Preparation method and application of artemisia rupestris dichloromethane component

Abstract

The invention relates to a preparation method and application of an artemisia rupestris dichloromethane component, and the method comprises the following steps: crushing the whole herb of the artemisia rupestris, adding 95% ethanol, uniformly mixing, carrying out reflux extraction, filtering, combining extracting solutions, carrying out reduced pressure recovery and concentration, extracting by using ethyl acetate, extracting the residual liquid after extraction by using dichloromethane, and concentrating to obtain a dichloromethane extracting solution; and adding saturated sodium bicarbonate, extracting until the layer is colorless, adjusting the pH value to 3-4 by using hydrochloric acid, extracting by using dichloromethane, concentrating under reduced pressure, and drying in vacuum to obtain the artemisia rupestris dichloromethane component. Through general observation of the component on non-alcoholic fatty liver disease rats induced by high fat diet, blood biochemistry and other related indexes and histopathology, it is proved that the artemisia rupestris dichloromethane component has activity in the aspect of effectively relieving the non-alcoholic fatty liver disease; a theoretical basis is laid for further developing and utilizing the dichloromethane component of the artemisia rupestris to treat the non-alcoholic fatty liver disease, and a new way is provided.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically to a method for preparing a dichloromethane component of Artemisia striata and its application Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) refers to a chronic metabolic disease that excludes other causes (excessive drinking, drugs, etc.), characterized by hepatic lipid accumulation and metabolic disorders. NAFLD ranges from the initial simple fatty liver to nonalcoholic steatohepatitis, and even fibrosis, cirrhosis and cancer. The prevalence of NAFLD is increasing worldwide, with a global prevalence of about 32.4% and a high prevalence of about 29% in my country. Current studies have found that the clinical prevention and treatment of NAFLD mainly include single-target drug therapy, surgical treatment and lifestyle changes. There is only one FDA-approved therapeutic drug, but it has side effects such as nausea and vomiting. These treatments cannot completely solve the complex pathogenesis of liver damage. Therefore, the development of highly effective and less adverse reaction drugs for the treatment of nonalcoholic fatty liver disease is an urgent problem that technicians in this field need to solve.

[0003] Artemisia rupestris L. is the whole herb of Artemisia rupestris L., a plant of the genus Artemisia in the Asteraceae family. The whole herb is used as medicine and has multiple functions such as anti-allergy, stomachic, detoxification, antiviral, antibacterial and liver protection. It is clinically used to treat indigestion, colds and fevers, hepatitis, urticaria, allergic diseases, etc. At present, the research, development and utilization of Artemisia rupestris in China have a history of nearly 40 years. Its chemical components mainly include sesquiterpenes, flavonoids, volatile oils and other compounds. Zhang et al. studied the in vitro anti-liver cancer effect and mechanism of Artemisia rupestris alcohol extract. Recent studies have shown that the dichloromethane component of Artemisia rupestris has liver protection, enzyme reduction, anti-inflammatory and antioxidant effects on acute liver injury. This application intends to verify the therapeutic effect of the dichloromethane component of Artemisia rupestris on non-alcoholic fatty liver disease. Summary of the invention

[0004] The object of the present invention is to provide a preparation method and application of a dichloromethane component of Artemisia striata. The method comprises the following steps: crushing the whole herb of Artemisia striata, adding 95% ethanol to mix evenly, performing reflux extraction, filtering, combining the extracts, recovering and concentrating under reduced pressure, and then extracting with ethyl acetate. The remaining liquid after extraction is extracted with dichloromethane, concentrated to obtain a dichloromethane extract, and then adding saturated sodium bicarbonate to extract until the layer is colorless, adjusting the pH to 3-4 with hydrochloric acid, and then extracting with dichloromethane, concentrating under reduced pressure, and vacuum drying to obtain the dichloromethane component of Artemisia striata. The dichloromethane component of Artemisia striata obtained by the method of the present invention is used in the preparation of a drug for treating non-alcoholic fatty liver disease; and a theoretical foundation is laid for further developing and utilizing the dichloromethane component of Artemisia striata to treat non-alcoholic fatty liver disease.

