Lactone ring-type forsythia diterpenoids and their preparation method and anti-inflammatory and antioxidant applications

Abstract

The present invention discloses a lactone ring type forsythia diterpene and its preparation method and anti-inflammatory and antioxidant applications, belonging to the technical field of pharmaceutical chemistry. The lactone ring type forsythia diterpene is a semi-floral type diterpene containing a five-membered lactone ring and a molecular formula of C 20 H 30 The benefits of the present invention lie in: using an innovative extraction and separation process, forsythia diterpenes containing a five-membered lactone ring structural unit are efficiently isolated from the fruit of Forsythia suspensa. This five-membered lactone ring structural unit is extremely rare among the hemiolean diterpenes, making it the first such discovery in the genus Forsythia. Studies have found that this lactone ring-type forsythia diterpenoid has excellent anti-inflammatory and antioxidant activities, providing a new material basis for the study of the anti-inflammatory and antioxidant activities of Forsythia suspensa.

Description

Technical Field

[0001] The present invention relates to a compound, a preparation method and an application thereof, and in particular to a cymene-type diterpene containing a five-membered lactone ring structural unit separated from the fruit of Forsythia suspensa, a preparation method thereof, and anti-inflammatory and antioxidant applications thereof, belonging to the technical field of medicinal chemistry. Background Art

[0002] Forsythia suspensa Forsythia suspensa ) is a shrub of the genus Forsythia in the family Oleaceae. Its dried fruit is included in the Chinese Pharmacopoeia as a traditional Chinese medicinal material, with the effects of clearing away heat and detoxifying, reducing swelling and dispersing nodules. Modern pharmacological studies have shown that Forsythia has outstanding performance in anti-inflammatory, antiviral and antioxidant aspects. At present, research on the material basis of the efficacy of Forsythia is mostly focused on components such as phenylpropanoids (such as forsythiaside), flavonoids (such as rutin), triterpenes and phenylethanoid glycosides. These compounds have been proven to have anti-inflammatory and antiviral activities. There is also a class of diterpenoid compounds with significant activity in Forsythia. Due to the low content of such compounds in Forsythia and the lack of mature methods for the efficient separation of diterpenoid monomers from Forsythia in existing technologies, their research has been neglected for a long time.

[0003] Diterpenoids have attracted much attention in the development of natural product drugs due to their complex structures and diverse biological activities. Cynanchalcan-type diterpenes are a type of bicyclic diterpenes with decahydronaphthalene as the parent nucleus. They have a rich variety of structural types, the most representative of which are andrographolide and sclerotin. Cynanchalcan-type diterpenes in Leonurus plants can be divided into furan ring type, dihydrofuran ring type, tetrahydrofuran ring type, lactone ring type and α, β-unsaturated lactone ring type according to the type of five-membered ring attached to C12. In recent years, studies have found that cynanchalcan-type diterpenes have significant biological activities in antibacterial, anti-inflammatory and anti-tumor aspects.

[0004] For the helianthane-type diterpenes from Forsythia plants, it has been found that C12 in most of their structures is connected to a but-2-enoic acid structural unit. Helianthane-type diterpenes with special structures such as a five-membered lactone ring connected to C12 have not been reported yet because of their extremely small number and the difficulty in separation and purification. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention aims to provide a forsythia suspensa-type diterpenoid containing a five-membered lactone ring structural unit and having good anti-inflammatory and antioxidant activities, as well as a method for efficiently isolating the diterpenoid from the fruit of Forsythia suspensa, and also to provide the pharmaceutical application of the diterpenoid.

[0006] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0007] Lactone ring type forsythia diterpenoid, the lactone ring type forsythia diterpenoid is a semi-floral type diterpenoid, containing a five-membered lactone ring structural unit, the molecular formula is C20 H 30 O3, the structural formula is as follows:

[0008] .

