CN107951946B - Preparation method of cortex phellodendri processed product extract for resisting oral ulcer - Google Patents
Preparation method of cortex phellodendri processed product extract for resisting oral ulcer Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicine preparation, and particularly relates to a preparation method of an extract of an anti-oral ulcer phellodendron amurense processed product. The preparation method defines the processing technology of honey phellodendron amurense, and extracts the honey phellodendron amurense with ethyl acetate, and the prepared extract of the processed phellodendron amurense product has the drug effect of resisting canker sore, thereby providing a foundation for researching and developing new drugs.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine preparation, and particularly relates to a preparation method of an extract of an anti-oral ulcer phellodendron amurense processed product.
Background
Cortex Phellodendri is a commonly used Chinese medicinal material with effects of clearing heat, eliminating dampness, clearing pathogenic fire, removing steam, removing toxic substance and treating sore, and is called as cortex Phellodendri, and is a Rutaceae plant cortex PhellodendriPhellodendron chinense Schneid, dried bark, originally named "Berberis amurensis", listed as the top grade, was recorded in Shen nong's herbal Jing. The processed products of the traditional Chinese medicine phellodendron include raw products, salt products, honey products, human milk fried products, calcined products, wine products and the like. At present, the preparation process of the processed phellodendron bark products which is well-known in pharmacopoeia adopts salt roasting and charcoal making. Yuan- 'Tang Ye (decoction) records that the honey is fried into fine powder to treat aphtha, such as Shen, Yuan-Lu (treasure decoction) records that the honey is fried into fine powder to treat aphtha paralysis, and Qing-' Zhen Yi (differentiation) records thatStir-baked with honey decoction, it is collected to lie on the septum but not drop, and indicated for symptoms of feverish sensation in the chest, palms and soles, eye pain and aphtha. According to the ancient records, honey-fried phellodendron bark has a great effect on treating aphtha, and in patent CN103784847, "an external powder for treating oral ulcer", honey-fried phellodendron bark is used, and the honey-fried method is shown in Lei gong Bao Zhi Lun, and the method has the following defects that in Lei gong Bao Zhi Lun, the mentioned processing method has no specific parameter setting, the mentioned frying method is a method of Wen Wu fire Bao, and in the earlier experiments, the effective component berberine in phellodendron bark is found to have thermal instability at high temperature, so the firepower in the processing process cannot be too high, and Wu fire cannot be selected in the processing process, otherwise, the berberine component is destroyed in a large amount to further reduce the drug effect, so the original record mode needs to be updated and improved. The literature of honey-roasting and clinical application examination of phellodendron refers to the process of honey-roasting method of phellodendron, and the monograph of stir-roasting in the literature of herbal medicine of the past generations is recorded, but only the existing literature of processing science of traditional Chinese medicine and Chinese pharmacopoeia are not recorded, and the related reports are few. The honey-roasting process of the phellodendron amurense is recorded according to the herbal literature: every jin of the phellodendron shreds is prepared with honey three to two (150 g), a small amount of water is added, uniformly stirred with the phellodendron shreds, moistened thoroughly, fried with slow fire to moderate degree. The process has the following defects that the temperature used in the processing process of the golden cypress is not related, but in the research process, the processing temperature factor is found to have a significant influence on the quality of the golden cypress processed with honey, so that the processing temperature for processing the golden cypress processed with honey is necessary to be set, in addition, the patent also specifically limits the polar part of the honey product, and can treat the symptoms of the oral ulcer more specifically and effectively, and the original documents have related records.
Therefore, the development of an economical, convenient and simple-to-operate method for preparing an extract of a processed phellodendron amurense product for resisting oral ulcer is a new problem to be solved at present.
The invention content is as follows:
the invention aims to provide a method for processing phellodendron amurense to resist canker sore, which defines the processing technology of honey phellodendron amurense and the drug effect of resisting canker sore and provides a foundation for researching and developing new drugs. The honey phellodendron amurense processed product extract obtained by the method has good effect of treating aphtha.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing cortex Phellodendri processed product extract for resisting oral ulcer comprises the following steps:
step 1: adding honey water (the mass ratio of honey to water =0.5-2:1, preferably honey to water =1: 1) into the clean phellodendron amurense decoction pieces, uniformly stirring, moistening until the honey water is absorbed completely, completely permeating the honey water into the decoction piece tissues, and frying at the temperature of 120-130 ℃ for 2-6 min, preferably 4 min. And cooling, and removing impurities, wherein 10-30Kg of honey is used for every 100Kg of phellodendron amurense decoction pieces, and preferably 30Kg of honey is used for every 100Kg of phellodendron amurense decoction pieces.