[0005] The method for preparing the dichloromethane component of Artemisia sphaerocephala described in the present invention is carried out according to the following steps:

[0006] a. After crushing the whole herb of Artemisia striata, add 10 mL of 95% ethanol per 1 g of the crushed Artemisia striata and mix well to obtain a mixed solution;

[0007] b. Reflux the mixed solution obtained in step a at room temperature for 2 times, each time for 2 hours, filter, and combine the reflux extracts to obtain a filtrate;

[0008] c. recovering the filtrate obtained in step b under reduced pressure and concentrating it to obtain a concentrated solution of the alcohol extract of Artemisia annua;

[0009] d. extracting the concentrated Artemisia annua alcohol extract obtained in step c with ethyl acetate, and collecting the remaining liquid after extraction;

[0010] e. Extract the remaining liquid of Artemisia argyi obtained in step d with dichloromethane and concentrate it to obtain a dichloromethane extract;

[0011] f. Add saturated sodium bicarbonate to the dichloromethane extract of Artemisia sphaerocephala obtained in step e, and extract until the sodium bicarbonate layer is colorless to obtain a sodium bicarbonate extract;

[0012] g. The sodium bicarbonate extract obtained in step f is adjusted to pH 3-4 with hydrochloric acid, and then extracted with dichloromethane. The dichloromethane extract is collected, concentrated under reduced pressure, and dried in vacuo to obtain the dichloromethane component of Artemisia striata.

[0013] The artemisia affine dichloromethane component obtained by the method is used in preparing medicine for treating non-alcoholic fatty liver disease.

[0014] The preparation method and application of the Artemisia annua dichloromethane component of the present invention are to observe the effects of the Artemisia annua dichloromethane component on the body weight, organ index and fat index of rats through a high-fat diet-induced non-alcoholic fatty liver rat model, and to observe the changes in the levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total protein (TP), albumin (ALB) and total bile acid (TBA) in rat serum, perform liver tissue pathological examination and detect liver tissue inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α); and to verify the efficacy of the Artemisia annua dichloromethane component in treating non-alcoholic fatty liver through determination:

[0015] The Artemisia annua dichloromethane component obtained by the method of the present invention is used in non-alcoholic fatty liver disease to reduce the body weight, organ coefficient and obesity index of rats with non-alcoholic fatty liver model; reduce the levels of TG, TC, ALT, AST, ALP and LDH in the serum of rats with non-alcoholic fatty liver model; increase the level of HDL-C in the serum of rats with non-alcoholic fatty liver model; and reduce the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the liver of rats with non-alcoholic fatty liver model. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A is the appearance image of each group of rats in Example 2 of the present invention, Figure 1 B is a line graph of weight changes of rats in each group in Example 2 of the present invention;

[0017] Figure 2 The test results of TC, TG, HDL-C, ALT, AST, T-Bil, ALP, LDH, TP, ALB and TBA in the serum of each group of rats in Example 2 of the present invention;

[0018] Figure 3 These are the results of hematoxylin-eosin (HE) staining and Oil Red O staining of the liver tissues of the rats in each group in Example 2 of the present invention; the results of the detection of inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the liver tissues of the rats in each group in Example 2 of the present invention. Figure 4 The invention discloses the effect of the dichloromethane component of Artemisia annua on the levels of inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the liver of NAFLD rats. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. All other embodiments obtained by ordinary technicians in the field without making creative work based on the embodiments of the present invention shall fall within the scope of protection of the present invention.

