Extraction method and application of cortex juglandis mandshuricae extract

Abstract

The invention discloses an extraction method and application of a cortex juglandis mandshuricae extract, relates to the extraction method and application of the cortex juglandis mandshuricae extract, and relates to the field of traditional Chinese medicine extraction and chemical separation. In order to solve the problems of high dependence, high addiction and remarkable adverse reaction of the current clinically common anti-helicobacter pylori medicine, the method comprises the following steps: 1, weighing 20kg of cortex juglandis mandshuricae, adding ethanol with the volume concentration of 80% according to a solid-liquid ratio of 1: 10, soaking for 7 days, extracting for three times, combining extracting solutions, recovering a solvent, and freeze-drying to obtain BQLY-Z; and 2, adding water into 100g of the total extract of the cortex juglandis mandshuricae obtained in the step 1 for dissolving, and eluting by adopting a macroporous resin D101 chromatographic column, ultrapure water and ethanol with the volume concentration of 20%, 40%, 60% and 80% to obtain BQLY-0, BQLY-20, BQLY-40, BQLY-60 and BQLY-80. The cortex juglandis mandshuricae extract directly acts on helicobacter pylori and can inhibit the growth of the helicobacter pylori and increase the diameter of an inhibition zone.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of north blue dragon clothes extraction method and its application, it relates to the field of traditional Chinese medicine extraction and chemical separation. BACKGROUND

[0002] Helicobacter pylori (HP) is a highly infectious bacteria, with a global infection rate of up to 50%, and is associated with gastric cancer, gastric ulcer, gastritis and other diseases, and is listed as a class I risk factor for gastric cancer. Eradication of HP can significantly reduce the incidence and mortality of gastric cancer. Currently, the quadruple therapy based on proton pump inhibitors (PPI) is a common clinical treatment for gastric ulcer. However, due to the irrational use of antibiotics, drug resistance in patients is increasing, resulting in a decline in the overall effectiveness of quadruple therapy. Therefore, at this stage, finding a low drug resistance, stable and effective bacteriostatic alternative drug is a problem to be solved.

[0003] Traditional Chinese medicine has the advantages of multi-target and multi-path regulation in fighting against Helicobacter pylori, with low drug resistance risk, small adverse reactions, and the ability to improve the function of gastric mucosa as a whole. It has a broad prospect in the clinical treatment of Helicobacter pylori positive. SUMMARY

[0004] The present application aims to solve the technical problems of current clinical anti-Helicobacter pylori drugs, which are highly dependent and addictive, and have significant adverse reactions. The present application provides a method for extracting north blue dragon clothes extract and its application.

[0005] The extraction method of north blue dragon clothes extract is as follows:

[0006] Take 20 kg of north blue dragon clothes and soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days. Extract three times, combine the extract, recover the solvent, and freeze-dry to obtain north blue dragon clothes total extract.

[0007] The extraction method of north blue dragon clothes extract is as follows:

[0008] I. Take 20 kg of north blue dragon clothes and soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days. Extract three times, combine the extract, recover the solvent, and freeze-dry to obtain north blue dragon clothes total extract (BQLY-Z).

[0009] II. Dissolve 100 g of north blue dragon clothes total extract (BQLY-Z) obtained in step I in water, and use a macroporous resin D101 chromatographic column to elute with ultrapure water to obtain north blue dragon clothes ethanol extract water eluate (BQLY-0).

[0010] The extraction method of north blue dragon clothes extract is as follows:

[0011] I. Take 20 kg of North Qinglongyi, add 80% ethanol with a volume concentration of 1:10, soak for 7 days, extract 3 times, combine the extract, recover the solvent, freeze-dry, and obtain North Qinglongyi total extract (BQLY-Z);

[0012] II. Dissolve 100 g of North Qinglongyi total extract (BQLY-Z) obtained in step I in water, use a macroporous resin D101 chromatographic column, elute with 20% ethanol with a volume concentration, and obtain North Qinglongyi ethanol extract 20% ethanol eluate (BQLY-20).

