Terpenic acid compound, method for separating terpenic acid compound from honeysuckle and application of terpenic acid compound

Abstract

The invention relates to the technical field of natural product extraction, in particular to application of a terpenic acid compound separated from honeysuckle to preparation of an antiviral drug and a separation method of the terpenic acid compound. The novel terpene acid compound has the beneficial effects that the novel terpene acid compound is separated from honeysuckle for the first time, the separation and purification methods are simple, and the compound is found to have certain anti-hepatitis C virus activity and potential medicinal value after an activity test.

Description

[0001] The present application is a divisional application of application No. 2023102760639, filed on March 21, 2023, with the title of "Terc acid compound and method for separating the same from honeysuckle and application thereof". TECHNICAL FIELD

[0002] The present application relates to the technical field of natural product extraction, and particularly relates to a method for separating terc acid compounds from honeysuckle and application thereof. BACKGROUND

[0003] Honeysuckle is the dried flower buds or early opened flowers of Lonicera japonica Thunb. of Caprifoliaceae. The plant Lonicera japonica Thunb. is mainly distributed in East China, South China, Southwest China, Hebei, Shanxi, Liaoning, Shaanxi and Gansu; Lonicera japonica Thunb. in South China is mainly distributed in Guangdong, Guangxi and Hainan; Lonicera japonica Thunb. in Zhejiang, Anhui, Fujian, Jiangxi, Hubei, Hunan, Guangdong, Guangxi, Sichuan, Guizhou and Yunnan; Lonicera japonica Thunb. in Guangxi, Guizhou and Yunnan. It 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.

[0004] Honeysuckle has 3-4 flowering periods in a year, and the first batch of flowers in April to June accounts for about 70% of the annual yield. The flower buds are slightly curved, with a rough lower part, about 2-3 cm long, and the flowers are white at the beginning, and turn 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 shape 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, and the highest growth altitude can reach 1500 m. 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, and the accumulation of functional components is affected by the production area, altitude, variety and processing method.

[0005] 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.

[0006] 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.

[0007] 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

[0008] The purpose of the present application is to provide a preparation method of a terpenoid compound, specifically, using honeysuckle as raw material, and preparing it by extraction, separation and purification. Another purpose of the present application is to test the anti-hepatitis C virus (HCV) activity of the obtained terpenoid compound, in order to analyze its medicinal value.

[0009] The application discloses a terpenoid compound separated from honeysuckle as an application to preparation of an antiviral medicine, and a structural formula of the terpenoid compound is shown as (A1):

[0010] (A1).

[0011] Preferably, the virus is hepatitis C virus (HCV).

[0012] The application further discloses a preparation method of the terpenoid compound, comprising the following steps: (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; (2) n-butanol extraction: the crude extract extract obtained in the 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 an n-butanol partial extract; (3) silica gel column chromatography: the n-butanol partial extract obtained in the step (2) is subjected to column chromatography separation, 200-300 mesh silica gel is used as a filler, chloroform-methanol with a volume ratio of 15 / 1-1 / 5 is used as an eluent, 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; (4) reverse phase high performance liquid chromatography purification: the silica gel column chromatography fraction obtained in the step (3) is further subjected to separation 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; (5) separation of the target compound: the fraction obtained in the step (4) is further subjected to separation by means of a gel column LH-20, and then the compound (A1) is obtained through semi-preparative reverse phase HPLC purification.

[0013] Preferably, the specific operation method of the step (1) is as follows: dried honeysuckle is extracted by means of ethanol, the mass / volume ratio between the honeysuckle and the ethanol is 1 / 2-1 / 5 (Kg / L), extraction is performed at normal temperature for 1-7 days, then filtration is performed to obtain an ethanol extract, 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 a crude extract extract.

[0014] Preferably, the concentration of the extraction solvent ethanol used in the step (1) is 95%.

[0015] Preferably, the extraction times of n-butanol in the step (2) are 2-4 times, more preferably 3 times, the n-butanol extraction phases are combined, the solvent is recovered under reduced pressure to obtain the n-butanol partial extract.

[0016] Preferably, the eluent for the gel column LH-20 in step (5) is 50% methanol.

[0017] Preferably, the mobile phase of the semi-preparative reversed-phase HPLC in step (5) is MeCN-H2O, 15:85, v / v.

[0018] The beneficial effects of this invention are as follows: This invention is the first to isolate compound (A1) from honeysuckle. The isolation and purification methods are simple. After activity testing, it was found that the compound has certain anti-hepatitis C virus activity and has potential medicinal value. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described below with reference to the examples. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0020] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating the orientation or positional relationship shown, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0022] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0023] In Example 1, 20 kg of naturally dried honeysuckle was extracted by soaking in 80 L of 95% ethanol at room temperature for 3 days each time, for a total of 3 soaks. After filtration, the filtrates were combined, and the resulting filtrates were concentrated under reduced pressure using a rotary evaporator at a temperature below 45°C to obtain 3.2 kg of total extract. The obtained total extract was suspended in 2500 ml of distilled water, and then extracted sequentially with petroleum ether (2500 ml × 3), ethyl acetate (2500 ml × 3), and n-butanol (2500 ml × 3). After combining the extracts with the same extraction solvent, the n-butanol extract was concentrated under reduced pressure to recover the solvent and obtain 315 g of extract. This extract was first subjected to rough separation by column chromatography on 200-300 mesh silica gel, with gradient elution using chloroform-methanol (v / v, 15:1 to 1:5) as the eluent, to obtain 7 fractions Fr.1-Fr.7. Fr.5 (67.6 g) was further separated by preparative reversed-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 purified by semi-preparative reversed-phase HPLC (MeCN-H2O, 15:85, v / v) after separation by dextran gel LH-20 (solvent: 50% methanol) to obtain compound (A1) 55 mg.

[0024]

[0025] (A1) Analysis revealed that compound (A1) has the molecular formula C20H32O4 and is a white solid powder. 1 H and 13 The spectral data of CNMR (DMSO-D6) are shown in Table 1 below:

[0026] Table 1: NMR data of compound (A1)

[0027] To further illustrate the beneficial effects of the present invention, the following application examples are provided: Application Example 1 Detection of the in vitro anti-HCV activity of compound (A1) We constructed in vitro replication subsystems for the full-length HCV genome containing HCV genotypes 1a (H77) and 1b (Con1), and a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection method. Different concentrations of compound A1 and the same volume of dimethyl sulfoxide (DMSO, negative control) were added to the above viral replication systems, and after incubation for 48 hours, qRT-PCR was performed for quantitative detection of viral nucleic acid.

[0028] In the pharmacological activity experiments, compound (A1) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The initial concentration of the compound was set at 500 nM, and the final concentration was 1.0 nM. Each treatment was performed in 3 independent replicates, with 5 replicates per replicate. The experimental data were statistically analyzed.

[0029] Testing revealed that compound (A1) exhibited a certain inhibitory effect on the replication efficiency of both H77 and Con1 HCV isoforms. Specifically, this compound significantly reduced the IC50 of the Con1 HCV isoform replication. 50 The IC50 for H77 subtype HCV replication is approximately 185 nM, with an IC50 of approximately 108 nM.

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a terpene acid compound isolated from honeysuckle in the preparation of an antiviral drug, characterized in that, The structural formula of the terpene acid compound is shown in (A1): （A1）。 2. The application according to claim 1, characterized in that, The virus in question is the hepatitis C virus.

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