Sesquiterpenoids isolated from the roots of Piper melongena and their anti-inflammatory applications

Abstract

The present invention discloses sesquiterpenoid compounds isolated from the roots of Piper melongena and their anti-inflammatory applications, belonging to the field of medical technology. The sesquiterpenoid compounds are selected from any one of Compound 1, Compound 2, Compound 3, and Compound 4 shown in the following formula. The method for isolating the sesquiterpenoid compounds is as follows: extracting the dried roots of Piper melongena with ethanol, combining the extracts and concentrating them to obtain an extract, extracting the extract with petroleum ether, and subjecting the extracted components to silica gel column chromatography, normal phase column chromatography separation, and HPLC purification, or silica gel column chromatography, normal phase column chromatography separation, reverse phase column chromatography separation, and HPLC purification to obtain compounds 1-4. The sesquiterpenoid compounds provided in the present invention are all optically pure compounds with determined stereoconfiguration, especially compounds 1 and 4, which are potential anti-inflammatory drug leads and have prospects for further development of anti-inflammatory drugs. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more particularly to sesquiterpenoid compounds separated from the root of Piper melongena and anti-inflammatory applications thereof. Background Art

[0002] Zanthoxylum bungeanum has a long history of being used as medicine. The "Newly Revised Materia Medica" records that Zanthoxylum bungeanum is "spicy, hot, and non-toxic," and "treats heart and stomach pain, coldness in the middle, and breaks stagnation." It has the effects of dispelling wind, unblocking meridians, regulating qi and activating blood circulation, and is often used by the people to treat rheumatoid arthritis, heart and stomach pain, trauma, etc. The extract of the root of Zanthoxylum bungeanum contains many active ingredients, such as santhemin, santheminic acid, alkaloids, etc., which have multiple active effects such as anti-tumor and analgesia. For example, the Chinese patent document with publication number CN105481770A discloses a preparation method for extracting alkaloids from the root of Zanthoxylum bungeanum, which includes the following steps: raw material treatment; acidic alcohol aqueous solution percolation extraction; preparation of a concentrated solution of the total alkaloid extract of the root of Zanthoxylum bungeanum; preparation of an eluent; and preparation and purification of compounds. The invention uses a low-concentration alcohol-acidic alcohol aqueous solution as the extraction solvent, and repeatedly recycles and utilizes the extraction, which can significantly improve the extraction efficiency. The extracted alkaloids are of great significance for the development of new anti-tumor drugs.

[0003] Terpenoids can be divided into monoterpenes, sesquiterpenes, diterpenes, ester terpenes, triterpenes and polyterpenes according to their chemical structure. As one of the most numerous and diverse natural products, terpenoids have received extensive attention in recent years due to their important biological activities such as anticancer, antioxidant, antiviral and anti-inflammatory. Among them, sesquiterpenes are the most widely distributed class of terpenoids, and their pharmacological activities are very rich, with good application prospects in the pharmaceutical field. The Chinese patent document with publication number CN116621854A discloses terpenoids in Ailanthus altissima leaves and a method for preparing the same. The invention extracts and separates 4 terpenoids from Ailanthus altissima leaves, a plant of the genus Ailanthus in the family Simaroubaceae, and the corresponding terpenoids have good anti-liver cancer activity.

[0004] The inflammatory response is a complex biological response, generally associated with inflammatory factors, inflammatory mediators, inflammasomes, and related targets. Among them, inflammatory mediators play a key role in the inflammatory pathway. However, overexpression of inflammatory mediators can lead to immune system dysfunction and induce inflammatory diseases. Furthermore, recent research indicates that acute inflammation caused by trauma and infection can transform into chronic inflammation. Long-term inflammatory responses can damage the liver, kidneys, heart, intestines, brain, and other organs. Common diseases associated with inflammation include cardiovascular disease, arthritis, Alzheimer's disease, diabetes, gastroenteritis, and autoimmune diseases. In short, the inflammatory response requires precise and targeted regulation. Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used antipyretic and analgesic drugs. Their anti-inflammatory mechanism primarily involves inhibiting cyclooxygenase to reduce the production of inflammatory mediators. However, these drugs have significant side effects, and emerging biologics are very expensive. Therefore, there is a need to develop anti-inflammatory drugs with lower costs and fewer side effects. Summary of the Invention

[0005] The invention provides sesquiterpenoid compounds separated from the roots of Piper melongena. The separation method is simple, reliable, efficient and operable. The separated sesquiterpenoid compounds have anti-inflammatory activity.

