A sesquiterpene lactone compound with antibacterial activity

Abstract

The present application relates to the technical field of natural product extraction, and particularly relates to application of a sesquiterpene lactam compound separated from honeysuckle to preparation of an antiviral drug and a separation method thereof.The present application has the beneficial effect that a novel sesquiterpene lactam compound is separated from honeysuckle for the first time, the separation and purification method is simple, and after activity testing, it is found that the compound has certain antibacterial activity and has potential medicinal value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction, and particularly relates to a method for separating sesquiterpene lactam compounds from honeysuckle and application thereof. BACKGROUND

[0002] Honeysuckle is the dried flower buds or early opened flowers of Lonicera japonica Thunb. of Caprifoliaceae. Lonicera japonica Thunb. is mainly distributed in East China, South China, Southwest China, Hebei, Shanxi, Liaoning, Shaanxi and Gansu. Lonicera confusa is mainly distributed in Guangdong, Guangxi and Hainan. Lonicera macgyllivrayi is distributed in Zhejiang, Anhui, Fujian, Jiangxi, Hubei, Hunan, Guangdong, Guangxi, Sichuan, Guizhou and Yunnan. Lonicera fulvotomentosa is distributed in Guangxi, Guizhou and Yunnan. Honeysuckle has the effects of clearing heat and detoxifying, and is used for treating exogenous wind-heat or warm disease fever, heatstroke, toxic dysentery, carbuncle, sore, laryngalgia and various infectious diseases.

[0003] Honeysuckle has 3-4 flowering periods in a year, and the first flowering period is from April to June, accounting for about 70% of the annual yield. The flower bud is slightly curved, with a rough upper part and a thin lower part, and is 2-3 cm long. The flower is white at the beginning, and turns yellow after 1-2 days due to the rapid increase of carotenoid content. According to the color and shape, it is usually divided into four main periods: green bud period, two white period, silver flower period and gold flower period. The fruiting period is relatively late, from October to November, and the fruit is round or oval, about 3 mm long, and blue-black when mature. Honeysuckle has high tolerance to the growth environment, mainly due to its high content of phenolic substances. The highest growth altitude can reach 1500 m, and Shandong and Henan are the main production areas of honeysuckle. Honeysuckle is rich in various bioactive substances, and has the effects of regulating blood sugar, antioxidant, antitumor and antiviral. The accumulation of functional components is affected by the production area, altitude, variety and processing method.

[0004] At present, the secondary metabolites of honeysuckle and their related activities have been studied. Among them, Chinese patent application CN201710610649.9 discloses a method for isolating luteolin from honeysuckle. The extraction reagent contains not only ethanol but also sodium hydroxide solution. Sodium hydroxide plays a role in cell lysis, accelerating the lysis of cell wall and cell membrane. Under the combined action of ethanol and sodium hydroxide, the efficiency of extracting luteolin from honeysuckle is higher. Chinese patent application CN200510019402.7 discloses a method for extracting honeysuckle polysaccharide from honeysuckle. Water is used to extract crude polysaccharide from honeysuckle, sevag reagent method is used to remove protein, chloroform, petroleum ether and other solvents are used to remove fat-soluble impurities, and finally ethanol is used to precipitate and separate the polysaccharide from the water extract. After drying, solid polysaccharide is obtained, filling the gap of honeysuckle polysaccharide extraction method, with the advantages of fast, simple, low cost and high yield, which is conducive to further study of the application of honeysuckle polysaccharide in medicine. Chinese patent application CN201110303723.5 discloses a process for extracting chlorogenic acid from honeysuckle. The process provides a short-time, low-energy and high-yield extraction process for extracting chlorogenic acid from honeysuckle. Ultrasonic method is used to extract chlorogenic acid from honeysuckle. The process steps are as follows: taking the crushed honeysuckle powder in a test tube, adding 60-80% ethanol solution with a material-liquid ratio of 1:10 for 24h, adjusting the pH value of the system to 2-6, and ultrasonic extraction for 2-4 times.

[0005] In addition, Luo et al. (Components and processing of honeysuckle, Food and machinery, April 2022, Vol. 38, No. 246, No. 4, pp. 228-233) studied the active components, drying processing methods, functional component extraction and processing products of honeysuckle in recent years, and discussed the future development direction. It is pointed out that honeysuckle contains flavonoids, phenolic compounds, volatile oils, iridoid glycosides and many other compounds, and it is pointed out that these compounds have the functions of regulating blood sugar, antioxidant, antitumor and antiviral.