[0009] The preparation method of the aforementioned lactone ring-type Forsythia diterpenoid comprises the following steps:

[0010] (1) Take the fruit of Forsythia suspensa, extract it with methanol or 95% ethanol under reflux, recover the organic solvent, and obtain extract I;

[0011] (2) Dispersing extract I with deionized water, then extracting with chloroform or ethyl acetate, combining the organic solvent portions, and recovering the organic solvent to obtain extract II;

[0012] (3) Dissolve extract II in n-butanol, then extract with 3%-4% hydrochloric acid, combine the n-butanol fractions, and recover the organic solvent to obtain the diterpene fraction;

[0013] (4) The diterpene fraction was loaded onto a normal phase silica gel column and gradient eluted using a cyclohexane-ethyl acetate system or a cyclohexane-acetone system as an eluent. The eluate was detected by TLC. The eluate containing the diterpene was intercepted and concentrated under reduced pressure to obtain a concentrate I.

[0014] (5) The concentrate I was subjected to chromatography separation using a preparative thin layer chromatography to intercept the diterpene component, which was eluted with acetone and concentrated under reduced pressure to obtain the concentrate II;

[0015] (6) Separate the concentrated solution II using a Sephadex LH-20 gel column or preparative high performance liquid chromatography, collect the diterpene components, concentrate under reduced pressure, crystallize, and dry to obtain the aforementioned lactone ring-type Forsythia diterpene.

[0016] Preferably, in step (1), when methanol is used for reflux extraction, the ratio of Forsythia suspensa fruit to methanol is 1 kg:23 L, and the methanol is divided into 10 parts, 8 parts, and 5 parts, each of which is refluxed and extracted once, and each reflux extraction is 1 hour; when 95% ethanol is used for reflux extraction, the ratio of Forsythia suspensa fruit to 95% ethanol is 1 kg:10 L, and the reflux extraction is performed twice, the first time is 2 hours, and the second time is 1 hour.

[0017] Preferably, in step (4), when the eluent adopts a cyclohexane-ethyl acetate system, the eluent is eluted in a gradient according to a volume ratio of (100:0) to (50:50); when the eluent adopts a cyclohexane-acetone system, the eluent is eluted in a gradient according to a volume ratio of (95:5) to (60:40).

[0018] Preferably, in step (5), the developing solvent is a mixture of methanol, acetone and chloroform, V 甲醇 :V 丙酮 :V 氯仿=1:1:8, intercept the component with Rf=0.6.

[0019] Preferably, in step (6), when Sephadex LH-20 gel column is used to separate the concentrated solution II, the eluent is a mixture of 95% ethanol and acetone, V 95%乙醇 :V 丙酮 =1:1; when the concentrated solution II is separated by preparative high performance liquid chromatography, the chromatographic conditions are: C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 甲醇 :V 水 =75:25, flow rate 5.0 mL / min, injection volume 2 mL, detection wavelength 255 nm, and components with retention time between 16 min and 18 min were collected.

[0020] Application of the aforementioned lactone ring-type Forsythia diterpenoids in the preparation of anti-inflammatory drugs and / or antioxidant drugs.

[0021] The benefits of the present invention lie in that an innovative extraction and separation process is used to efficiently separate a helianthane-type diterpene from the fruit of Forsythia suspensa. The C12 and C15 of the helianthane-type diterpene are connected by an oxygen atom to form a five-membered lactone ring structural unit. The number of helianthane-type diterpenes with this five-membered lactone ring structural unit is extremely small, and this is the first time it has been discovered in Forsythia suspensa plants. Experimental verification shows that the lactone-ring forsythia diterpene has good anti-inflammatory and antioxidant activities, providing a new material basis for the study of the anti-inflammatory and antioxidant activities of Forsythia suspensa. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HR-MS spectrum of the lactone ring-type Forsythia diterpenoid provided by the present invention;

[0023] Figure 2 The lactone ring type forsythia diterpenoid provided by the present invention 1 H-NMR spectrum;

[0024] Figure 3 The lactone ring type forsythia diterpenoid provided by the present invention 13 C-NMR spectrum;