[0013] The extraction method of North Qinglongyi extract is as follows:

[0014] I. Take 20 kg of North Qinglongyi, add 80% ethanol with a volume concentration of 1:10, soak for 7 days, extract 3 times, combine the extract, recover the solvent, freeze-dry, and obtain North Qinglongyi total extract (BQLY-Z);

[0015] II. Dissolve 100 g of North Qinglongyi total extract (BQLY-Z) obtained in step I in water, use a macroporous resin D101 chromatographic column, elute with 20% ethanol with a volume concentration, and obtain North Qinglongyi ethanol extract 20% ethanol eluate (BQLY-20).

[0016] The extraction method of North Qinglongyi extract is as follows:

[0017] I. Take 20 kg of North Qinglongyi, add 80% ethanol with a volume concentration of 1:10, soak for 7 days, extract 3 times, combine the extract, recover the solvent, freeze-dry, and obtain North Qinglongyi total extract (BQLY-Z);

[0018] II. Dissolve 100 g of North Qinglongyi total extract (BQLY-Z) obtained in step I in water, use a macroporous resin D101 chromatographic column, elute with 20% ethanol with a volume concentration, and obtain North Qinglongyi ethanol extract 20% ethanol eluate (BQLY-20).

[0019] The extraction method of North Qinglongyi extract is as follows:

[0020] I. Take 20 kg of North Qinglongyi, add 80% ethanol with a volume concentration of 1:10, soak for 7 days, extract 3 times, combine the extract, recover the solvent, freeze-dry, and obtain North Qinglongyi total extract (BQLY-Z);

[0021] II. Dissolve 100 g of North Qinglongyi total extract (BQLY-Z) obtained in step I in water, use a macroporous resin D101 chromatographic column, elute with 20% ethanol with a volume concentration, and obtain North Qinglongyi ethanol extract 20% ethanol eluate (BQLY-20).

[0022] The north green dragon clothes extract is used for inhibiting the growth of helicobacter pylori.

[0023] The north green dragon clothes is the dry pericarp of immature fruit of Juglans mandshurica Max. of Juglandace walnut genus. It is recorded in Song Dynasty “Kai Bao Ben Cao” for the first time, and is called walnut green dragon, and then “Shandong Chinese Herbal Medicine Handbook” calls it “green dragon clothes”. A large number of researches at home and abroad show that the north green dragon clothes has analgesic, anti-inflammatory, antibacterial and other pharmacological effects, and can be used for treating gastritis, stomachache, digestive tract cancer, bacterial dysentery, enteritis and other diseases. And helicobacter pylori mainly colonizes in gastric mucosa, especially in antrum, so the north green dragon clothes has broad prospects in the field of anti-helicobacter pylori.

[0024] The north green dragon clothes extract of the present application directly acts on helicobacter pylori, can inhibit the growth of helicobacter pylori, and increase the diameter of the bacteriostatic circle. The intervention of helicobacter pylori positive mice by intragastric administration proves that it can also reduce the expression level of inflammatory factors of helicobacter pylori positive mice, so the present application has good application prospect in the preparation of related drugs for intervention of helicobacter pylori positive gastric ulcer, and can be further used for the development of related drugs. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a photo of the growth of helicobacter pylori intervened by BQLY-Z; Figure 2 It is a photo of the growth of helicobacter pylori intervened by BQLY-0; Figure 3 It is a photo of the growth of helicobacter pylori intervened by BQLY-20; Figure 4 It is a photo of the growth of helicobacter pylori intervened by BQLY-40; Figure 5 It is a photo of the growth of helicobacter pylori intervened by BQLY-60; Figure 6 It is a photo of the growth of helicobacter pylori intervened by BQLY-80; Figure 7 It is a photo of the growth of helicobacter pylori intervened by 2-hydroxy-1,4-naphthoquinone; Figure 8 It is a photo of the comparison of the diameters of the bacteriostatic circles of the north green dragon clothes extracts; Figure 9 It is a comparison chart of the expression levels of inflammatory factors (IL-6) of mice; Figure 10 It is a comparison chart of the expression levels of inflammatory factors (IL-8) of mice. DETAILED DESCRIPTION

[0026] The technical scheme of the present application is not limited to the following specific embodiments, and also includes any combination of the specific embodiments.

[0027] Specific embodiment one: the extraction method of the north green dragon clothes extract in the present embodiment is as follows:

[0028] I. Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the total extract of North Qinglongyi (BQLY-Z).

