Limonin compound as well as preparation method and application thereof

Abstract

The invention discloses a series of limonin compounds as well as a preparation method and application thereof. According to the invention, two limonin compounds, namely, methyl kihadanin D and kihadanin D, are extracted and separated from dried root bark of dictamnus dasycarpus, wherein the methyl kihadanin D is a limonin compound with a novel structure. The anti-inflammatory effect of the monomeric compound is evaluated by adopting LPS-induced RAW264.7 cell NO release, and the two compounds are proved to have an obvious inhibiting effect on NO release and have a great application prospect as a potential anti-inflammatory drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to limonene compounds, their preparation methods, and applications. Background Technology

[0002] Limonins are a class of highly oxidized tetracyclic triterpenoids with a 17β-furan ring. Their novel and diverse structures and significant biological activities make them an important source for the discovery of natural drugs. In 1841, Bernay discovered the first limonin compound in citrus seeds and named it limonin. To date, approximately 2700 limonin compounds have been discovered in plants. In terms of their basic carbon skeleton structure, limonins are formed by the loss of four terminal carbons from the 17-position side chain of biogenic precursors such as euphorbia and euphorbia, which have a cyclopentane-polyhydrophenanthrene skeleton (A / B / C / D tetracyclic rings). Therefore, they are also called tetra-lower triterpenoids. The A, B, C, and D rings can undergo various ring-cleaving, epoxidation, rearrangement, and migration processes to form a wide variety of highly bioactive limonins. Currently, the accepted classification standard, based on the characteristics of the cyclopentane-polyhydrophenanthrene skeleton, divides limonins into three categories: intact ring type, open ring type, and rearranged ring type, with the rearranged type accounting for the largest proportion.

[0003] Dictamnus dasycarpus is a plant belonging to the genus Dictamnus in the family Rutaceae. Dictamnus dasycarpus Dried root bark of *Dictamnus dasycarpus*, mainly produced in Northeast and North China. It has a bitter taste and cold nature, and enters the spleen, stomach, and bladder meridians. It has the effects of clearing heat and drying dampness, dispelling wind and detoxifying. In traditional Chinese medicine, *Dictamnus dasycarpus* is an important medicine for treating skin diseases. Formulas such as *Dictamnus dasycarpus* decoction, *Dictamnus dasycarpus* powder, and *Dictamnus dasycarpus* drink are clinically used to treat damp-heat sores, weeping eczema, urticaria, scabies, and rheumatic arthralgia with significant efficacy. Literature reports that the chemical components of *Dictamnus dasycarpus* mainly include limonene and alkaloids. Limonene, due to its structural diversity, exhibits a wide range of biological activities, such as anti-inflammatory, antibacterial, and antitumor effects.

[0004] Lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, is a potent immune activator that can significantly induce macrophage activation. Studies have shown that LPS, as a classic inflammatory inducer, primarily triggers the inflammatory cascade by activating the Toll-like receptor 4 (TLR-4)-dependent nuclear factor kappa B (NF-κB) signaling pathway. The LPS-induced RAW264.7 mouse macrophage model is widely used in inflammatory response research. Nitric oxide (NO) is an important mediator and regulator in the inflammatory process. When macrophages are stimulated by external LPS, it can promote the hypersecretion of inflammatory mediators such as NO, causing severe inflammatory responses. Therefore, NO overexpression plays a key role in the development of inflammatory diseases. Literature reports that limonin compounds derived from Dictamnus dasycarpus root bark, such as dictamlimonol D, fraxinellone, and dasylactone A, have inhibitory activity against LPS-induced NO release from RAW264.7 cells.