[0025] Figure 4 This is the HSQC spectrum of the lactone ring type Forsythia diterpenoid provided by the present invention;

[0026] Figure 5 This is the HMBC spectrum of the lactone ring-type Forsythia diterpenoid provided by the present invention;

[0027] Figure 6 The lactone ring type forsythia diterpenoid provided by the present invention 1 H- 1 H COSY spectrum;

[0028] Figure 7 The NOESY spectrum of the lactone ring type Forsythia diterpenoid provided by the present invention;

[0029] Figure 8 The single crystal X-ray diffraction pattern of the lactone ring type Forsythia diterpenoid provided by the present invention;

[0030] Figure 9 It is the main lactone ring type forsythia diterpenoid provided by the present invention 1 H- 1 H COSY and HMBC correlation diagram, where the bold line represents 1 H- 1 H COSY, arrowed lines indicate HMBC;

[0031] Figure 10 This is the main NOESY correlation diagram of the lactone ring type Forsythia diterpenoids provided by the present invention. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] 1. Structure of lactone-ring forsythia diterpenoids

[0034] The lactone ring type forsythia diterpenoid provided by the present invention is a semi-floral type diterpenoid containing a five-membered lactone ring structural unit, and the molecular formula is C 20 H 30 O3, the structural formula is as follows:

[0035] .

[0036] 2. Preparation method of lactone ring type forsythia diterpenoids

[0037] Example 1

[0038] The present invention provides a method for preparing the lactone ring-type Forsythia diterpenoid, which specifically comprises the following steps:

[0039] 1. Take 10 kg of Forsythia suspensa fruit and extract it twice with 100 L of 95% (v / v) ethanol under reflux, with the first extraction period lasting 2 h and the second extraction period lasting 1 h. Combine the extracts and then concentrate under reduced pressure to recover the organic solvent to obtain extract I.

[0040] 2. Disperse extract I with an equal volume of deionized water, then extract with an equal volume of ethyl acetate three times, combine the organic solvent portions, and then concentrate under reduced pressure to recover the organic solvent to obtain extract II;

[0041] 3. Dissolve extract II with 2 volumes of n-butanol, then extract with an equal volume of 3% (v / v) hydrochloric acid five times, combine the n-butanol fractions, and then concentrate under reduced pressure to recover the organic solvent to obtain the diterpene fraction;

[0042] 4. The diterpene fraction was sequentially separated by silica gel column chromatography, preparative thin layer chromatography, and preparative high performance liquid chromatography. Specifically:

[0043] (1) The diterpene fraction was loaded onto a normal phase silica gel column and eluted with a cyclohexane-acetone system in a gradient elution ratio of (95:5) to (60:40) by volume. The eluate was detected by thin layer chromatography (TLC). The eluate containing the diterpene was intercepted and concentrated under reduced pressure to obtain a concentrate I.

[0044] (2) The concentrate I was subjected to further chromatography using a preparative thin layer chromatography (TLC) with a mixture of methanol, acetone, and chloroform as the developing solvent. 甲醇 :V 丙酮 :V 氯仿 =1:1:8, intercept the component with Rf=0.6, elute with acetone, and concentrate under reduced pressure to obtain concentrate II;

[0045] (3) Separate the components in concentrate II using preparative HPLC. The chromatographic conditions are: C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase: V 甲醇 :V 水 =75:25, flow rate 5.0 mL / min, injection volume 2 mL, detection wavelength 255 nm, collect the components with retention time between 16 min and 18 min, concentrate under reduced pressure, crystallize, and dry to obtain 187.84 mg of colorless needle-shaped crystals with a purity of 99.8%.

[0046] The colorless needle-shaped crystals are easily soluble in chloroform and acetone, and the specific rotation is [α] 25 D +37.4 (c0.30, methanol).

[0047] In high-resolution mass spectrometry (HR-MS) detection ( Figure 1 ),exist m / z 341.2081[M+ Na] + The quasi-molecular ion peak of the compound was obtained, and the molecular formula of the colorless needle-shaped crystal was determined to be C 20 H 30 O3.