Step 2: and (2) adding 70% ethanol solvent in an amount which is 5-15 times that of the honey-fried phellodendron bark, extracting twice with reflux, wherein each time is 1 hour, combining the two extracting solutions, and recovering ethanol under reduced pressure until no ethanol smell exists, so as to obtain an ethanol extract. Dispersing the extract with appropriate amount of water, extracting with ethyl acetate, extracting the residual water layer with ethyl acetate, recovering solvent to obtain ethyl acetate extract, dissolving with water, and adjusting concentration to 0.11 g/mL.
The phellodendron amurense processed product extract for resisting oral ulcer is applied to the medicine for treating oral ulcer. The prepared cortex Phellodendri processed product extract for resisting oral ulcer can improve NO, SOD and MDA level contents of damaged oral fibroblast, and can repair oxidative damage of oral cell.
Compared with the prior art, the invention has the beneficial effects.
The existing technology has no exact parameters in the honey-roasting process of part of phellodendron amurense, and easily causes unexpected effect in the roasting process, and meanwhile, in order to highlight related treatment effects, the technology conducts the research on the effect of resisting canker sore on honey-roasted phellodendron amurense with different polarities, finds that the effect of an ethyl acetate layer in the honey-roasted phellodendron amurense is the most prominent, and thus selects a more effective treatment scheme for diseases.
According to the application, the influence of honey-fried golden cypress on oxidative stress reaction of oral cells is investigated from a cell level by establishing a mouse oral fibroblast oxidative damage model, so that the treatment effect of honey-fried golden cypress is verified, and a certain experimental basis is provided for clinical medication. The subject group firstly discovers that the oxidative stress reaction of phellodendron honey-fried, especially ethyl acetate layer polar part, on oral cells is obviously enhanced when phellodendron honey-fried before and after the phellodendron honey-processing is carried out on pharmacodynamics comparison study. The invention of the technology can improve the medicinal value of the processed phellodendron bark product, finds the medicinal efficacy of honey phellodendron bark, and can be further developed into the anti-canker sore medicine.
The honey phellodendron amurense processed product prepared by the preparation method and the application of the honey phellodendron amurense processed product provided by the invention are not recorded in the existing research reports, the preparation method has the characteristics of high efficiency, stable process, simplicity and convenience in operation and low cost, and the honey phellodendron amurense has a good effect of resisting oral ulcer and has a stronger treatment effect compared with a crude phellodendron amurense product.
Drawings
FIG. 1 is a high performance liquid chromatogram of a cortex Phellodendri reference sample and a sample.
Wherein, 1 is phellodendrine hydrochloride, 2 is berberrubine, and 3 is berberine hydrochloride.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples, and all the techniques realized based on the above-described contents of the present invention are within the scope of the present invention.
Example 1.
1. Instruments and materials.
1.1 Instrument: waters e2695-2998 high performance liquid chromatograph (Waters corporation, USA), model METTLERAE240 type one hundred thousand analytical balance (METTLER, Switzerland), KQ-250DB type numerical control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan), FA1004B electronic balance (Shanghai precision scientific instruments, Inc.), oven (WS 70B type), double single-side ultra-clean workbench (Shanghai Intelligent science instruments, Inc.), CO 1004B electronic balance (Shanghai precision scientific instruments, Inc.), double-side ultra-clean workbench (Shanghai Intelligent science instruments, Inc.), and the like2Incubator (Japan Sanyo MCO-15 AC), microplate reader (American Sammer Feishell science and technology Co., Ltd.), TGL-16C type high-speed desk centrifuge (Shanghai' an Tingsu scientific Instrument Co., Ltd.), inverted microscope(Ningbo Yongxin NIB-100), a vertical pressure steam sterilizer (Shanghai Bo science instruments Co., Ltd.) -an 80 ℃ ultra-low temperature refrigerator (Qingdao Heire medical equipment Co., Ltd.), an ultraviolet-visible spectrophotometer (Beijing Pujingyu general instruments Co., Ltd.), a micropipette (Sammer Feishel instruments Co., Ltd.), an electronic balance (Shanghai Yao New electronic technology Co., Ltd.), and a vortex mixer (Shanghai Qingpu Huxi instruments Co., Ltd.).