[0005] The novel limonene involved in this invention is derived from dandelion (… Dictamnus dasycarpus The compound was extracted and isolated from the dried root bark of *Turcz.*, and its anti-inflammatory effect was evaluated by measuring the release of NO in LPS-induced mouse RAW264.6 macrophage culture medium. The application of compound 1 and compounds 1-2 in the LPS-induced RAW264.7 cell model has not been reported. Summary of the Invention

[0006] The main objective of this invention is to provide a novel method for extracting and isolating limonin and a series of limonin from the dried root bark of Dictamnus dasycarpus, and to demonstrate their activity in inhibiting LPS-induced NO release from RAW264.7 cells.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A method for preparing a limonene compound includes the following steps: Step 1) After pulverizing the dried root bark of Dictamnus dasycarpus, extract it by heating and reflux with ethanol solution, and collect the extract; concentrate the extract under reduced pressure to obtain the total extract, disperse the total extract in an appropriate amount of water, extract with ethyl acetate, concentrate under reduced pressure to obtain the ethyl acetate extract fraction. Step 2) The ethyl acetate extract obtained in Step 1) is subjected to silica gel column chromatography with a mesh size of 200-300, and eluted sequentially with a petroleum ether-dichloromethane-methanol gradient. The eluents are collected and concentrated under reduced pressure, and named Fr.A~Fr.F respectively; the volume ratio of petroleum ether-dichloromethane-methanol is 1:0:0~0:0:1. Step 3) Fr.C was subjected to ODS column chromatography with methanol-water gradient elution. Five eluent fractions were collected, concentrated under reduced pressure, and named Fr.C1 to Fr.C5. The methanol-water volume ratio was 10:90 to 90:10. Step 4) Separate Fr.C5 by MCIGEL CHP20P small-well resin column chromatography, elute with methanol-water gradient, and collect the eluent to obtain 6 fractions, named Fr.C51~Fr.C56; the methanol-water volume ratio is 0:1~90:10; Step 5) Fr.C54 was subjected to Sephadex LH-20 gel column chromatography with methanol as the eluent. The eluent was concentrated under reduced pressure and then prepared by preparative HPLC. The preparative HPLC conditions were as follows: detection wavelength: 225 nm, eluent: methanol-water volume ratio 55:45, flow rate: 6 mL / min. After preparation, the eluent was concentrated and then prepared by preparative HPLC. Limonene compound 1 was obtained by elution with acetonitrile-water at a volume ratio of 50:50 at 6 mL / min, named methylkihadanin D. Step 6) Fr.D was subjected to ODS column chromatography with methanol-water gradient elution. Five eluent fractions were collected, concentrated under reduced pressure, and named Fr.D1 to Fr.D5 respectively. The methanol-water volume ratio was 10:90 to 90:10. Step 7) Fr.D4 was subjected to chromatographic chromatography on a 200-300 mesh silica gel column, and eluted sequentially with a chloroform-methanol gradient. Nine eluent fractions were collected, concentrated under reduced pressure, and named Fr.D41 to Fr.D49 respectively; the volume ratio of chloroform to methanol was 98:2 to 0:1. Step 8) Fr.D41 was prepared by gradient preparative HPLC with a detection wavelength of 225 nm and methanol-water as the eluent. The gradient elution was carried out at a flow rate of 5 mL / min, and the methanol-water volume ratio was 40:60 for 0-5 min and 80:20 for 65 min. After separation, preparative HPLC was used to prepare the limonene compound 2, which was named kihadanin D.

[0008] In step 1, the ethanol solution has a volume fraction of 70%, and the amount used is 3 times the mass of the dried root bark of Dictamnus dasycarpus. The extraction is performed 5 times, each time for 1.5 hours.

[0009] In step 2, the mass ratio of ethyl acetate extract to silica gel is 1:7, and the volume ratio of petroleum ether-dichloromethane-methanol is 1:0:0, 10:1:0, 5:1:0, 2:1:0, 1:1:0, 0:1:0, 0:1000:1, 0:100:1, 0:50:1, 0:30:1, 0:15:1, 0:8:1, 0:4:1, 0:1:1, 0:0:1. The column volume is denoted as BV, and 10 BV is eluted for each ratio.

[0010] In step 3, the methanol-water volume ratio is 10:90, 30:70, 50:50, 70:30, and 90:10. The column volume is denoted as BV, and 6 BV is eluted for each ratio.

[0011] In step 4, the methanol-water volume ratio is 0:1, 10:90, 30:70, 50:50, 70:30, 90:10, and the column volume is denoted as BV. Each ratio elutes 6 BV.