[0048] In the H NMR spectrum ( 1 H-NMR) detection ( Figure 2, 400 MHz), 3 methyl signals, 1 hydroxyl hydrogen signal, 1 methylene hydrogen signal of hydroxymethyl and 3 unsaturated hydrogen signals were found.

[0049] In the carbon NMR spectroscopy ( 13 C-NMR) detection ( Figure 3 , 100 MHz), and 20 carbon signals were found.

[0050] Heteronuclear single quantum correlation (HSQC) detection ( Figure 4 ), it was determined that these 20 carbon signals belonged to 3 methyl groups, 8 methylene groups, 4 methine groups and 5 quaternary carbon groups.

[0051] through 1 Heteronuclear multicarbon correlation spectroscopy (HMBC) detection of H Figure 5 ), the colorless needle-shaped crystals were determined to be hemipentane-type diterpenes.

[0052] In the homonuclear heterotopic correlation spectrum ( 1 H- 1 H COSY) detection ( Figure 6 ), three spin systems were discovered. The first spin system included three methylene groups, the second spin system included one methine group and two methylene groups, and in the third spin system, one methylene group was observed to be coupled to two different methine groups.

[0053] Comprehensive analysis of HSQC, HMBC and 1 H- 1 H COSY analysis confirmed that the planar structure of the colorless needle-shaped crystals was 18-hydroxyhexadecane-8(17),13-diene-15,12-lactone.

[0054] In two-dimensional nuclear Overhauser effect spectroscopy (NOESY) detection ( Figure 7 ), it was found that H-5 was correlated with CH3-19 and H-9, H-9 was correlated with H-5 and H-11a, CH3-20 was correlated with H2-18 and H-11b, and H-11b was correlated with H-12 and CH3-20. This indicates that CH3-19, H-5 and H-9 are in the same plane and have an α configuration, while H2-18, CH3-20 and H-12 are located on the other side of the molecular plane and have a β configuration.

[0055] Detected by single crystal X-ray diffraction (Cu target) ( Figure 8 ), the absolute configurations of the chiral carbon atoms C-4, C-5, C-9, C-10, and C-12 were determined to be S, R, S, R, and R, respectively.

[0056] From the above test results, it can be determined that the colorless needle-shaped crystals are (4S, 5R, 9S, 10R, 12R)-18-hydroxy-1 ... 20 H 30 O3, the structural formula is:

[0057] .

[0058] Because the colorless needle-shaped crystals were separated from Forsythia suspensa, they are a type of hemipentane diterpene, and C12 and C15 are connected by oxygen atoms to form a five-membered lactone ring structural unit, so they are named lactone ring-type Forsythia suspensa diterpene.

[0059] The lactone ring type forsythia diterpenoids 1 H-NMR and 13 C-NMR data are detailed in Table 1-1 and Table 1-2, and related spectra are detailed in Figures 1 to 10 .

[0060] Table 1-1 NMR data of lactone ring type forsythia diterpenoids (solvent is DMSO- d 6 )

[0061]

[0062] Table 1-2 NMR data of lactone ring type forsythia diterpenoids (solvent is DMSO- d 6 )

[0063]

[0064] Example 2

[0065] Another method for preparing the forsythia diterpenoid containing a five-membered lactone ring provided by the present invention specifically comprises the following steps:

[0066] 1. Take 10 kg of Forsythia suspensa fruit and extract it with 100 L, 80 L, and 50 L of methanol respectively, each time for 1 hour. Combine the extracts and then concentrate under reduced pressure to recover the organic solvent to obtain extract I;

[0067] 2. Disperse extract I with an equal volume of deionized water, then extract with an equal volume of chloroform three times, combine the organic solvent portions, and then concentrate under reduced pressure to recover the organic solvent to obtain extract II;

[0068] 3. Dissolve extract II with 3 times the volume of n-butanol, then extract with an equal volume of 4% (v / v) hydrochloric acid 5 times, combine the n-butanol fractions, and then concentrate under reduced pressure to recover the organic solvent to obtain the diterpene fraction;

[0069] 4. The diterpene fraction was separated by silica gel column chromatography, preparative thin layer chromatography and Sephadex LH-20 gel column chromatography in sequence. Specifically:

[0070] (1) The diterpene fraction was loaded onto a normal phase silica gel column and eluted with a cyclohexane-ethyl acetate system in a gradient elution ratio of (100:0) to (50:50). The eluate was detected by TLC. The eluate containing the diterpene was intercepted and concentrated under reduced pressure to obtain a concentrate I.