[0020] Example 1

[0021] a. Take 15 kg of whole Artemisia striata and crush it, add 10 mL of 95% ethanol per 1 g of the crushed Artemisia striata and mix well to obtain a mixed solution;

[0022] b. Reflux the mixed solution obtained in step a twice, each time for 2 hours, filter, and combine the reflux extracts to obtain a filtrate;

[0023] c. recovering the filtrate obtained in step b under reduced pressure and concentrating it to obtain a concentrated solution of the alcohol extract of Artemisia annua;

[0024] d. extracting the concentrated Artemisia annua alcohol extract obtained in step c with ethyl acetate, and collecting the remaining liquid after extraction;

[0025] e. Extract the remaining liquid of Artemisia argyi obtained in step d with dichloromethane and concentrate it to obtain a dichloromethane extract;

[0026] f. Add saturated sodium bicarbonate to the dichloromethane extract of Artemisia sphaerocephala obtained in step e, and extract until the sodium bicarbonate layer is colorless to obtain a sodium bicarbonate extract;

[0027] g. The sodium bicarbonate extract obtained in step f was adjusted to pH 3-4 with hydrochloric acid, and then extracted with dichloromethane. The dichloromethane extract was collected, concentrated under reduced pressure, and dried in vacuo to obtain 0.42% of dichloromethane component of Artemisia striata.

[0028] Example 2

[0029] Application of the Artemisia truncatula dichloromethane component obtained by the method in preparing a drug for treating non-alcoholic fatty liver disease:

[0030] 1. Effects of dichloromethane fraction of Artemisia striata on high-fat diet-induced non-alcoholic fatty liver disease rat model:

[0031] In this experiment, a high-fat diet was used to replicate the non-alcoholic fatty liver model.

[0032] After SD rats were adaptively fed for 7 days, they were randomly divided into blank control group and model group according to body weight. During the modeling period, the blank control group was fed normally without modeling. The model group was fed with high-fat diet for 12 weeks. After the modeling, the model group rats were randomly divided into model group, positive control silybin capsule group 21.88 mg / kg / d, Artemisia selengensis dichloromethane component low-dose group 3.75 mg / kg / d, medium-dose group 7.50 mg / kg / d and high-dose group 15.00 mg / kg / d according to body weight. The blank control group and model group were gavaged with distilled water, and the other positive drug control group, Artemisia selengensis dichloromethane component low-dose, medium-dose and high-dose groups were gavaged with 10 mL / kg for 5 weeks.

[0033] 1.1 Situation observation:

[0034] The changes in the fur, emotional state, behavioral state, excitement level, activity level, and feces of rats in each group were observed daily before and after modeling and drug administration;

[0035] 1.2 Weight changes of rats:

[0036] The body weight of rats in each group was measured after modeling and every week during the administration period, and the average body weight change of rats in each group was calculated.

[0037] 1.3 Organ index and obesity index determination:

[0038] The liver, kidney, spleen, epididymal white adipose tissue and perirenal white adipose tissue of the rats were collected and weighed, the body length was measured and the Lee's index was calculated according to the following formula:

[0039] Organ coefficient = organ weight (g) / body weight (g) × 100%;

[0040] Obesity index = body fat weight (g) / body weight (g) × 100%;

[0041] Lee's index = body weight (g) × 1000 / 3 / body length (cm);

[0042] 1.4 Serum index detection:

[0043] The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total protein (TP), albumin (ALB), and total bile acid (TBA) in the rat serum were determined using an automatic biochemical instrument;

[0044] 1.5 Histopathological examination:

[0045] (1) HE staining:

[0046] Rat liver tissues were collected, washed with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. Subsequently, these liver tissue sections were stained with hematoxylin-eosin;

[0047] (2) Oil Red O staining:

[0048] Preparation of frozen sections: fix fresh tissue in 4% formaldehyde for 24h, first dehydrate in 15% sucrose solution at 4℃, then dehydrate in 30% sucrose solution at 4℃; slightly dry the tissue, put it on the embedding table, drop OCT agent, and freeze it quickly until hardened; fix the embedding table, slice it roughly and flat, with a thickness of 8-10μm, stick it on a slide, and store it at -20℃ after marking; rewarm and dry the slices, fix them for 15min, wash them with water, and then stain them with oil red for 8-10min after drying, wash them with water; slightly differentiate them with 75% alcohol, and wash them with distilled water; dye the slices with hematoxylin solution for 3-5min, wash them with tap water, differentiate them with differentiation solution, wash them with tap water, blue them with bluing solution, rinse them with running water, seal them, examine them with a microscope, and collect images;