[0029] II. Dissolve 100 g of the total extract of North Qinglongyi obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with ultrapure water to obtain the water eluate of the ethanol extract of North Qinglongyi (BQLY-0).

[0030] Specific embodiment II: The extraction method of the North Qinglongyi extract in this embodiment is as follows:

[0031] Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the total extract of North Qinglongyi.

[0032] Specific embodiment III: The extraction method of the North Qinglongyi extract in this embodiment is as follows:

[0033] I. Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the total extract of North Qinglongyi;

[0034] II. Dissolve 100 g of the total extract of North Qinglongyi obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the 20% ethanol eluate of the ethanol extract of North Qinglongyi (BQLY-20).

[0035] Specific embodiment IV: The extraction method of the North Qinglongyi extract in this embodiment is as follows:

[0036] I. Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the total extract of North Qinglongyi;

[0037] II. Dissolve 100 g of the total extract of North Qinglongyi obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 40% ethanol to obtain the 40% ethanol eluate of the ethanol extract of North Qinglongyi (BQLY-40).

[0038] Specific embodiment V: The extraction method of the North Qinglongyi extract in this embodiment is as follows:

[0039] I. Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract 3 times, combine the extract, recover the solvent, and freeze-dry to obtain North Qinglongyi total extract;

[0040] II. Dissolve 100 g of the North Qinglongyi total extract obtained in step I in water, and use a macroporous resin D101 column to elute it with 60% ethanol to obtain North Qinglongyi ethanol extract 60% ethanol eluate (BQLY-60).

[0041] Specific embodiment six: the extraction method of the North Qinglongyi extract of the present embodiment is as follows:

[0042] I. Take 20 kg of North Qinglongyi, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract 3 times, combine the extract, recover the solvent, and freeze-dry to obtain North Qinglongyi total extract;

[0043] II. Dissolve 100 g of the North Qinglongyi total extract obtained in step I in water, and use a macroporous resin D101 column to elute it with 60% ethanol to obtain North Qinglongyi ethanol extract 60% ethanol eluate (BQLY-60).

[0044] Specific embodiment seven: the North Qinglongyi extract of the present embodiment is used to inhibit the growth of Helicobacter pylori.

[0045] The following experiments are used to verify the effect of the present application:

[0046] Experiment I:

[0047] I. Materials and methods

[0048] 2-Hydroxy-1,4-naphthoquinone standard was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (item number: S24529-25g).

[0049] 1. Bacterial culture

[0050] Initial expansion:

[0051] Coat the plate with 200 μL of concentrated glycerol bacteria of Helicobacter pylori. Incubate at 37°C for 24 h in 5% oxygen, 10% carbon dioxide, and 85% nitrogen. The next day, use a cell scraper to take the bacteria on the blood plate for Gram staining and microscopic examination. The microscopic examination result is Gram-negative bacteria (see Figure 2 ), and there is no mixed bacteria, confirming that it is Helicobacter pylori bacteria.

[0052] Subculture expansion:

[0053] Take 2 mL centrifuge tube, add 1 mL PBS, scrape the cultured H. pylori with cell scraper, collect in PBS, take 0.1 mL H. pylori liquid, dilute 10 times to measure OD value, 1 OD = 2.11 x 108 cfu / mL. And calculate the final concentration.

[0054] Quantitative:

[0055] Take 2 mL centrifuge tube, add 1 mL PBS, scrape the cultured H. pylori with cell scraper, collect in PBS, take 0.1 mL H. pylori liquid, dilute 10 times to measure OD value, 1 OD = 2.11 x 108 cfu / mL. And calculate the final concentration.

[0056] The concentration calculation formula is as follows (OD600-OD value; k-dilution times; c-bacterial liquid concentration).