[0071] (2) The concentrate I was subjected to further chromatography using a preparative thin layer chromatography (TLC) with a mixture of methanol, acetone, and chloroform as the developing solvent. 甲醇 :V 丙酮 :V 氯仿 =1:1:8, intercept the component with Rf=0.6, elute with acetone and concentrate under reduced pressure to obtain concentrate II;

[0072] (3) The concentrated solution II was chromatographed on a Sephadex LH-20 gel column, and the eluent was a mixture of 95% (v / v) ethanol and acetone. 95%乙醇 :V 丙酮 =1:1, collect the diterpene components, concentrate under reduced pressure, crystallize, and dry to obtain 172.46 mg of colorless needle-shaped crystals with a purity of 99.7%.

[0073] Upon testing, the colorless needle-shaped crystals were found to be lactone-ring forsythia diterpenoids, which had the same structure as the lactone-ring forsythia diterpenoids in Example 1.

[0074] 3. Anti-inflammatory and antioxidant activities of lactone-ring forsythia diterpenoids

[0075] Mouse macrophage RAW264.7 cells were prepared using conventional methods, and then the cells were washed twice with RPMI-1640 culture medium, counted, and the cell concentration was adjusted to 2×10 6 cells / mL to obtain a cell suspension.

[0076] The cell suspension was seeded into a 24-well plate, 1 mL per well, and incubated in a 37°C, 5% CO2 incubator for 2 hours. Non-adherent cells were then washed with phosphate-buffered saline (PBS). 1 mL of RPMI-1640 culture medium containing varying concentrations (0.1 μmol / L, 1.0 μmol / L, 10 μmol / L, and 100 μmol / L) of the lactone-type forsythia diterpenoids prepared in Example 1 was then added to each well. Wells without lactone-type forsythia diterpenoids served as solvent controls and were incubated in a 37°C, 5% CO2 incubator for 0.5 hours. Then, 1 μL of 1 mg / mL lipopolysaccharide (LPS) was added and incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the supernatant was collected and the levels of TNF-α, IL-1β, IL-6, and ROS were measured using corresponding ELISA kits. The data were statistically analyzed and expressed as mean ± SD.

[0077] The inhibition rates of different concentrations of lactone-type forsythia diterpenoids on the release of ROS, TNF-α, IL-1β, and IL-6 from mouse macrophages RAW264.7 were calculated according to the following formula:

[0078] Inhibition rate = [(C 刺激组 -C 给药组 ) / (C 刺激组 -C 溶剂对照组 )]×100%

[0079] Among them, C 刺激组 is the amount of TNF-α, IL-1β, IL-6 or ROS contained in the supernatant collected from the wells to which only LPS was added, C 给药组 is the amount of TNF-α, IL-1β, IL-6 or ROS contained in the supernatant collected from the wells added with lactone ring type forsythia diterpenoids and lipopolysaccharide, C 溶剂对照组 The amounts of TNF-α, IL-1β, IL-6, or ROS contained in the supernatant collected from the wells to which no lactone ring-type forsythia diterpenes were added.

[0080] After calculation, the inhibition rates of different concentrations of lactone ring-type forsythia diterpenoids on the release of ROS, TNF-α, IL-1β, and IL-6 from mouse macrophage RAW264.7 are as follows:

[0081]

[0082] Note: Different letters in the same column indicate significant differences, p<0.05.