[0006] The specific technical solutions adopted are as follows:

[0007] The sesquiterpenoid compound isolated from the root of Piper melongena is selected from any one of Compound 1, Compound 2, Compound 3, and Compound 4 shown in the following formula:

[0008]

[0009] Specifically, the blue pepper is the blue pepper plant of the genus Blue Pepper in the Lauraceae family (Lindera glauca (Siebold & Zucc.) Blume)).

[0010] The present invention also provides a method for separating the sesquiterpenoid compounds, which specifically comprises the following steps:

[0011] (1) The dried roots of Piper melongena were extracted with ethanol, the combined extracts were concentrated to obtain an extract, the extract was extracted with petroleum ether, and the extracted components were subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system for gradient elution to obtain 7 fractions Fr.1-Fr.7;

[0012] (2) Fraction Fr.4 was purified by a normal phase silica gel column using a petroleum ether-ethyl acetate gradient elution system. The eluate was analyzed by thin layer chromatography and the same components were combined to obtain 21 fractions Fr.4.1-Fr.4.21.

[0013] (3) Fraction Fr.4.8 was purified by semi-preparative HPLC to obtain compound 4;

[0014] (4) Fraction Fr.4.12 was purified by reverse phase silica gel column with a methanol-water gradient elution system to obtain 45 fractions Fr.4.12.1-Fr.4.12.45;

[0015] (5) Fraction Fr.4.12.7 was purified by semi-preparative HPLC to obtain compound 2;

[0016] (6) Fraction Fr.4.12.8 was purified twice by semi-preparative HPLC to obtain compound 3;

[0017] (7) Fraction Fr.4.12.9 was purified by semi-preparative HPLC to obtain compound 1.

[0018] Preferably, in step (1), the ethanol used is industrial ethanol with a concentration of 80-95%, the ethanol extraction method is hot immersion method, the extraction temperature is 50-60° C., and the extraction is performed 6-7 times, each time for 20-24 hours.

[0019] More preferably, the ethanol used is industrial ethanol with a concentration of 95%, the extraction temperature is 55° C., and the extraction is performed 7 times, each time for 24 hours.

[0020] Preferably, in step (1), the volume ratio of petroleum ether and ethyl acetate during elution changes to 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1; in step (2), the volume ratio of petroleum ether and ethyl acetate during elution changes to 12:1, 10:1, 8:1, 5:1, 3:1, 0:1; in step (4), the volume ratio of methanol and water during elution changes to 20:80, 40:60, 60:40, 80:20, 100:0.

[0021] Preferably, in step (3), the conditions for the semi-preparative HPLC method are: a Welch Ultimate XB-C18 liquid chromatography column is selected, isocratic elution separation is performed, the mobile phase is acetonitrile / water with a volume ratio of 70:30, and the flow rate is 2.0 mL / min.

[0022] Preferably, in step (5), the conditions for the semi-preparative HPLC method are: a Welch Ultimate XB-C18 liquid chromatography column is selected, isocratic elution separation is performed, the mobile phase is methanol / water with a volume ratio of 24:76, and the flow rate is 2.0 mL / min.

[0023] Preferably, in step (6), the conditions for the first semi-preparative HPLC method are: selecting a Welch Ultimate XB-C18 liquid chromatography column, isocratic elution separation, the mobile phase is acetonitrile / water with a volume ratio of 35:65, and the flow rate is 2.0 mL / min; the conditions for the second semi-preparative HPLC method are: selecting a Welch Ultimate XB-C18 liquid chromatography column, isocratic elution separation, the mobile phase is methanol / water with a volume ratio of 40:60, and the flow rate is 2.0 mL / min.

[0024] Preferably, in step (7), the conditions for the semi-preparative HPLC method are: a Welch Ultimate XB-C18 liquid chromatography column is selected, isocratic elution separation is performed, the mobile phase is acetonitrile / water with a volume ratio of 36:64, and the flow rate is 2.0 mL / min.

[0025] The invention also discloses the application of the sesquiterpenoid compound in the preparation of anti-inflammatory drugs.

[0026] The invention also discloses an anti-inflammatory drug, which comprises the sesquiterpenoid compound.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The sesquiterpenoid compounds provided in the present invention are all optically pure compounds with a defined stereoconfiguration, which have not been reported in the prior art. They have anti-inflammatory activity, and compounds 1 and 4, in particular, are potential anti-inflammatory drug leads, with prospects for further development of anti-inflammatory drugs.