1.2 reagent: cortex Phellodendri is purchased from Sichuan province medicinal materials; acetonitrile and methanol are chromatographically pure, water is double distilled water, and other reagents are analytically pure. The berberine hydrochloride reference substance is purchased from China institute for drug and biological products (lot No. 0713-. The phellodendrine hydrochloride reference substance is purchased from Kyormant biotechnologies Limited and used for content determination, and the purity is more than 98 percent. Sophora japonica Honey (Liu's bee product, Limited liability company, Inula province, Susong county, Anhui province). DMEM high-glucose medium (Thermo corporation), newborn fetal bovine serum (Thermo corporation), diabody (penicillin and streptomycin, Gibco corporation), non-essential amino acids (Gibco corporation), pancreatin (realta corporation), PBS (Solarbio corporation), 75% medical sterilized alcohol, CCK8 kit (nanjing has been built into biotechnology limited company), BCA protein concentration determination kit (picyunnan biotechnology institute), superoxide dismutase (SOD) determination kit (nanjing has been built into bioengineering institute), Nitric Oxide (NO) kit (nanjing has been built into bioengineering institute), cellular Malondialdehyde (MDA) kit (nanjing has been built into bioengineering institute), water is purified water, and the rest of the reagents are analytically pure.
2. The embodiment provides a preparation method of a honey phellodendron amurense processed product, which comprises the following steps:
adding honey water (honey: water =1: 1) into the cleaned cortex Phellodendri decoction pieces, stirring, moistening until the honey water is absorbed completely, and parching at 120-130 deg.C for 4 min. And (6) cooling and removing impurities. Extracting the honey-fried phellodendron bark with 10 times of 70% ethanol solvent, reflux-extracting twice for 1 hour each time, mixing the two extracting solutions, recovering ethanol under reduced pressure until no alcohol smell exists to obtain an alcohol extract, dispersing the alcohol extract with a proper amount of water, extracting with ethyl acetate, extracting the residual water layer with ethyl acetate, recovering the solvent to obtain an ethyl acetate extract, dissolving with water, and adjusting the concentration to 0.11g/mL to obtain the phellodendron bark honey-fried phellodendron bark extract.
3. A preparation process of the processed honey phellodendron amurense.
Taking the content of berberine hydrochloride and phellodendrine hydrochloride as evaluation indexes, selecting honey adding amount, frying temperature and frying time as investigation factors, and adopting orthogonal design L9(34) The best processing technique of honey-fried phellodendron is preferred.
Orthogonal experiments, as follows:
the index measuring method comprises the following steps: high performance liquid chromatography is adopted. Chromatographic conditions are as follows: column, Ecosil-C18 (250 mm x 4.6mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid solution (50: 50, 0.1g sodium dodecyl sulfate per 100 mL); the detection wavelength is 284 nm; the flow rate is 1 mL/min; the column temperature was 25 ℃. The chromatogram is shown in FIG. 1.
Solution preparation of control solution preparation: precisely weighing 4.82mg of berberine hydrochloride reference substance, 4.58mg of phellodendrine hydrochloride reference substance and 3.14mg of berrubine in 50mL volumetric flasks respectively, adding chromatographic methanol to dilute to scale, and shaking up to obtain 0.0964mg/mL berberine hydrochloride reference substance solution, 0.0916mg/mL phellodendrine hydrochloride reference substance solution and 0.0628 mg/mL berberine hydrochloride reference substance solution. Preparation of a test solution: taking about 0.1g of the powder (passing through a third sieve), precisely weighing, placing in a100 mL measuring flask, adding 80mL of mobile phase, performing ultrasonic treatment (power of 250W, frequency of 40 KHz) for 40min, and cooling. Diluting with mobile phase to scale, shaking, filtering, and collecting filtrate.
Composite score =0.6 × (Xi/Xmax) +0.2 × (Yi/Ymax) +0.2 × (Zi/Zmax)
The above formula X is berberine hydrochloride content, Y is phellodendrine hydrochloride content, Z is berrubine content, and the parameters of the two indexes of the comprehensive score of the phellodendron honey-fried product are respectively set as 60%, 20% and 20%, the same is given below.