[0057] c=k*OD600*1OD

[0058] 2, coating plate, punching and adding drug

[0059] Take 0.1 mL, 1 x 108 cfu / mL of H. pylori suspension, quickly coat evenly on sterile solid medium (Columbia blood plate), use 1 mL of sterile pipette gun head to drill 4 holes evenly in the blood plate, respectively, take 200 uL of BQLY-Z solution, BQLY-0 solution, BQLY-20 solution, BQLY-40 solution, BQLY-60 solution, BQLY-80 solution and 2-hydroxy-1,4-naphthoquinone solution dissolved in DMSO with a concentration of 200 mg / mL, then gently inject into the hole; under the condition of 4℃, the extract is diffused on the medium for 12h, then the bacterial plate is placed in a 37℃ electric heating three gas (5% oxygen, 10% carbon dioxide, 85% nitrogen) constant temperature incubator for micro-aerobic culture for 24h, the diameter of the inhibition zone is measured by cross method, and the results are recorded (the measurement results need to be subtracted by the diameter of the gun head). The corresponding extraction solvent is used as a blank control, DMSO control is set, and other operations are the same as above. Each treatment sets 3 repeated holes.

[0060] II. Experimental results

[0061] In the in vitro antibacterial experiment of North Qinglongyi extract, as shown in Figure 1 , 2 , 3, 4, 5, 6, 7, 8, the inhibition zone results show that BQLY-20 has the best antibacterial effect, 2-hydroxy-1,4-naphthoquinone has significant antibacterial effect, and other components also have certain analgesic effect.

[0062] Experiment two:

[0063] I. Materials and methods

[0064] 1. Experimental materials

[0065] SPF level male C57BL / 6 mice, 6 weeks old, weighing 18-22 g, 36, the temperature of the room is 22±2°C, the humidity is 55±5%, 12 h day and night alternation, mice free feeding and drinking water and feed.

[0066] 2. Animal grouping

[0067] The 36 SPF level male C57BL / 6 mice were randomly divided into 5 groups: control group (9), model group (9), model+BQLY-Z group (6), model+BQLY-20 group (6), model+amoxicillin group (6).

[0068] After 7 days of adaptive feeding, NCTC11673 H. pylori (Helicobacter pylori) bacterial solution was administered by gavage, 0.2 mL per mouse (about 1×10 9 cfu / mL of NCTC11673 H. pylori), all animals were fasted for 12 h before gavage, and then H. pylori bacterial suspension was given, 1 time / day, 3 times in 5 days, of which 9 mice in the negative control group were replaced with sterile saline instead of H. pylori bacterial solution, 8 weeks after gavage, 3 mice in the control group and model group were randomly killed, and the gastric mucosa was taken to evaluate H. pylori infection and inflammation, to exclude the natural infection of mice with H. pylori.

[0069] According to the crude drug dosage of Qinglongyi (30g / person / day), the extraction rate of crude drug (BQLY-Z extraction rate 14.6%, BQLY-20: 4.45g obtained by loading and eluting 100g of BQLY-Z), and the gavage amount of mice (300μL / 30g), the drug solution concentration was calculated as follows: BQLY-Z: 85.30mg / mL, BQLY-20: 3.80mg / mL. The drug solution was prepared using ultrapure water. The drug solution concentration was prepared as follows: BQLY-Z: 85.30mg / mL, BQLY-20: 3.80mg / mL, amoxicillin: 2.49mg / mL.

[0070] ① Control group (9): After 8 weeks of gavage with equal amount of sterile saline, gavage with saline, 300μL / 30g of mouse body weight, 1 time / day, for 4 weeks

[0071] ② Model group (9): After 8 weeks of H. pylori bacterial infection model establishment, gavage with saline, 300μL / 30g of mouse body weight, 1 time / day, for 4 weeks

[0072] ③ Model + BQLY-Z group (6): after 8 weeks of H. pylori infection model, BQLY-Z was given by gavage, 300 μL / 30 g of mouse body weight, once a day, for 4 weeks

[0073] ④ Model + BQLY-20 group (6): after 8 weeks of H. pylori infection model, BQLY-20 was given by gavage, 300 μL / 30 g of mouse body weight, once a day, for 4 weeks

[0074] ⑤ Model + amoxicillin group (6): after 8 weeks of H. pylori infection model, amoxicillin was given by gavage, 300 μL / 30 g of mouse body weight, once a day, for 4 weeks

[0075] After four weeks of administration, the mouse gastric mucosa was taken, and the expression of IL-6 and IL-8 in the gastric mucosa was detected by ELISA

[0076] After the antral tissue was thawed, the IL-6 and IL-8 contents were determined according to the ELISA kit instructions, the absorbance value of the sample (detection wavelength was 450 nm) was detected by the enzyme marker, and the concentration of IL-6 and IL-8 in the sample was calculated by using the standard curve.