[0012] In step 6, the methanol-water volume ratio is 10:90, 30:70, 50:50, 70:30, and 90:10. The column volume is denoted as BV, and 6 BV is eluted for each ratio.

[0013] In step 7, the volume ratio of chloroform to methanol is 98:2, 96:4, 94:6, 0:1, and the column volume is denoted as BV. Each ratio elutes 5 BV.

[0014] A limonene compound, named methyl kihadanin D, has the following structural formula: ; In formula (I), ring B is a six-membered ring, ring C is a six-membered ring, ring D is a six-membered lactone ring, ring E is a five-membered lactone ring, and F is an epoxy three-membered ring.

[0015] A pharmaceutical composition comprising the limonene compound 1 or 2 and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient.

[0016] The pharmaceutical composition is used in the preparation of anti-inflammatory drugs.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention is the first to isolate and purify a new compound limonin 1 from Dictamnus dasycarpus root bark.

[0018] 2) This invention uses LPS-induced NO release in RAW264.7 cells to evaluate the anti-inflammatory effects of monomeric compounds, confirming that compounds 1 and 2 have a significant inhibitory effect on NO release, and they have great application prospects as potential anti-inflammatory drugs. Attached Figure Description

[0019] Figure 1 The 1H NMR spectrum of compound 1 in Example 1 of this invention; Figure 2 The carbon NMR spectrum of compound 1 in Example 1 of this invention; Figure 3 This is the high-resolution mass spectrum of compound 1 in Example 1 of the present invention; Figure 4 The 1H NMR spectrum of compound 2 in Example 1 of this invention; Figure 5 The carbon NMR spectrum of compound 2 in Example 1 of this invention; Figure 6 This is the high-resolution mass spectrum of compound 2 in Example 1 of the present invention; Figure 7 A is the inhibition curve of compound 1 in Example 2 of the present invention on LPS-induced NO release in RAW264.7 cells; Figure 7 B is a graph showing the inhibition of NO release from LPS-induced RAW264.7 cells by compound 2 in Example 2 of this invention. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. These embodiments are merely exemplary and do not limit the scope of protection of the present invention.

[0021] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0022] Example 1: Method for separating and purifying limonene compounds from Dictamnus dasycarpus root bark 1) White Dictamnus ( Dictamnus dasycarpus 6.0 kg of dried root bark of *Turcz.* was pulverized and extracted five times by reflux with three times the volume of 70% ethanol solution, each time for 1.5 h. The extracts were combined, and the solvent was recovered under reduced pressure at 35 °C. The extract was then concentrated to obtain the total extract. The total extract was dispersed in 2 L of water and extracted three times with an equal volume of ethyl acetate. The extract was then concentrated under reduced pressure to obtain the ethyl acetate extract fraction.

[0023] 2) The ethyl acetate extract (175 g) was mixed with 1.5 times the amount of silica gel (100-200 mesh), with a mass ratio of ethyl acetate extract to silica gel of (1:7). Separation was performed by silica gel column chromatography (200-300 mesh), and the column volume was recorded as BV. Elution was performed sequentially using a petroleum ether-dichloromethane-methanol gradient (1:0:0, 10:1:0, 5:1:0, 2:1:0, 1:1:0, 0:1:0, 0:1000:1, 0:100:1, 0:50:1, 0:30:1, 0:15:1, 0:8:1, 0:4:1, 0:1:1, 0:0:1, V / V), with 10 BV eluted for each ratio. The elution fractions were developed using silica gel thin-layer chromatography GF254 with petroleum ether:dichloromethane = 1:3, dichloromethane:methanol = 90:1, 30:1, 15:1 and 8:1, respectively. Similar components were combined and six eluents were collected. The resulting eluents were concentrated under reduced pressure and named (Fr.AF).

[0024] 3) The fraction Fr.C (22 g) was eluted by an ODS column with a methanol-water gradient (10:90, 30:70, 50:50, 70:30, 90:10, V / V). The column volume was recorded as BV. Each fraction was eluted by 6 BV. The eluents obtained were collected into 5 fractions, concentrated under reduced pressure, and named as (Fr.C1, Fr.C2, Fr.C3, Fr.C4, Fr.C5).