[0049] 1.6 Detection of inflammatory factors in liver tissue:

[0050] Interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by ELISA according to the instructions of the corresponding kits;

[0051] 1.7 Statistical data processing method: All experimental data were expressed as x ± SD, two-sample t-test was used for pairwise comparison, one-way analysis of variance was used for inter-group comparison, P < 0.05 was considered statistically significant, and the statistical software was SPSS 21.0 version;

[0052] 1.8 Experimental results:

[0053] 1.8.1 Behavioral Observation:

[0054] Before modeling, the rats in each group were in normal condition and activity; after modeling, the rats in the model group appeared listless, with obese abdomen, slow reaction, rough and shiny hair, sticky feces, and yellowish bedding color; the rats in the blank control group showed good responsiveness, white and smooth hair, normal appetite and excretion function; after 5 weeks of drug intervention, the rats in each drug group showed different degrees of relief from the above conditions;

[0055] 1.8.2 Weight changes:

[0056] During the modeling period, the weight gain rate of the rats in the model group was significantly higher than that in the blank group. The weight of the model group and each drug-treated group gradually decreased after drug intervention. Figure 1 ;

[0057] 1.8.3 Effects of dichloromethane components of Artemisia ternata on body length, organs and fat changes in NAFLD rats:

[0058] The experimental results showed that compared with the blank control group, the Lee's index, liver wet weight, liver coefficient, epididymal fat index and perirenal fat index of the model group rats were significantly increased (P<0.001); compared with the model group, the Lee's index of each dose group of ARA-D was reduced but there was no statistical difference, the liver wet weight was significantly reduced (P<0.01), the liver index was significantly reduced (P<0.05 or P<0.001), the epididymal fat index of the low dose group was significantly reduced (P<0.01), the perirenal fat index of the positive drug group and the low and medium dose groups was significantly reduced (P<0.05 or P<0.001); there was no statistical significance in spleen index and kidney index, the results are shown in Table 1;

[0059] Table 1 Changes in body length, organs and fat of rats in each group (n=6)

[0060]

[0061] Note: Compared with the blank control group # P<0.05, ## P<0.01, ### P<0.001; compared with the model group * P<0.05, ** P<0.01, *** P < 0.001;

[0062] 1.8.4 Effects of dichloromethane fraction of Artemisia annua on serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total protein (TP), albumin (ALB) and total bile acid (TBA) in rats with NAFLD:

[0063] The experimental results showed that compared with the blank control group, the serum triglyceride (TG) level of the rats in the model group was significantly increased (P<0.001), and the triglyceride (TG) level of the ARA-D dose groups was significantly decreased compared with the model group (P<0.001); compared with the blank control group, the serum total cholesterol (TC) level of the rats in the model group was significantly increased (P<0.05), and the ARA-D medium and high dose groups were significantly lower than the model group (P<0.01); compared with the blank control group, the serum high-density lipoprotein cholesterol (HDL-C) level of the rats in the model group was significantly decreased (P<0.001); the ARA-D positive drug group and high dose group significantly increased the high-density lipoprotein cholesterol (HDL-C) level (P<0.05 or P<0.01);

[0064] Compared with the blank control group, the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in the model group were significantly increased, and the difference was statistically significant (P<0.001); compared with the model group, the levels of alkaline phosphatase (ALP) and alanine aminotransferase (ALT) in each drug administration group were lower than those in the model group, and the difference was statistically significant (P<0.001). The levels of aspartate aminotransferase (AST) in the low, medium and high dose drug administration groups were lower than those in the model group, and the difference was significant (P<0.05 or P<0.001). The levels of lactate dehydrogenase (LDH) in the low, medium and high dose drug administration groups were decreased, and there was a significant statistical difference (P<0.05 or P<0.01).