[0006] On this basis, it is hoped that the secondary metabolites of honeysuckle will be further studied, so as to discover compounds with new skeleton structure, study their pharmacological activities, and analyze their potential medicinal value. SUMMARY

[0007] The purpose of the present application is to provide a preparation method of sesquiterpene lactam compounds, specifically, using honeysuckle as raw material, and preparing by extraction, separation and purification. Another purpose of the present application is to test the antibacterial activity of the obtained sesquiterpene lactam compounds, in order to analyze their medicinal value.

[0008] The application discloses a sesquiterpene lactam compound separated from honeysuckle as an application to preparation of an antiviral medicine, and a structural formula of the sesquiterpene lactam compound is shown as (A1):

[0009]

[0010] Preferably, the bacteria are gram-positive bacteria.

[0011] More preferably, the gram-positive bacteria are at least one of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

[0012] The application further discloses a preparation method of the sesquiterpene lactam compound, comprising the following steps:

[0013] (1) ethanol extraction: natural dried honeysuckle is added into ethanol for extraction, filtration is performed, and the filtrate is concentrated under reduced pressure to obtain a crude extract extract;

[0014] (2) n-butanol extraction: the crude extract extract obtained in the step (1) is suspended in water, and then sequentially extracted with petroleum ether, ethyl acetate and n-butanol; the n-butanol extract is concentrated under reduced pressure to obtain an n-butanol partial extract;

[0015] (3) silica gel column chromatography: the n-butanol partial extract obtained in the step (2) is subjected to column chromatography separation, the filler is 200-300 mesh silica gel, the eluent is chloroform-methanol with a volume ratio of 15 / 1-1 / 5, gradient elution is performed, the eluent containing the target compound is collected, and the solvent is removed under reduced pressure to obtain a silica gel column chromatography fraction;

[0016] (4) reverse phase high performance liquid chromatography purification: the silica gel column chromatography fraction obtained in the step (3) is further separated on a preparative reverse phase high performance liquid chromatography (MeOH-H2O, 40:60, v / v), the eluent containing the target compound is collected, and the solvent is removed under reduced pressure to obtain a reverse phase high performance liquid chromatography purification fraction;

[0017] (5) separation of the target compound: the fraction obtained in the step (4) is purified by semi-preparative reverse phase HPLC to obtain the compound shown as the formula (A1).

[0018]

[0019] Preferably, the specific operation method of the step (1) is as follows: the dried honeysuckle is extracted by ethanol, the mass / volume ratio between the honeysuckle and the ethanol is 1 / 2-1 / 5 (Kg / L), the extraction is performed at normal temperature for 1-7 days, then the ethanol extract is obtained through filtration, the filter residue is repeatedly extracted for 2-4 times, the ethanol extract obtained in each time is combined, the solvent is recovered through concentration under reduced pressure to obtain the crude extract extract.

[0020] Preferably, the concentration of the extraction solvent ethanol used in step (1) is 95%.

[0021] Preferably, the extraction of n-butanol in step (2) is 2-4 times, more preferably 3 times, and the n-butanol extraction phase is combined to recover the solvent under reduced pressure to obtain the n-butanol partial extract.

[0022] Preferably, the mobile phase of semi-preparative reverse-phase HPLC in step (5) is MeCN-H2O, 40:60, v / v.

[0023] The beneficial effects of the present application are:

[0024] The compound of formula (A1) is isolated from honeysuckle for the first time, the separation and purification method is simple, and the compound is found to have certain antibacterial activity after activity testing, and has potential medicinal value. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application will be further described below in conjunction with examples, and the following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0026] The technical solutions of the present application will be described below in conjunction with examples, obviously, the described examples are part of the examples of the present application, not all examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the examples, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0028] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0029] Example 1

[0030] The natural dried honeysuckle 20 kg was soaked with 80 L of 95% ethanol at room temperature for extraction, 3 days each time, 3 times in total, and the filtrate was combined after filtration. The obtained filtrate was concentrated under reduced pressure using a rotary evaporator at a temperature lower than 45°C to obtain a total extract 3.2 kg. The obtained total extract was suspended with 2500 ml of distilled water, and then sequentially extracted with petroleum ether (2500 ml x 3), ethyl acetate (2500 ml x 3), and n-butanol (2500 ml x 3). After combining the extracts of the same extraction solvent, the n-butanol extract was concentrated under reduced pressure to recover the solvent to obtain an extract 315 g. The extract was first subjected to column chromatography using silica gel with a particle size of 200-300 mesh, and gradient elution was performed using chloroform-methanol (v / v, 15:1 to 1:5) as the eluent to obtain 7 fractions Fr.1-Fr.7 in sequence. Fr.5 (67.6 g) was further separated on a preparative reverse-phase high-performance liquid chromatograph (MeOH-H2O, 40:60, v / v) to obtain 10 fractions (Fr.5.1 to Fr.5.10). Fr.5.2 (10.5 g) was further purified by semi-preparative reverse-phase HPLC (MeCN-H2O, 40:60, v / v) to obtain compound (A1) 182 mg.