The result of the variance analysis shows that the influence factor of honey-fried phellodendron is A (frying temperature)>B (honey adding quantity)>C (stir-frying time), and the stir-frying time and the honey addition amount of the three factors have significant influence on the content of the alkaloid of the phellodendron honey-fried. Determining the optimal processing process A by combining the visual analysis table and considering the actual production2B3C1Adding honey (30 Kg of honey is used for every 100Kg of decoction pieces, and the ratio of honey to water is 1: 1) to thoroughly moisten the decoction pieces, and frying for 4min at the bottom temperature of a pot of 120-130 ℃.
4. To further verify the beneficial effects of the invention, the following test case of "honey phellodendron against canker sore" is provided.
4.1 measurement method.
After L929 cells in logarithmic growth phase were digested, scraped, blown, adjusted to a cell density of 1X 107 cells/mL, and seeded in 96-well culture plates in a volume of 100. mu.L per well. After incubation at 37 ℃ in a 5% CO2 incubator for 24 h, the supernatant was discarded. Group 3, i.e. blank control: adding 200 μ L of blank culture solution; model control group: 2. mu.g/mL of H was added202100 μ L, and diluting with blank culture solution to a final volume of 200 μ L; administration group: adding different cortex Phellodendri crude product extractive solutions into medicated culture solution with dosage of 100 μ L and H2 μ g/mL202A final volume of 200. mu.L of 100. mu.L (final concentration of H202 of 1. mu.g/mL) was prepared in 3 duplicate wells per concentration. After culturing for 24 h in a 5% CO2 incubator at 37 ℃, determining a standard curve of a protein standard substance by using a microplate reader according to the operation of the BCA protein concentration determination kit instruction, and calculating the protein concentration of the sample according to the standard curve. According to the operation of the NO kit specification, the content of NO in the cells is determined by a nitric acid reductase method and an ultraviolet spectrophotometer. According toThe kit for measuring superoxide dismutase (SOD) and cell Malondialdehyde (MDA) is operated by an instruction book, the absorbance value of each hole is measured by an enzyme-labeling instrument, and the SOD content and the MDA content in the cell are calculated by using the measured protein concentration of the sample.
Statistical software SPSS17.0 is adopted for statistical treatment, data are represented by x ̅ +/-s, one-way anova is adopted for comparison among groups, P <0.05 is different, and the statistical significance is achieved.
4.2 results of measurement
4.2.1 Effect of cortex Phellodendri before and after stir-frying with Mel on NO content in L929 cells with oxidative damage
Compared to the blank groupP<0.05，**P<0.01; in comparison to the set of models,# P<0.05，## P<0.01; compared with the raw product, the honey-fried product with the same dosage:a P<0.05，aa P<0.01。
4.2.2 Effect of phellodendron bark on SOD content of oxidative damage L929 cells before and after honey roasting:
compared to the blank groupP<0.05，**P<0.01; in comparison to the set of models,# P<0.05，## P<0.01; compared with the raw product, the honey-fried product with the same dosage:a P<0.05，aa P<0.01。
4.2.3 Effect of phellodendron bark on MDA content of oxidative damage L929 cells before and after honey roasting:
in comparison to the blank set, the data is,* P<0.05，** P<0.01; in comparison to the set of models,# P<0.05，## P<0.01; compared with the raw product, the honey-fried product with the same dosage:a P<0.05，aa P<0.01。
from the experimental results, it can be known that the phellodendron honey-fried product can obviously increase the SOD activity and NO content of oxidative damage L929 cells (in comparison with the model group) ((P<0.05 orP<0.01), reducing the content of MDA (P<0.05 orP<0.01), which indicates that the phellodendron bark stir-baked with honey has certain effect of treating the oral ulcer, and the polar part of the ethyl acetate layer has the best effect of treating the oral ulcer among the different polar parts. Therefore, the ethyl acetate dosage form part is adopted for extraction in the preparation process of the technology, and the oral ulcer symptom is treated.
Example 2
100g of phellodendron amurense decoction pieces are added with honey water and stirred evenly (honey: water =1: 1), the mixture is moistened until the honey water is absorbed completely, stir-fried for 4 minutes at the temperature of 120-130 ℃, cooled naturally, and impurities are removed to obtain honey phellodendron amurense. Extracting cortex Phellodendri processed with honey with 10 times of 70% ethanol under reflux for 2 times (each time for 1 hr), filtering, mixing filtrates, and recovering ethanol under reduced pressure to obtain ethanol extract. Dispersing the extract with appropriate amount of water, extracting with ethyl acetate, extracting the residual water layer with petroleum ether, recovering solvent to obtain ethyl acetate extract, dissolving with water, and adjusting concentration to 0.11 g/mL.
