CN109939103B

CN109939103B - Use of coriander lactone-1 and preparation method thereof

Info

Publication number: CN109939103B
Application number: CN201910325219.1A
Authority: CN
Inventors: 高红伟; 苑仁祎坤; 黄莉婷; 许琼明; 杨世林; 奉建芳; 赵立春
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2019-04-22
Filing date: 2019-04-22
Publication date: 2021-06-25
Anticipated expiration: 2039-04-22
Also published as: CN109939103A

Abstract

本发明公开了一种芫荽内酯‑1的用途及其制备方法。本发明的化合物芫荽内酯‑1对受损肾组织具有防治作用，用于制备治疗受损肾组织药物的用途。The invention discloses a use of coriander lactone-1 and a preparation method thereof. The compound coriander lactone-1 of the present invention has a preventive effect on damaged renal tissue, and is used for preparing a medicine for treating damaged renal tissue.

Description

Application and preparation method of coriander lactone-1

Technical Field

The invention belongs to the technical field of application of coriander lactone-1, and particularly relates to new application of coriander lactone-1 and a preparation method thereof.

Background

Acute Kidney Injury (AKI) is a clinically common critical condition, and the incidence rate of the AKI is on an increasing trend year by year and is one of the important causes of death of patients. Sepsis is a systemic inflammatory response syndrome caused by infectious agents and is a common cause of AKI. Approximately 42% of sepsis patients develop AKI, and the mortality rate after AKI in sepsis patients reaches 70%. AKI caused by sepsis is one of the problems which need to be solved urgently in critical emergency medicine and kidney pathology. The inventor continuously researches the kidney tissue damage mechanism, applies the compound (HG-1) which is separated and extracted from coriander fruits by the inventor in the early period to the research of preventing and treating the kidney tissue damage, and searches for a new finding.

Disclosure of Invention

The invention aims to provide a new application of coriander lactone-1, wherein the coriander lactone-1 has a prevention and treatment effect on damaged kidney tissues and is used for preparing a medicine for treating the damaged kidney tissues.

Preferably, the structural formula of the coriander lactone-1 is as follows:

preferably, the coriander lactone-1 is prepared by the following steps:

crushing 30kg of dried coriander fruit medicinal material, extracting volatile oil of coriander fruit by using petroleum ether through a Soxhlet extraction method, removing 4.5L of volatile oil, carrying out Soxhlet extraction on the residual medicine dregs by using methanol, heating and volatilizing until no alcohol smell exists to obtain a medicinal material methanol extract liquid extract, dispersing the liquid extract by using 0.5-1.5L of distilled water, then sequentially extracting by using petroleum ether and dichloromethane, recycling a solvent from the extract liquid under reduced pressure, removing 605.0g of petroleum ether extract to obtain 125.0g of dichloromethane extract;

and step two, performing reduced pressure silica gel column chromatography on the dichloromethane extract, and sequentially performing reaction on the dichloromethane extract with petroleum ether: eluting ethyl acetate at the volume ratio of 90:10, 80:20 and 70:30 to obtain a first elution part Fr.A1, a second elution part Fr.A2 and 20.0g of a third elution part Fr.A3, and purifying the third elution part Fr.A3 by reduced pressure silica gel column chromatography, and then eluting with petroleum ether: the volume ratio of ethyl acetate was 80:20 to give 10.5g of a fourth elution site fr.b1, which was then passed through an ODS reversed-phase medium-pressure column sequentially eluting with methanol: eluting with water at a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30 and 80:20 to obtain 7 fractions, loading the 7 fractions onto a plate, combining the plate with the same main spot component to obtain 2.00g of methanol fraction Fr.C, passing through ODS reversed phase medium pressure column, sequentially eluting with methanol: eluting with water at a volume ratio of 20:80, 30:70, 40:60, 50:50 and 60:40 to obtain a first fraction, a second fraction, a third fraction, a fourth fraction and a fifth fraction, respectively, combining the fourth fraction and the fifth fraction to obtain a 126mg combined fraction part Fr.D, and finally purifying with a semi-preparative high performance liquid phase to obtain a compound coriander lactone-1;

wherein the reduced pressure column of the silica gel column chromatography is 200-300 meshes.

