A terpenoid compound, a method for separating the same from honeysuckle and application of the terpenoid compound

Abstract

The present application relates to the technical field of natural product extraction, and particularly relates to application of a terpenoid 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 terpenoid 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 activity of resisting hepatitis C virus 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 terpene acid 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, anti-tumor and anti-virus. 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 in the extraction method of honeysuckle polysaccharide, and having the advantages of rapidity, simplicity, 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 terpenoid compounds, specifically, using honeysuckle as raw material, and preparing by extraction, separation and purification. Another purpose of the present application is to test the activity of the obtained terpenoid compounds against hepatitis C virus (HCV) to analyze their medicinal value.

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

[0009]

[0010] Preferably, the virus is hepatitis C virus (HCV).

[0011] The application further discloses a preparation method of the terpenoid compound, comprising the following steps:

[0012] (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;

[0013] (2) n-butanol extraction: the crude extract extract obtained in the step (1) is suspended in water, and then petroleum ether, ethyl acetate and n-butanol are sequentially used for extraction; the n-butanol extract is concentrated under reduced pressure to obtain an n-butanol partial extract;

[0014] (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;

[0015] (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;

[0016] (5) separation of the target compound: the fraction obtained in the step (4) is further separated by gel column LH-20, and then the compound (A1) is obtained through semi-preparative reverse phase HPLC purification.

[0017] 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, the ethanol extract is obtained through filtration, the filter residue is repeatedly extracted for 2-4 times, the ethanol extracts obtained in each time are combined, the solvent is recovered through concentration under reduced pressure to obtain the crude extract extract.

[0018] Preferably, the concentration of the extraction solvent ethanol used in the step (1) is 95%.

[0019] Preferably, the extraction times 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.

[0020] Preferably, the eluent of gel column LH-20 in step (5) is 50% methanol.

[0021] Preferably, the mobile phase of semi-preparative reverse-phase HPLC in step (5) is MeCN-H2O, 15:85, v / v.

[0022] The present application has the following beneficial effects:

[0023] The compound (A1) is isolated from honeysuckle for the first time, the separation and purification method is simple, and the compound is found to have certain anti-hepatitis C virus activity through activity testing, and has potential medicinal value. DETAILED DESCRIPTION

[0024] 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.

[0025] The technical solutions of the present application will be described below clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0026] In the description of the present application, it should be noted that the orientations or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like are the orientations or positional relationships shown, 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 indicated 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 description purposes and cannot be understood as indicating or implying relative importance.

[0027] 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 fixed connection, or detachable connection, or integrally connected; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or 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.

[0028] Example 1

[0029] 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. After filtration, the filtrate was combined and concentrated under reduced pressure using a rotary evaporator at a temperature below 45°C to obtain a total extract 3.2 kg. The total extract was suspended in 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 solvent, the n-butanol extract was concentrated under reduced pressure to recover the solvent, and then 315 g of extract was obtained. The extract was first subjected to column chromatography using 200-300 mesh silica gel, and then gradient eluted with 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 by preparative reverse phase high performance liquid chromatography (MeOH-H2O, 40:60, v / v) to obtain 10 fractions (Fr.5.1 to Fr.5.10). Fr.5.5 (15 g) was further separated by Sephadex LH-20 (50% methanol as the solvent) and then purified by semi-preparative reverse phase HPLC (MeCN-H2O, 15:85, v / v) to obtain compound (A1) 55 mg.

[0030]

[0031] The compound (A1) was analyzed to have a molecular formula of C20H 32 O4, in the form of a white solid powder, which 1 H and 13 The spectral data of1H NMR (DMSO-D6) and13C NMR (DMSO-D6) of compound (A1) are shown in Table 1 below:

[0032]

[0033] Table 1: NMR data of compound (A1)

[0034]

[0035]

[0036] In order to further illustrate the beneficial effects of the present application, the following application examples are provided:

[0037] Application Example 1

[0038] Detection of in vitro anti-HCV activity of compound (A1)

[0039] The full-length in vitro replicon system containing HCV gene subtypes 1a (H77) and 1b (Con1) was constructed, and a real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection method was established. Different concentrations of compound (A1) and the same volume of dimethyl sulfoxide (DMSO, negative control) were added to the above virus replication system, and the culture was continued for 48 hours. Then, qRT-PCR viral nucleic acid quantitative detection was performed.

[0040] In the pharmacological activity experiment, compound (A1) was dissolved in DMSO and prepared into a 10 mM stock solution. The starting concentration of the compound was set at 500 nM, and the final concentration was 1.0 nM. Each treatment was performed in three independent repeated experiments, with five replicate wells for each repetition. The experimental data were statistically analyzed.

[0041] It was detected that compound (A1) had a certain inhibitory effect on the replication efficiency of H77 and Con1 subtypes of HCV. The IC50 of the compound for Con1 subtype HCV replication was about 108 nM, and the IC50 for H77 subtype HCV replication was about 185 nM. 50

[0042] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. A method for preparing a terpene acid compound (A1), characterized in that, The preparation method includes the following steps: (1) Ethanol extraction: The naturally dried honeysuckle was added to ethanol for extraction, filtered, and the filtrate was concentrated under reduced pressure to obtain crude extract; the concentration of the extraction solvent ethanol was 95%. (2) n-Butanol extraction: The crude extract obtained in step (1) is suspended in water and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence. The n-butanol extract is concentrated under reduced pressure to obtain n-butanol extract. (3) Silica gel column chromatography: The n-butanol extract obtained in step (2) was separated by column chromatography. The packing material was silica gel of 200-300 mesh. Chloroform-methanol with a volume ratio of 15 / 1 to 1 / 5 was used as the eluent for gradient elution. The eluent containing the target compound was collected. The solvent was removed by vacuum distillation to obtain the silica gel column chromatography fraction. (4) Reversed-phase high performance liquid chromatography purification: The silica gel column chromatography fraction obtained in step (3) is further separated on a preparative reversed-phase high performance liquid chromatography (RPLC) with MeOH-H2O, 40:60, v / v. The eluent containing the target compound is collected, and the solvent is removed by vacuum distillation to obtain the purified fraction by reversed-phase high performance liquid chromatography. (5) Separation of target compounds: The fraction obtained in step (4) was separated again by a gel column LH-20, and then purified by semi-preparative reversed-phase HPLC to obtain terpene acid compounds (A1).

2. The preparation method according to claim 1, characterized in that, The specific operation method of step (1) is as follows: the dried honeysuckle is extracted with ethanol, the mass-volume ratio between honeysuckle and ethanol is 1 / 2 to 1 / 5, Kg / L, and the extraction is carried out at room temperature for 1 to 7 days. Then the ethanol extract is obtained by filtration. The filter residue is extracted 2 to 4 times. The ethanol extracts obtained each time are combined, and the solvent is recovered by vacuum concentration to obtain crude extract.

3. The preparation method according to claim 1, characterized in that, In step (2), the n-butanol is extracted 2-4 times.

4. The preparation method according to claim 1, characterized in that, In step (2), the n-butanol is extracted three times.

5. The preparation method according to any one of claims 2-4, characterized in that, In step (5), the eluent for the gel column LH-20 is 50% methanol; in step (5), the mobile phase for the semi-preparative reversed-phase HPLC is MeCN-H2O, 15:85, v / v.

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