[0077] III. Experimental results

[0078] This experiment, as shown in Figure 9 、 10 compared with the control group, the expression of inflammatory factors in the model group increased, indicating that the modeling was successful. Compared with the model group, the expression of inflammatory factors in the administration intervention group decreased. As shown in Figure 9 、 10 , among which BQLY-20 had the best effect on reducing inflammatory factors, and other components also had certain analgesic effect. It is proved that the extract of North Qinglongyi can not only directly kill Helicobacter pylori, but also repair inflammation in the stomach.

[0079] Table 1 Expression level of inflammatory factors in mice

[0080]

[0081] Experiment two:

[0082] Liquid chromatography operation method and spectrum results:

[0083] Take BQLY-Z, BQLY-0, BQLY-20, BQLY-40, BQLY-60, BQLY-80, each 0.1 g, respectively dissolved in 10 mL of methanol. The following chromatographic conditions and mass spectrum conditions were used for sample injection.

[0084] 1. Chromatographic conditions

[0085] Waters ACQUITY TM UPLC liquid chromatograph; ACQUITY TM BEH C 18 Chromatographic column (100 mm x 2.1 mm, 1.7 μm); ACQUITY TM UPLC BEH C 18 Pre-column (5.0 mm x 2.1 mm, 1.7 μm); column temperature 35℃; mobile phase 0.1% formic acid aqueous solution (A) - 0.1% formic acid acetonitrile (B), gradient elution (0 min, 5% B; 5 min, 10% B; 15 min, 35% B; 25 min, 70% B: 30 min, 100% B; 32 min, 100% B); flow rate 0.3 mL·min -1 ; injection volume 2 μL; automatic injector temperature 4℃.

[0086] 2. Mass spectrometry conditions

[0087] Electrospray positive and negative ion modes, positive and negative ion source voltages 5500 V and -4500 V, ion source temperature 550℃, atomizing gas N2, atomizing gas and auxiliary gas pressures 379.2 kPa, curtain gas pressure 241.3 kPa, cracking voltage ±80 V, collision energy ±40 eV, collision energy expansion 20 eV. TOF / MS scan range m / z 100-1500, IDA setting response value of 8 highest peaks over 100 cps for secondary mass spectrometry scanning, Product Ion scan range m / z 50-1000, dynamic background deduction (DBS) turned on. Data acquisition software Analyst TF 1.6 software, data processing software Peakview 2.0-masterview 1.0.

[0088] BQLY-Z liquid chromatography-mass spectrometry results as follows:

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[0107] BQLY-0 Liquid Mass Results are as follows:

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[0119] BQLY-20 Liquid Mass Results are as follows:

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[0133] BQLY-40 liquid chromatography results are as follows:

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[0153] BQLY-60 liquid chromatography results are as follows:

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[0183] BQLY-80 liquid mass results are as follows:

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Claims

1. A method for extracting an extract from the root of Polygonum orientale L., characterized by The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate.

2. A method for extracting an extract from the North Korean ginseng root, characterized by The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; 3. A method for extracting an extract from the North Korean ginseng fruit, characterized by II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate. The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; 4. A method for extracting an extract from the North Korean ginseng fruit, characterized by II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate. The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; 5. A method for extracting an extract from the North Korean ginseng fruit, characterized by II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate. The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; 6. A method for extracting an extract from the North Korean ginseng fruit, characterized by II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate. The extraction method of the north green dragon skin extract is as follows: I. Take 20 kg of north green dragon skin, soak it in 80% ethanol with a solid-liquid ratio of 1:10 for 7 days, extract it 3 times, combine the extract, recover the solvent, freeze-dry, and obtain the north green dragon skin total extract; 7. Use of an extract of Hedychium flavescens, characterized in that II. Dissolve 100 g of the north green dragon skin total extract obtained in step I in water, use a macroporous resin D101 chromatographic column, and elute it with 20% ethanol to obtain the north green dragon skin ethanol extract 20% ethanol eluate. The north green dragon skin extract is used to inhibit the growth of Helicobacter pylori and reduce the expression level of inflammatory factors.

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