The invention at least comprises the following beneficial effects: the inventor further researches in the coriander lactone-1 (HG-1) with antioxidant effect separated and extracted from coriander fruits under the analysis of kidney tissue damage mechanism, and finds that the coriander lactone-1 has new application and good prevention and treatment effect on damaged kidney tissues through analysis and experiments.

Drawings

FIG. 1 is a high resolution mass spectrum of compound HG-1 of the present invention;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of compound HG-1 of the present invention;

FIG. 3 is a nuclear magnetic resonance carbon spectrum of compound HG-1 of the present invention;

FIG. 4 is an HSQC spectrum of compound HG-1 of the present invention;

FIG. 5 is a HMBC spectrum of compound HG-1 of the present invention;

FIG. 6 is a COSY spectrum of the compound HG-1 of the present invention;

FIG. 7 is a bar graph of BUN content in serum of various groups of mice in the experiment;

FIG. 8 is a bar graph of Creatinie levels in the serum of groups of mice in the experiment;

FIG. 9 is a bar graph of the serum TNF- α content of groups of mice in the assay;

FIG. 10 is a bar graph of IL-6 levels in serum of groups of mice in the experiment;

FIG. 11 is a bar graph of IL-1. beta. content in serum of mice in each group of the experiment.

In the column diagrams of FIGS. 7 to 8, from left to right, the first column is a control group, the second column is an LPS group, the third column is an LPS +5mg/kg HG-1 group, the fourth column is an LPS +10mg/kg HG-1 group, the fifth column is an LPS +20mg/kg HG-1 group, and the sixth column is an LPS + DEX group.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

The invention discloses a new application of coriander lactone-1 (HG-1), which has a prevention and treatment effect on damaged kidney tissues and is used for preparing a medicine for treating the damaged kidney tissues. The structural formula of the coriander lactone-1 is as follows:

the structure identification of the coriander lactone-1 (HG-1) is as follows:

the coriander lactone-1 is light yellow powder, and its high resolution mass spectrogram (HR-ESI-MS) of FIG. 1 shows its excimer ion peak M/z 193.1234[ M-H [ -H ]₂O+H]⁺Two-dimensional combining fig. 2 and 3Nuclear magnetic resonance spectrum of which molecular formula is presumed to be C₁₂H₁₈O₃(calculated value 193.1229[ M-H)₂O+H]⁺) The unsaturation degree is 4.

The nmr spectrum of fig. 3 shows a total of 12 carbon signals, and the combination of the nmr spectrum of fig. 2 and the HSQC spectrum of fig. 4 identifies HG-1 compounds having a total of 2 methyl carbons, 3 methylene carbons, 5 methine carbons, and 2 quaternary carbons. Further observing the low field region of the NMR spectrum of FIG. 3, it is presumed that the compound contains an ester group and a carbon-carbon double bond. It is presumed that the compound further contains two rings, based on the unsaturation degree of the compound being 4.

In the NMR chart of FIG. 2, the HSQC chart data of FIG. 4 is combined, wherein the hydrogen signal delta of 1 obvious double-bonded carbon can be observed in the low field region_H6.54(1H, td, J ═ 3.1,0.8Hz), 2 distinct hydrogen on oxygen-containing carbons signals δ were also observed_H4.02(1H,ddd,J＝8.9,7.3,5.4Hz)、δ_H4.4(1H, qd, J ═ 6.4,3.6Hz), Hydrogen signals on 2 methyl carbons δ_H1.40(3H,m)、δ_H0.94(3H, m), combined with the data in the HSQC spectrum of fig. 4 and the compound formula, it was determined that the compound contained one ester group, one carbon-carbon double bond, and, based on the unsaturation of the compound being 4, it was determined that the compound contained two rings.