[0025] 4) Fr.C5 (11 g) was eluted with a methanol-water gradient (0:1, 10:90, 30:70, 50:50, 70:30, 90:10, V / V) on an MCIGEL CHP20P small-pore resin column, with 6 BV of eluent eluted in each ratio. The eluents were collected and concentrated under reduced pressure and named as (Fr.C51, Fr.C52, Fr.C53, Fr.C54, Fr.C55, Fr.C56).

[0026] 5) Fr.C54 (4 g) was eluted with methanol on a Sephadex LH-20 column for 3 BV. The eluent was concentrated under reduced pressure and then prepared by preparative HPLC. The detection wavelength was 225 nm, and the elution was methanol-water (55:45, V / V, 6 mL / min). After preparation, the eluent was concentrated and then prepared by preparative HPLC. The preparative HPLC conditions were as follows: detection wavelength: 225 nm, acetonitrile-water (50:50, V / V, 6 mL / min) to obtain compound 1.

[0027] 6) Fr.D (26 g) was eluted by an ODS column with a methanol-water gradient (10:90, 30:70, 50:50, 70:30, 90:10, V / V), with 6 BV eluted in each ratio. Five eluent fractions were collected, concentrated under reduced pressure, and named (Fr.D1, Fr.D2, Fr.D3, Fr.D4, Fr.D5).

[0028] 7) Fr.D4 (6 g) was subjected to silica gel column chromatography (200-300 mesh), with column volume denoted as BV. A gradient elution of chloroform-methanol (98:2, 96:4, 94:6, 0:1, V / V) was used, with 5 BV eluted at each ratio. The eluent fractions were developed using silica gel thin-layer chromatography on a GF254 silica gel column with a chloroform:methanol ratio of 95:5. Similar components were combined, and nine eluent fractions were collected. These fractions were concentrated under reduced pressure and named (Fr.D41, Fr.D42, Fr.D43, Fr.D44, Fr.D45, Fr.D46, Fr.D47, Fr.D48, Fr.D49).

[0029] 8) Fr.D41 (495 mg) was prepared by gradient preparative HPLC with a detection wavelength of 225 nm and elution by a methanol-water gradient (0-5 min 40:60; 65 min 80:20, V / V, 5 mL / min); then compound 2 was prepared by preparative HPLC with a detection wavelength of 225 nm and elution by methanol-water (45:55, V / V, 5 mL / min).

[0030] The proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 1 are as follows: Figure 1 , 2 As shown in Figures 1 and 3; the proton NMR spectrum, carbon NMR spectrum, and high-resolution mass spectra of compound 2 are shown in Figures 3 and 4 respectively. Figure 4 , 5 As shown in Figures 6 and 7. The structures of compounds 1 and 2 were determined using physicochemical constants and modern spectroscopic techniques. Compound 1 is a novel compound not previously reported in the literature. Its structural formula is shown below:

[0031] Compound 1: methyl kihadanin D White amorphous powder (methanol); α ] 25 D +10.6 ( c 0.1, MeOH); UV (MeOH) λ max (log e) 210(3.57) nm; ECD ( c 1.8 × 10 -4 M, MeOH) λ max (Δ ε ) 208 (+2.13), 234 (-6.12), 261 (-0.29), 291 (-4.51) nm; HRESIMS m / z 536.2489 [M + NH4] + (calcd for C 27 H 38 O 10 N, 536.2490), the molecular formula of compound 1 was determined to be C 27 H 34 O 10 ; 1 H (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3), data are shown in Table 1; the structural formula is shown in Equation I, which contains two double bonds, and C-1 and C-2 are... Z The configuration is as follows: C-3 is a methyl ester, C-4 is a hydroxyl group, C-7 is a ketone carbonyl group, C-16 and C-21 are ester groups, and C-23 is a methoxy group.