[0065] Compared with the blank control group, the serum albumin (ALB) and total bilirubin (T-Bil) levels of the model group rats were significantly decreased, with significant statistical differences (P<0.05 or P<0.001); there was no statistical difference in the levels of total bile acid (TBA) and total protein (TP); compared with the model group, the serum albumin (ALB) levels of the ARA-D medium, high dose group and positive drug group were higher than those of the model group, with statistical differences (P<0.01 or P<0.001); the serum total bilirubin (T-Bil) level of the positive drug group was higher than that of the model group, with statistical differences (P<0.05), and the serum total bilirubin (T-Bil) levels of the ARA-D low, medium and high dose groups increased, but there was no statistical difference; the serum total protein (TP) levels of the positive drug and high dose groups were lower than those of the model group, with statistical differences (P<0.05). Figure 2 ;

[0066] 1.8.5 Histopathological examination:

[0067] HE staining results: The liver structure of rats in the blank control group was intact, the hepatocytes were arranged in an orderly manner, the cytoplasm was uniform, the nucleus was centered, and no abnormalities were found; the liver structure of rats in the model group was disordered, the hepatocytes were swollen, the fat vacuoles increased, the nucleus was shifted, and there was focal inflammation and necrosis; the fat vacuoles, inflammation and necrosis of the liver of rats in the treatment group were alleviated, as shown in Figure 3 ;

[0068] Oil red O staining results: There was almost no red fat deposition in the liver cells of the rats in the normal group, the cell morphology was regular, the boundaries were clear, and the nucleus was centered; a large number of red lipid droplets appeared in the liver cells of the rats in the model group, with irregular morphology, blurred boundaries, and offset nuclei. The lipid droplets in the liver of the rats in the treatment group decreased, and the cell morphology and boundaries were improved. Figure 3 ;

[0069] 1.8.6 Effects of dichloromethane fraction of Artemisia annua on the levels of inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the liver of NAFLD rats:

[0070] The experimental results showed that compared with the blank control group, the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the liver of rats in the model group increased sharply, with significant differences (P < 0.001); compared with the model group, low, medium and high doses of Artemisia annua dichloromethane components could significantly reduce the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the liver of NAFLD rats, with significant differences (P < 0.001). Figure 4 .

Claims

1. A method for preparing a dichloromethane component of Artemisia striata, characterized in that Follow these steps: a. After crushing the whole herb of Artemisia striata, add 10 mL of 95% ethanol per 1 g of the crushed Artemisia striata and mix well to obtain a mixed solution; b. Reflux the mixed solution obtained in step a at room temperature for 2 times, each time for 2 h, filter, and combine the reflux extracts to obtain a filtrate; c. recovering the filtrate obtained in step b under reduced pressure and concentrating it to obtain a concentrated solution of the alcohol extract of Artemisia annua; d. extracting the concentrated Artemisia annua alcohol extract obtained in step c with ethyl acetate, and collecting the remaining liquid after extraction; e. Extract the remaining liquid of Artemisia argyi obtained in step d with dichloromethane, and concentrate to obtain a dichloromethane extract; f. Add saturated sodium bicarbonate to the dichloromethane extract of Artemisia sphaerocephala obtained in step e, and extract until the sodium bicarbonate layer is colorless to obtain a sodium bicarbonate extract; g. The sodium bicarbonate extract obtained in step f is adjusted to pH 3-4 with hydrochloric acid, and then extracted with dichloromethane. The dichloromethane extract is collected, concentrated under reduced pressure, and dried in vacuo to obtain the dichloromethane component of Artemisia striata.

2. Use of the dichloromethane component of Artemisia affine obtained by the method as claimed in claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease.

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