and blood stasis. By calculating the TXB2 / 6-keto-PGF1α ratio, it can be seen that compared with the normal group, the TXB2 / 6-keto-PGF1α ratio of the model group was significantly increased. Compared with the model group, the ratios of the drug administration groups were significantly decreased, indicating that the coagulation state of the rats was improved after drug administration. In summary, the therapeutic effects of each drug administration group in rats with cold coagulation and blood stasis syndrome were new vinegar myrrh>compound danshen dripping pills>vinegar myrrh>myrrh.

[0132] (3) Determination of the content of oxidative stress indicators SOD and MDA

[0133] The collected blood samples were placed for 1 hour and then centrifuged at 3500 r / min for 10 minutes. The serum was transferred to an EP tube and the levels of inflammatory indicators SOD and MDA were determined strictly according to the steps in the kit instructions.

[0134] Compared with the normal group, the serum SOD activity of rats in the model group was significantly decreased, and the MDA content was significantly increased, indicating that the redox balance of rats with cold stagnation and blood stasis syndrome was disturbed. Compared with the model group, the SOD activity of vinegar myrrh group, new vinegar myrrh group and compound Danshen dripping pill group was significantly increased, and the MDA content was significantly decreased, indicating that different preparations can improve the symptoms of cold stagnation and blood stasis syndrome in rats by inhibiting oxidative stress. Compared with the myrrh group, the SOD activity of the new vinegar myrrh group was significantly increased, and the MDA content was significantly decreased, indicating that the new myrrh vinegar preparation process can effectively alleviate the oxidative stress damage of rats with cold stagnation and blood stasis syndrome.

[0135] (4) Histopathological examination

[0136] After blood was collected from the rats, 10% chloral hydrate was injected intraperitoneally for anesthesia, and the common carotid artery of the rats was obtained and placed in precooled phosphate buffered saline solution to wash away the blood, fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced, stained with H&E, and observed and photographed under an optical microscope at different magnifications.

[0137] The intima of the abdominal aorta of rats in the normal group was smooth, the structure of the smooth muscle cells in the media was basically clear, the direction was basically consistent, and the arrangement was relatively neat; the endothelial layer of the abdominal aorta of rats in the model group fell off and broke, the vascular endothelial cells protruded into the lumen in a nail-like column, the smooth muscle cells were arranged irregularly, and the cell nuclei were shrunken. The endothelial structure of rats in each drug-treated group was basically intact and clear, the smooth muscle cells were arranged in the order of new vinegar myrrh> vinegar myrrh> myrrh, and the degree of nail-like columnar protrusion of vascular endothelial cells into the lumen was myrrh> vinegar myrrh> new vinegar myrrh, but the degree of damage was lower than that of the model group.

[0138] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing vinegar-processed frankincense and myrrh to improve the bioavailability and efficacy of frankincense and myrrh, the method comprising the following steps: (1) Purification: Weigh a certain amount of frankincense and myrrh respectively, remove impurities, and grind the obtained pure frankincense and pure myrrh once to obtain coarse frankincense powder and coarse myrrh powder; (2) Preparation of frankincense vinegar: Weigh the frankincense coarse powder prepared in step (1), sieve it, and dry it in a constant temperature forced air drying oven preheated at 100° C.-150° C. for 30-90 minutes to obtain partially deoiled frankincense. After taking the prepared partially deoiled frankincense out of the drying oven, immediately spray 2.5%-30% by weight of rice vinegar on it, and then immediately put it into a colloid mill added with water and grind it in a cycle to a fineness of 10-50 μm to obtain a suspension dispersion. (3) Myrrh vinegar preparation: Myrrh is prepared by the method described in step (2) above, or the following method is used to prepare myrrh vinegar: the coarse myrrh powder prepared in step (1) is weighed and sieved, and then 2.5% to 15% by weight of rice vinegar is sprayed on the powder and stirred evenly, and then the powder is dried in a constant temperature forced air drying oven preheated at 80 to 120° C. for 30 to 90 minutes to obtain partially degreased myrrh, and then the powder is immediately put into a colloid mill for circulation grinding to a certain fineness to obtain a myrrh suspension dispersion; (4) Freeze-drying: The suspension dispersion prepared in step (2) and step (3) is cooled to room temperature and then freeze-dried, crushed and sieved, and the finished freeze-dried powder is stored.

2. The preparation method according to claim 1, characterized in that: In step (2), a 20-mesh screen is used, the preheating temperature is 120° C., the baking time is 60 min, the rice vinegar used is 5% by weight, and the colloid mill is set to 1-5 gears at 10-50 μm scale.

3. The preparation method according to claim 1, characterized in that: In step (3), a 20-mesh screen is used, the preheating temperature is 100° C., the baking time is 60 min, the rice vinegar used is 5% by weight, and the colloid mill is set to 1-5 gears at 10-50 μm scale.

4. The preparation method according to any one of claims 1 to 3, characterized in that: In step (4), freeze-drying is performed using a vacuum freeze dryer and the product is crushed and passed through a 60-80 mesh sieve.

5. A vinegar-processed frankincense and myrrh prepared by the preparation method according to any one of claims 1 to 4, characterized in that: Compared with the vinegar-processed frankincense and myrrh prepared by adopting the traditional processing method, the vinegar-processed frankincense and myrrh have higher equilibrium solubility, smaller particle size and larger specific surface area.

6. A vinegar-processed frankincense and myrrh prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The vinegar-processed frankincense and myrrh have higher bioavailability than those prepared by traditional processing methods.

7. A vinegar-processed frankincense and myrrh prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The vinegar-processed frankincense has stronger anti-inflammatory and analgesic effects than the vinegar-processed frankincense prepared by the traditional processing method, and the vinegar-processed myrrh has stronger therapeutic effect on cold coagulation and blood stasis than the vinegar-processed myrrh prepared by the traditional processing method.

8. The vinegar-processed frankincense and myrrh according to any one of claims 5 to 7, characterized in that: The traditional preparation method of vinegar-made frankincense or myrrh comprises the following steps: weighing frankincense or myrrh, frying in a hot pot over low heat until the surface is melted, spraying 5% by weight of rice vinegar, and frying until dry.

9. The vinegar-processed frankincense and myrrh according to claim 8, characterized in that: The traditional preparation method of vinegar-made frankincense comprises the following steps: breaking raw frankincense into small pieces, placing the pieces in a pot and heating them over a low heat, stir-frying until the surface is slightly melted into a spherical shape, spraying 5% rice vinegar by weight when smoke comes out of the pot, stir-frying while spraying to ensure that the frankincense is heated evenly and fully in contact with the rice vinegar, and quickly taking the pieces out when the surface of the frankincense has an oily luster, and collecting and setting aside the pieces after the temperature drops to room temperature.

10. The vinegar-processed frankincense and myrrh according to claim 8, characterized in that: The traditional preparation method of vinegar-based myrrh comprises the following steps: removing bark and branches from the myrrh, breaking it into small pieces, placing it in a pot and heating it over a low heat, stir-frying it until the surface is slightly melted into a spherical shape, spraying 5% rice vinegar by weight when smoke comes out of the pot, and stir-frying it while spraying to ensure that the heat is even and the myrrh is in full contact with the rice vinegar, and quickly taking it out when the surface of the myrrh has an oily luster, and collecting it for later use after the temperature drops to room temperature.

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