[0032] Compound 2: kihadanin D Colorless oily substance (methanol); α ] 25 D + 30.4 ( c 0.1, MeOH); UV (MeOH) λ max (log e ) 210(3.71) nm; ECD ( c 1.6 × 10 -4 M, MeOH) λ max (Δ ε ) 220 (+7.25), 252 (-3.38), 262 (-2.73), 292 (-3.74) nm; HRESIMS m / z 501.2118 [M + H] + (calcd for C 27 H 33 O9,501.2119), the molecular formula of compound 2 was determined to be C 27 H 32 O9; 1H (600 MHz, CDCl3) and 13 C-NMR (150 MHz, CDCl3), data are shown in Table 1; the structural formula is shown in Formula II, which contains two double bonds, with ester groups at C-3, 16, and 21, a ketone carbonyl group at C-7, and a methoxy group at C-23.

[0033] Table 1. 1H and 1C NMR spectra of compounds 1 and 2 ( δ in ppm, J (in Hz, CDCl3).

[0034] Example 2: Inhibitory activity of limonene compounds 1 and 2 against LPS-induced NO in RAW254.7 cells. Mouse RAW264.7 mononuclear macrophages were cultured in RAW264.7 cell culture medium (Wuhan Pronosei Life Sciences Co., Ltd.) at 37 ℃ in a cell culture incubator with 5% CO2. RAW264.7 cells in the logarithmic growth phase were seeded into 96-well plates (4 × 10⁶ cells / well). 4 The plates were incubated at 37°C and 5% CO2. Once approximately 70% confluence was reached, the test drugs (compounds 1 and 2) were dissolved in DMSO and added to the 96-well plates. After 1 hour, LPS was dissolved in PBS and added to the 96-well plates to achieve a final LPS concentration of 250 nM. The final concentrations of the test drugs were 20, 10, 5, 2.5, and 1.25 nM, respectively. μ M or 5, 2.5, 1.25, 0.625, 0.3125 μ M, after incubating at 37 °C for 24 hours, 50 μL of the sample was taken from each well. μ Add the L supernatant to a new 96-well plate, then add equal volumes of GriessReagent I and II reagents sequentially. Place the plate in a fluorescence microplate reader and detect the fluorescence intensity at a wavelength of 540 nm.

[0035] Experimental results are as follows Figure 7 As shown, the results indicate that limonene compounds 1 and 2 described in this invention inhibit LPS-induced NO release from RAW264.7 cells, with IC50 values ​​of 2.69±1.71 and 1.25±0.14, respectively. μ M has great potential as an anti-inflammatory drug.

[0036] The limonene compounds 1 or 2 described in this invention, along with their pharmaceutically acceptable salts or pharmaceutically acceptable carriers or excipients, can be formulated into dosage forms suitable for oral or injectable applications, such as tablets, capsules, injections, powders, etc. Each dosage form can be prepared using conventional methods.

[0037] The above embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A method for preparing a limonene compound, characterized in that, Includes the following steps: Step 1) After pulverizing the dried root bark of Dictamnus dasycarpus, extract it by heating and reflux with ethanol solution, and collect the extract; concentrate the extract under reduced pressure to obtain the total extract, disperse the total extract in an appropriate amount of water, extract with ethyl acetate, concentrate under reduced pressure to obtain the ethyl acetate extract fraction. Step 2) The ethyl acetate extract obtained in Step 1) is subjected to silica gel column chromatography with a mesh size of 200-300, and eluted sequentially with a petroleum ether-dichloromethane-methanol gradient. The eluents are collected and concentrated under reduced pressure, and named Fr.A~Fr.F respectively; the volume ratio of petroleum ether-dichloromethane-methanol is 1:0:0~0:0:

1. Step 3) Fr.C was subjected to ODS column chromatography with methanol-water gradient elution. Five eluent fractions were collected, concentrated under reduced pressure, and named Fr.C1 to Fr.C5. The methanol-water volume ratio was 10:90 to 90:

10. Step 4) Separate Fr.C5 by MCIGEL CHP20P small-well resin column chromatography, elute with methanol-water gradient, and collect the eluent to obtain 6 fractions, named Fr.C51~Fr.C56; the methanol-water volume ratio is 0:1~90:10; Step 5) Fr.C54 was subjected to Sephadex LH-20 gel column chromatography with methanol as the eluent. The eluent was concentrated under reduced pressure and then prepared by preparative HPLC. The preparative HPLC conditions were as follows: detection wavelength 225 nm, eluent: methanol-water volume ratio 55:45, flow rate: 6 mL / min. After preparation, the solution was concentrated and then prepared by preparative HPLC. Limonene compound 1 was obtained by elution with acetonitrile-water at a volume ratio of 50:50 at 6 mL / min, named methyl kihadanin D. Step 6) Fr.D was subjected to ODS column chromatography with methanol-water gradient elution. Five eluent fractions were collected, concentrated under reduced pressure, and named Fr.D1 to Fr.D5 respectively. The methanol-water volume ratio was 10:90 to 90:

10. Step 7) Fr.D4 was subjected to chromatographic chromatography on a 200-300 mesh silica gel column, and eluted sequentially with a chloroform-methanol gradient. Nine eluent fractions were collected, concentrated under reduced pressure, and named Fr.D41 to Fr.D49 respectively; the volume ratio of chloroform to methanol was 98:2 to 0:

1. Step 8) Fr.D41 was prepared by gradient preparative HPLC with a detection wavelength of 225 nm and methanol-water as the eluent. The gradient elution was carried out at a flow rate of 5 mL / min, and the methanol-water volume ratio was 40:60 for 0-5 min and 80:20 for 65 min. After separation, preparative HPLC was used with a detection wavelength of 225 nm and methanol-water with a volume ratio of 45:55 at a flow rate of 5 mL / min to obtain limonin compound 2, named kihadanin D.

2. The method for preparing limonene compounds according to claim 1, characterized in that, In step 1, the ethanol solution has a volume fraction of 70%, and the amount used is 3 times the mass of the dried root bark of Dictamnus dasycarpus. The extraction is performed 5 times, each time for 1.5 hours.

3. The method for preparing limonene compounds according to claim 1, characterized in that, In step 2, the mass ratio of ethyl acetate extract to silica gel is 1:7, and the volume ratio of petroleum ether-dichloromethane-methanol is 1:0:0, 10:1:0, 5:1:0, 2:1:0, 1:1:0, 0:1:0, 0:1000:1, 0:100:1, 0:50:1, 0:30:1, 0:15:1, 0:8:1, 0:4:1, 0:1:1, 0:0:

1. The column volume is denoted as BV, and 10 BV is eluted for each ratio.

4. The method for preparing limonin compounds according to claim 1, characterized in that, In step 3, the methanol-water volume ratio is 10:90, 30:70, 50:50, 70:30, and 90:

10. The column volume is denoted as BV, and 6 BV is eluted for each ratio.

5. The method for preparing limonene compounds according to claim 1, characterized in that, In step 4, the methanol-water volume ratio is 0:1, 10:90, 30:70, 50:50, 70:30, 90:10, and the column volume is denoted as BV. Each ratio elutes 6 BV.

6. The method for preparing limonin compounds according to claim 1, characterized in that, In step 6, the methanol-water volume ratio is 10:90, 30:70, 50:50, 70:30, and 90:

10. The column volume is denoted as BV, and 6 BV is eluted for each ratio.

7. The method for preparing limonin compounds according to claim 1, characterized in that, In step 7, the volume ratio of chloroform to methanol is 98:2, 96:4, 94:6, 0:1, and the column volume is denoted as BV. Each ratio elutes 5 BV.

8. A limonene compound, characterized in that, The compound was named methyl kihadanin D, and its structural formula is as follows: In formula (I), ring B is a six-membered ring, ring C is a six-membered ring, ring D is a six-membered lactone ring, ring E is a five-membered lactone ring, and F is an epoxy three-membered ring.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises limonene compound 1 or 2 as described in claim 1, and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient.

10. Use of the pharmaceutical composition of claim 9 in the preparation of an anti-inflammatory active drug.

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