The two-dimensional nuclear magnetic resonance spectrogram analysis of the compound shown in the figure 2 and the figure 3 is combined, the chemical shifts of each carbon and hydrogen in the compound are assigned (see table 1), and the specific analysis process is as follows:

TABLE 1 NMR data (CD) of HG-1₃OD,500MHz)

From the COSY spectrum of FIG. 6, it is found that_H4.4(1H, qd, J ═ 6.4,3.6Hz) and δ_H2.19(1H,m)、δ_H1.49(1H, m) correlation, δ_H6.54(1H, td, J ═ 3.1,0.8Hz) and δ_H2.62(1H, m) correlation, δ_H2.62(1H, m) and δ_H4.02(1H,ddd,J＝8.9,7.3,5.4Hz)、δ_H1.46(1H, m) correlation, δ_H4.02(1H, ddd, J ═ 8.9,7.3,5.4Hz) and δ_H1.76(1H, m) correlation, δ_H1.76(1H, m) and δ_H0.94(3H, m) correlation, δ_H2.1(1H, ddd, J ═ 11.9,5.1,2.3Hz) and δ_H1.43(1H, m) correlation, δ can be determined in conjunction with the hydrocarbon attribute information given in the HSQC spectrum of FIG. 4_C66.6 and delta_C31.0 connected, delta_C135.7 and delta_C43.2 connected, delta_C43.2 and delta_C85.6、δ_C25.8 connected, delta_C85.6 and delta_C33.8 connected, delta_C33.8 and delta_C13.0 connected, delta_C25.8 and delta_C27.3 are connected.

From the HMBC spectrum of FIG. 5 we also found that δ_H4.4(1H, qd, J ═ 6.4,3.6Hz) and δ_C31.0、δ_C133.3 correlation, δ_H2.19(1H,m)、δ_H1.49(1H, m) and δ_C135.7、δ_C133.3、δ_C66.6、δ_C43.2、δ_C22.2, correlating; delta_H6.54(1H, td, J ═ 3.1,0.8Hz) and δ_C170.3、δ_C43.2、δ_C31.0 correlation; delta_H2.62(1H, m) and δ_C135.7、δ_C133.3、δ_C85.6、δ_C33.8、δ_C25.8, correlating; delta_H4.02(1H, ddd, J ═ 8.9,7.3,5.4Hz) and δ_C43.2 correlation; by combining the information given by the COSY spectrogram of FIG. 6, δ can be determined_C66.6 with ester group, delta_C31.0 and the carbon is an oxocarbon, so that a hydroxyl group is attached to the carbon. Thus, we determined the C-7 bit delta_C170.3, C-8 bit delta_C66.6, C-9 bit delta_C31.0, C-10 bit delta_C133.3, C-11 bit delta_C135.7, C-4 bit delta_CDelta at position 43.2 and C-5_C85.6. Also because in the HSQC spectrum of FIG. 4 we found δ_C133.3 is quaternary carbon and in the HMBC spectrum of FIG. 5, δ_H1.40(1H, m) and δ_C43.2、δ_C31.0 correlation, therefore, determine δ_C22.2 at the C-10 position, so that the C-12 position is delta_C22.2。

From the HMBC spectrum of FIG. 5, it is found that delta_H4.02(1H, ddd, J ═ 8.9,7.3,5.4Hz) and δ_C27.3、δ_C25.8 correlation, δ_H2.1(1H, ddd, J ═ 11.9,5.1,2.3Hz) and δ_C135.7 correlation, δ_H1.76(1H,m)、δ_H1.43(1H, m) and δ_C13.0、δ_C33.3、δ_C43.2、δ_C85.6 correlation, and by combining the information given by the COSY spectrogram of FIG. 6, we determined the delta at the C-1 position_C33.8, delta at C-2_C27.3, C-3 position delta_C25.8, C-6 position delta_C13.0. In summary, we identify the following structural formulas:

the preparation method of the coriander lactone-1 comprises the following steps:

wherein the reduced pressure column of the silica gel column chromatography is 300 meshes.

The animal experiment that the coriander lactone-1 has the prevention and treatment effect on the damaged kidney tissues comprises the following steps:

1 Material

1.1 Experimental animals

The SPF-grade healthy BALB/C mice are 22-28 g in weight, 7-8 weeks in male sex, provided by Suzhou university experimental animal center, all experimental animals are raised in a controllable environment at the room temperature of 18-24 ℃ and the humidity of 40-50%, the animals eat and drink water freely during the experiment, and the circadian rhythm is normal.