[0083] As shown in Table 2, the lactone ring-type Forsythia diterpenoids provided by the present invention have a good inhibitory effect on the release of ROS, TNF-α, IL-1β, and IL-6 from mouse macrophages RAW264.7 induced by lipopolysaccharide.

[0084] The above results show that the lactone ring-type Forsythia diterpenoids provided by the present invention have good anti-inflammatory and antioxidant activities. As a natural product, it can be developed into new anti-inflammatory and / or antioxidant Chinese medicines, and has certain application prospects.

[0085] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. Lactone ring type Forsythia diterpenoid, characterized in that, The lactone ring type forsythia diterpenoid is a semi-floral type diterpenoid containing a five-membered lactone ring structural unit, and the molecular formula is C 20 H 30 O3, the structural formula is as follows: 。 2. The method for preparing the lactone ring type Forsythia diterpenoid according to claim 1, characterized in that: The following steps are involved: (1) Take the fruit of Forsythia suspensa, extract it with methanol or 95% ethanol under reflux, recover the organic solvent, and obtain extract I; (2) Dispersing extract I with deionized water, then extracting with chloroform or ethyl acetate, combining the organic solvent portions, and recovering the organic solvent to obtain extract II; (3) Dissolve extract II in n-butanol, then extract with 3%-4% hydrochloric acid, combine the n-butanol fractions, and recover the organic solvent to obtain the diterpene fraction; (4) The diterpene fraction was loaded onto a normal phase silica gel column and gradient eluted using a cyclohexane-ethyl acetate system or a cyclohexane-acetone system as an eluent. The eluate was detected by TLC. The eluate containing the diterpene was intercepted and concentrated under reduced pressure to obtain a concentrate I. (5) The concentrate I was subjected to chromatography separation using a preparative thin layer chromatography to intercept the diterpene component, which was eluted with acetone and concentrated under reduced pressure to obtain the concentrate II; (6) Separate the concentrated solution II using a Sephadex LH-20 gel column or preparative high performance liquid chromatography, collect the diterpene components, concentrate under reduced pressure, crystallize, and dry to obtain the lactone ring-type Forsythia diterpene described in claim 1.

3. The method for preparing the lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (1), methanol is used for reflux extraction. The ratio of Forsythia suspensa fruit to methanol is 1 kg:23 L. Methanol is divided into 10 parts, 8 parts, and 5 parts, and reflux extraction is performed once for each part. Each reflux extraction is performed for 1 hour.

4. The method for preparing the lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (1), 95% ethanol is used for reflux extraction, and the ratio of Forsythia suspensa fruit to 95% ethanol is 1 kg:10 L. Reflux extraction is performed twice, the first time for 2 hours and the second time for 1 hour.

5. The method for preparing lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (4), the eluent used is a cyclohexane-ethyl acetate system, and the eluent is eluted in a gradient manner of (100:0) to (50:50) by volume.

6. The method for preparing lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (4), the eluent used is a cyclohexane-acetone system, and the eluent is eluted in a gradient manner of (95:5) to (60:40) by volume.

7. The method for preparing lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (5), the developing solvent is a mixture of methanol, acetone and chloroform, V 甲醇 :V 丙酮 :V 氯仿 =1:1:8, intercept the component with Rf=0.

6.

8. The method for preparing lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (6), the concentrated solution II is separated using a Sephadex LH-20 gel column, and the eluent is a mixture of 95% ethanol and acetone. 95%乙醇 :V 丙酮 =1:

1.

9. The method for preparing lactone ring type Forsythia diterpenoid according to claim 2, characterized in that: In step (6), the concentrated solution II is separated by preparative high performance liquid chromatography, and the chromatographic conditions are: C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 甲醇 :V 水 =75:25, flow rate 5.0 mL / min, injection volume 2 mL, detection wavelength 255 nm, and components with retention time between 16 min and 18 min were collected.

10. Use of the lactone ring-type Forsythia diterpenoids according to claim 1 in the preparation of anti-inflammatory drugs and / or antioxidant drugs.

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