Example 3
100g of phellodendron amurense decoction pieces are added with honey water and stirred evenly (honey: water =2: 1), moistened until the honey water is absorbed completely, and fried for 6 minutes under medium fire to obtain honey phellodendron amurense. Extracting cortex Phellodendri processed with honey with 15 times of 70% ethanol under reflux for 2 times, each for 1 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure to obtain ethanol extract. Dispersing the extract with appropriate amount of water, extracting with ethyl acetate, extracting the residual water layer with ethyl acetate, recovering solvent to obtain ethyl acetate extract, dissolving with water, and adjusting concentration to 0.11 g/mL.
Example 4
100g of phellodendron amurense decoction pieces are added with honey water and stirred evenly (honey: water =0.5: 1), moistened until the honey water is absorbed completely, and fried for 2 minutes under strong fire to obtain honey phellodendron amurense. Extracting cortex Phellodendri processed with honey with 5 times of 70% ethanol under reflux for 2 times, each for 1 hr, filtering, mixing filtrates, and recovering ethanol under reduced pressure to obtain ethanol extract. Dispersing the extract with appropriate amount of water, extracting with n-butanol, extracting the residual water layer with ethyl acetate n-butanol, recovering solvent to obtain ethyl acetate extract, dissolving with water, and adjusting concentration to 0.11 g/mL.
EXAMPLE 5 typical cases
Case 1: zhangqi, male, age 29, suffered from canker sores for 3 days, had two sores on the upper lip, and was occasionally painful when speaking. The pain was significantly reduced after application of the extract of example 2 of the present invention and was cured after 2 days.
Case 2: old woman, 24 years old, suffered from oral ulcer for 4 days, had 3 bean-sized ulcers visible on the lower part of the tongue, and felt pain at eating, and the pain was relieved after 5 days of smearing with the extract of the present invention example 3, and was cured after 7 days.
Claims (4)
1. A preparation method of an anti-canker sore phellodendron amurense processed product extract is characterized by comprising the following steps:
step 1: adding honey water into the clean phellodendron amurense decoction pieces, uniformly stirring, wherein the mass ratio of honey to water is 0.5-2:1, moistening until the honey water is absorbed completely, completely permeating the honey water into decoction piece tissues, frying for 2-6 min at the temperature of 120-130 ℃, cooling, removing impurities, and using 10-30Kg of honey for every 100Kg of phellodendron amurense decoction pieces;
step 2: taking a honey-fried phellodendron bark, adding a 70% ethanol solvent in an amount which is 5-15 times that of the honey-fried phellodendron bark, extracting twice with reflux, wherein each time is 1 hour, combining extracting solutions obtained in two times, recovering ethanol under reduced pressure until no alcohol smell exists to obtain an alcohol extract, dispersing the alcohol extract with a proper amount of water, extracting with ethyl acetate, extracting the residual water layer with ethyl acetate, recovering the solvent to obtain an ethyl acetate extract, then dissolving with water, and adjusting the concentration to 0.11g/mL to obtain the phellodendron bark honey-fried phellodendron bark.
2. The method for preparing the extract of the processed phellodendron amurense for resisting the dental ulcer according to claim 1, wherein the mass ratio of honey to water in the honey water in the step 1 is 1:1, and 30Kg of honey is used for every 100Kg of decoction pieces and stir-frying for 4 min.
3. The preparation method of the oral ulcer-resisting cortex phellodendri processed product extract according to claim 1, wherein the oral ulcer-resisting cortex phellodendri processed product extract is used in a medicament for treating oral ulcer.
4. The method for preparing the phellodendron amurense processed product extract for resisting the oral ulcer according to claim 1, wherein the prepared phellodendron amurense processed product extract for resisting the oral ulcer can improve the content of NO, SOD and MDA of damaged oral fibroblasts and repair the oxidative damage of the oral cells.
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| CN105541831A (en) * | 2016-01-15 | 2016-05-04 | 辽宁中医药大学 | Medicine root berberrubine derived from amur cork-tree processed product and separation preparation method and application thereof |
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| CN105541831A (en) * | 2016-01-15 | 2016-05-04 | 辽宁中医药大学 | Medicine root berberrubine derived from amur cork-tree processed product and separation preparation method and application thereof |
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