1.2 major drugs and reagents

HG-1 (prepared by the preparation method of the invention); dexamethasone sodium phosphate injection (DEX) (chen xin pharmaceutical products limited, national standard H37021969); lipopolysaccharide (LPS) (Sigma, usa); 0.9% sodium chloride injection (Shandongdu pharmaceutical Co., Ltd.); tumor necrosis factor (TNF-alpha) kit, interleukin (IL-6, IL-1 beta) kit (eBioscience, USA).

An electronic balance FA2104S (Shanghai Seiki Seiko, Seikagaku Kogyo; digital pipette gun (Olympus CX31 microscope), a high-speed centrifuge (Hunan instrument laboratory development Co., Ltd.); a-20 ℃ refrigerator (Sanyo product)), and a microplate reader (Chengdou san and Measure Co., Ltd.).

2. Procedure of the test

2.1 establishment and grouping of animal models

SPF-grade BALB/C mice are 60, male, and have the weight of 22-28 g. Mice were randomly divided into 6 groups: control group, model group (LPS group), LPS +5mg/kg HG-1 group, LPS +10mg/kg HG-1 group, LPS +20mg/kg HG-1 group and positive drug group (dexamethasone, LPS + DEX group), six groups, 10 per group. LPS group, LPS +5mg/kg HG-1 group, LPS +10mg/kg HG-1 group, LPS +20mg/kg HG-1 group and LPS + DEX group are respectively injected into the abdominal cavity to be injected with 10mg/kg LPS for molding, and the control group is injected with the same amount of physiological saline; LPS +5mg/kg HG-1 group, LPS +10mg/kg HG-1 group and LPS +20mg/kg HG-1 group respectively according to 5, 10, 20mg/kg dose in 2h before moulding, 2h, 4h after moulding intraperitoneal injection, the control group correspondingly injects 10mg/kg normal saline; the LPS + DEX group was given a 5mg/kg dose of DEX by tail vein injection 2h before molding. The animals were fasted for 12h before molding and were sacrificed 12h after molding, with free access to water. Serum was collected for urea nitrogen (BUN), creatinine (Crea), TNF- α, IL-6, IL-1 β assays.

2.2 serum BUN and Crea level detection

After 12h of modeling, whole mouse eyeball blood is taken, centrifuged at 3500r/min for 15min, supernatant is taken, and the Blood Urea Nitrogen (BUN) and creatinine (Crea) levels in the serum are detected by a ModuleP800 biochemical analyzer.

2.3 detection of serum TNF-alpha, IL-6 and IL-1 beta.

Serum TNF-alpha, IL-6 and IL-1 beta levels were detected by ELISA strictly according to the kit instructions.

2.4 statistical treatment

STATA8.0 statistical software is adopted to carry out one-factor variance analysis and t-test data, the difference with P <0.05 is statistically significant, and the smaller the P, the better the effect.

3. Results

3.1 Effect of HG-1 on serum Cr and BUN levels in mice of various groups

Referring to the bar graph of BUN content in mouse serum of FIG. 7 and Creatinie content in mouse serum of FIG. 8, the levels of Crea and BUN in mouse serum of LPS group are obviously higher than those in control group after LPS treatment for 12h, which indicates that AKI model induced by LPS (10mg/kg, 12h) is successful; the BUN content of the mouse serum of each dose of HG-1 group and LPS + DEX group is obviously lower than that of LPS group (P is less than 0.05, P is less than 0.01, the asterisk on the bar indicates that the statistical significance is achieved), and the serum Crea content of LPS +20mg/kg HG-1 group and LPS + DEX group is obviously lower than that of LPS group, so that the treatment effect of LPS +20mg/kg HG-1 group is equivalent to that of positive control drug LPS + DEX group. Because DEX is hormone, the side effect is obvious, the dosage is large, and a series of problems of osteoporosis are caused, the HG-1 of the invention does not belong to hormone, has small side effect, can adjust the dosage according to serious conditions, and is favorable for market demand.