[0031]

[0032] Analysis showed that the molecular formula of compound (A1) was C20H 32 O4, which was in the form of a white solid powder, and its 1 H and 13 The spectral data of1H NMR (DMSO-D6) and13C NMR (DMSO-D6) of compound (A1) are shown in Table 1 below:

[0033]

[0034] Table 1: NMR data of compound (A1)

[0035]

[0036]

[0037] In order to further illustrate the beneficial effects of the present application, the following application examples are provided:

[0038] Application Example 1

[0039] Detection of the minimum inhibitory concentration (MIC) of the compound (A1) on part of gram-positive bacteria

[0040] Test strains: methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 and vancomycin-resistant Enterococcus (VRE). Experimental method: The determination of MIC was carried out according to the CLSI guidelines by broth microdilution method. The colonies were cultured in MHB broth medium containing calcium and magnesium ions to form a final concentration of 5*105CFU / mL inoculum. The compound was dissolved in DMSO to prepare a drug solution with a final concentration of 1152 μg / mL, and DMSO was used as a negative control. The MIC was determined by two-fold dilution method. The MIC value was determined after the compound and the inoculum were incubated at 35°C for 20 hours.

[0041] Experimental results: The negative control DMSO was detected to be stained, and had no additional bacteriostatic effect; the MIC value of the compound of formula (I) on methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 was 18 μg / mL, and the MIC value on vancomycin-resistant Enterococcus (VRE) was 72 μg / mL. It is shown that the compound (A1) has significant bacteriostatic activity.

[0042] Finally, it should be pointed out that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The use of a sesquiterpene lactone compound isolated from honeysuckle as an antibacterial drug, characterized in that, The sesquiterpene lactone compound has a structural formula as shown in (A1): The bacteria are gram-positive bacteria; the gram-positive bacteria are at least one of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

2. A method for producing a sesquiterpene lactone compound (Al) characterized by, The preparation method comprises the following steps: (1) ethanol extraction: natural dried honeysuckle is added into ethanol for extraction, filtration, and the filtrate is concentrated under reduced pressure to obtain a crude extract extract; the concentration of the extraction solvent ethanol used is 95%; (2) n-butanol extraction: the crude extract extract obtained in step (1) is suspended in water, and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and the n-butanol extract is concentrated under reduced pressure to obtain an n-butanol partial extract; (3) silica gel column chromatography: the n-butanol partial extract obtained in step (2) is subjected to column chromatography separation, the filler is 200-300 mesh silica gel, the eluent is chloroform-methanol with a volume ratio of 15 / 1-1 / 5, gradient elution is carried out, the eluent containing the target compound is collected, and the solvent is removed under reduced pressure to obtain a silica gel column chromatography fraction; (4) reverse phase high performance liquid chromatography purification: the silica gel column chromatography fraction obtained in step (3) is further separated by preparative reverse phase high performance liquid chromatography, MeOH-H2O, 40:60, v / v, and the eluent containing the target compound is collected, and the solvent is removed under reduced pressure to obtain a reverse phase high performance liquid chromatography purification fraction; (5) separation of the target compound: the fraction obtained in step (4) is purified by semi-preparative reverse phase HPLC to obtain the sesquiterpene lactone compound (A1); 3. The method of claim 2, wherein, The specific operation method of step (1) is: the dried honeysuckle is extracted with ethanol, the mass-volume ratio of honeysuckle to ethanol is 1 / 2-1 / 5, Kg / L, extraction is carried out at room temperature for 1-7 days, then filtration is carried out to obtain an ethanol extract, the filter residue is repeatedly extracted for 2-4 times, the ethanol extracts obtained in each time are combined, and the solvent is recovered by concentration under reduced pressure to obtain a crude extract extract.

4. The preparation method according to claim 2, characterized in that, The extraction of n-butanol in step (2) is carried out 2-4 times.

5. The preparation method according to claim 2, characterized in that, The extraction of n-butanol in step (2) is carried out 3 times.

6. The method of any one of claims 2-5, wherein, The mobile phase of semi-preparative reverse phase HPLC in step (5) is MeCN-H2O, 40:60, v / v.

Images