3.2 Effect of HG-1 on TNF- α, IL-6 and IL-1 β levels in mouse serum

Referring to FIG. 9, bar graphs of TNF-alpha content in mouse serum, IL-6 content in mouse serum of FIG. 10 and IL-1 beta content in mouse serum of FIG. 11 show that after LPS treatment for 12h, the results show that the levels of TNF-alpha, IL-6 and IL-1 beta in mouse serum of LPS group are significantly increased compared with the control group, indicating that the AKI model induced by LPS (10mg/kg, 12h) is successful. The TNF-alpha, IL-6 and IL-1 beta contents of LPS +10mg/kg HG-1, LPS +20mg/kg HG-1 and LPS + DEX were all significantly reduced compared to LPS group (P <0.05, P < 0.01, P < 0.001, asterisks on bars indicate statistical significance). The result indicates that HG-1 can reduce the levels of TNF-alpha, IL-6 and IL-1 beta in serum and can play a good role in protecting the renal tissue injury of AKI induced by LPS.

Modern medical studies have shown that sepsis, which results from sepsis, is a significant cause of death in many critically ill patients, with the kidney being one of the most vulnerable organs and leading to the production of AKI, with an incidence of about 20% to 35%. Clinical treatment can reverse AKI, but chronic renal failure or end-stage renal failure still has a certain probability. Sepsis caused by gram-negative bacteria accounts for 50% -60%. Endotoxin (LPS), which is a component of the outer membrane of the cell wall of gram-negative bacteria (G-), can initiate an inflammatory response, is a key causative factor for AKI as a result of infection with gram-negative bacteria, has been shown to cause systemic inflammatory response syndrome and AKI, and is widely used in the mechanism search of systemic inflammatory response syndrome. In the research, an LPS 10mg/kg is injected into the abdominal cavity to prepare a mouse sepsis AKI model for researching the LPS resistance of HG-1.

Crea and BUN in serum are common biochemical indexes for detecting renal function, BUN is mainly discharged out of a body along with urine after blood flows through glomeruli and is filtered, the change of BUN has large influence on non-protein nitrogen values, and Crea clearance is influenced by glomerular concentration function and is the most reliable index for reflecting the renal function. The levels of both in serum reflect mainly the glomerular filtration function. Experiments detect the levels of BUN and Crea in the serum of mice, and the HG-1 is found to be capable of obviously reducing the levels of Crea and BUN in the serum and has obvious kidney tissue protection effect.

The research result shows that HG-1 can reduce the levels of TNF-alpha, IL-6 and IL-1 beta in kidney tissues of rats with sepsis. Under physiological state, the human body has low TNF-alpha, IL-6 and IL-1 beta levels, and under pathological state, the TNF-alpha, IL-6 and IL-1 beta secretion in the human body is greatly increased to promote the activation of inflammatory cells and form a 'waterfall effect' in the body, so that the quantity of inflammatory mediators is continuously increased, and further, the tissue cells are damaged. Research results show that the expression levels of TNF-alpha, IL-6 and IL-1 beta in a mouse body are obviously increased on average during sepsis AKI, and the mouse body is not exceptional in kidney tissues, so that the kidney tissues are damaged. Subsequent studies have shown that TNF-. alpha.levels are elevated in vivo in response to systemic inflammation caused by sepsis, and the results of this study are identical. Therefore, TNF-alpha, IL-6 and IL-1 beta play an important role in sepsis acute AKI. The research result shows that HG-1 can down-regulate the expression of TNF-alpha, IL-6 and IL-1 beta, which is not only helpful for improving the condition of patients, but also has good guiding significance for subsequent treatment. HG-1 can regulate the expression level of TNF-alpha, IL-6 and IL-1 beta in kidney tissue, thereby playing a role in protecting sepsis kidney tissue injury.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. the purposes of coriander lactone-1, is characterized in that, described coriander lactone-1 has preventive effect on sepsis renal tissue damage, is used for preparing the purposes of treating sepsis renal tissue damage medicine;

Wherein, the structural formula of described coriander lactone-1 is:

2. the purposes of coriander lactone-1 as claimed in claim 1 is characterized in that, described coriander lactone-1 is prepared by following steps:

Step 1: Pulverize the dried coriander fruit medicinal materials, extract the volatile oil of the coriander fruit by the Soxhlet extraction method with petroleum ether, remove the volatile oil, perform Soxhlet extraction on the remaining medicinal residues with methanol, heat and evaporate to dryness until there is no alcohol smell, Obtaining the medicinal material methanol extraction liquid extract, dispersing the liquid extract with distilled water, then extracting with petroleum ether and dichloromethane in turn, recovering the solvent from the extract under reduced pressure, removing the petroleum ether extract, and obtaining the dichloromethane extract;

Step 2: After the dichloromethane extract is subjected to reduced pressure silica gel column chromatography, gradient elution is carried out with the volume ratio of petroleum ether: ethyl acetate for three times successively to obtain the first elution fraction Fr.A1, and the first elution fraction Fr. The second elution part Fr.A2 and the third elution part Fr.A3, after the third elution part Fr.A3 is subjected to silica gel column chromatography under reduced pressure, the volume ratio of petroleum ether: ethyl acetate is 70-90: 20 eluted to obtain the fourth elution site Fr.B1, and then passed through the ODS reversed-phase medium pressure column, followed by gradient elution with the volume ratio of methanol: water 7 times, to obtain 7 fractions, the 7 fractions After spotting, the same components as the main spots were combined to obtain methanol fraction Fr.C, and then passed through an ODS reversed-phase medium pressure column, followed by gradient elution with the volume ratio of methanol:water for 5 times, and the first fractions were obtained respectively. , the second fraction, the third fraction, the fourth fraction and the fifth fraction, the fourth fraction and the fifth fraction are combined to obtain the combined fraction part Fr.D, and finally the compound is obtained by semi-preparative high-performance liquid phase purification to obtain the compound coriander lactone-1 ;

Wherein, the reduced pressure column of silica gel column chromatography is 200-300 meshes.

3. purposes of coriander lactone-1 as claimed in claim 2 is characterized in that, described coriander lactone-1 is prepared by following steps:

Step 1. Pulverize 30 kg of dried coriander fruit medicinal materials, extract the volatile oil of coriander fruit with petroleum ether and Soxhlet extraction method, remove 4.5L of volatile oil, and then perform Soxhlet extraction on the remaining medicinal residues with methanol, and heat and evaporate to dryness. Alcoholic taste, get the medicinal material methanol extract liquid extract, disperse the liquid extract with 0.5-1.5L distilled water, then extract with petroleum ether and dichloromethane in turn, recover the solvent from the extract under reduced pressure, and remove the petroleum ether extract 605.0 g, to obtain 125.0 g of dichloromethane extract;

Step 2. After the dichloromethane extract is subjected to reduced pressure silica gel column chromatography, the volume ratios of petroleum ether and ethyl acetate are successively eluted at 90:10, 80:20 and 70:30 to obtain the first wash. Remove part of Fr.A1, the second elution part Fr.A2 and 20.0g of the third elution part Fr.A3, after the third elution part Fr.A3 is subjected to silica gel column chromatography under reduced pressure, with petroleum ether: ethyl acetate The volume ratio of the ester is 80:20 to elute to obtain 10.5g of the fourth elution site Fr.B1, which is then passed through an ODS reversed-phase medium pressure column, followed by methanol:water volume ratios of 20:80, 30:70, and 40: Elution was carried out at 60, 50:50, 60:40, 70:30 and 80:20 to obtain 7 fractions. After spotting the 7 fractions, the same components as the main spots were combined to obtain 2.00g methanol fraction Fr.C , and then pass through the ODS reversed-phase medium pressure column, and sequentially use methanol: water volume ratios of 20:80, 30:70, 40:60, 50:50 and 60:40 to elute to obtain the first fraction and the second fraction respectively. , the third fraction, the fourth fraction and the fifth fraction, the fourth fraction and the fifth fraction are combined to obtain 126mg of combined fractions Fr.D, and finally the compound coriander lactone-1 is obtained by purifying with semi-preparative high performance liquid phase;

Among them, the reduced pressure column of silica gel column chromatography is 300 meshes.