[0029] (2) The separation method of the present invention is simple, reliable, efficient and highly operable. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the HRESIMS spectrum of compound 1.

[0031] Figure 2 is the HSQC spectrum of compound 1.

[0032] Figure 3 For compound 1 1 H– 1 H COSY spectrum.

[0033] Figure 4 is the HMBC spectrum of compound 1.

[0034] Figure 5 is the NOESY spectrum of compound 1.

[0035] Figure 6 is the HRESIMS spectrum of compound 4.

[0036] Figure 7 is the HSQC spectrum of compound 4.

[0037] Figure 8 For compound 4 1 H– 1 H COSY spectrum.

[0038] Figure 9 is the HMBC spectrum of compound 4.

[0039] Figure 10 is the NOESY spectrum of compound 4.

[0040] Figure 11 is the X-ray single crystal diffraction pattern of compound 1-4.

[0041] Figure 12 This is a statistical diagram of the inhibitory effects of compounds 1 and 4 on NO in the LPS-induced RAW 264.7 cell inflammation model, where A is compound 1 and B is compound 4.

[0042] Figure 13 The graph shows the inhibitory effects of compounds 1 and 4 on iNOS and COX-2 protein expression in the LPS-induced RAW 264.7 cell inflammation model, wherein A and B are compounds 1, and C and D are compounds 4.

[0043] Figure 14 This is a statistical diagram showing the down-regulation effects of compounds 1 and 4 on inflammatory factors in the LPS-induced RAW 264.7 cell inflammation model, where A is compound 1 and B is compound 4.

[0044] Note: In the figure, ** indicates P < 0.01, *** indicates P < 0.001, compared with the control group; # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, compared with the LPS group; & indicates P < 0.05, compared with the positive drug Dex group. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the following examples and accompanying drawings. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] Specifically, the blue pepper in the embodiment is the blue pepper plant of the genus Blue Pepper in the Lauraceae family (Lindera glauca (Siebold & Zucc.) Blume)).

[0047] Example 1

[0048] (1) Preparation of Compound 1-4

[0049] Dried roots of Piper melongena (45 kg) were hot-macerated and extracted with 95% industrial ethanol at 55°C for 6 extractions, each for 24 hours. The combined extracts were concentrated to obtain an extract (3.4 kg). The extract was extracted with 25 L of petroleum ether. The fractions (1.4 kg) obtained by petroleum ether extraction were subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system (volume ratios of petroleum ether to ethyl acetate varied from 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1). A total of seven fractions, Fr.1 to Fr.7, were collected.

[0050] Fraction Fr.4 was passed through a normal phase silica gel column using a petroleum ether-ethyl acetate system (volume ratios of petroleum ether and ethyl acetate varied from 12:1, 10:1, 8:1, 5:1, 3:1, and 0:1) for gradient elution. The eluate was analyzed by thin-layer chromatography, and the same components were combined to obtain 21 fractions Fr.4.1-Fr.4.21.

[0051] Fraction Fr.4.8 was further separated and purified by semi-preparative HPLC to obtain compound 4 (7.9 mg, t R =20.8min), the semi-preparative HPLC conditions were as follows: a Welch Ultimate XB-C18 liquid chromatography column was used for isocratic elution, the mobile phase was acetonitrile / water with a volume ratio of 70:30, and the flow rate was 2.0mL / min;

[0052] Fraction Fr.4.12 was purified by reverse phase C 18 Silica gel chromatography column, gradient elution with a methanol-water system (volume ratio of methanol to water varied from 20:80, 40:60, 60:40, 80:20, 100:0) to obtain 45 fractions Fr.4.12.1-Fr.4.12.45;

[0053] Fraction Fr.4.12.7 was further separated and purified by semi-preparative HPLC to obtain compound 2 (31.8 mg, t R =29.6min), the semi-preparative HPLC conditions were as follows: a Welch Ultimate XB-C18 liquid chromatography column was used for isocratic elution, the mobile phase was methanol / water with a volume ratio of 24:76, and the flow rate was 2.0mL / min;

[0054] Fraction Fr.4.12.8 was further separated by semi-preparative HPLC to obtain liquid chromatography peaks Fr.4.12.8.1-4.12.8.18. The conditions of the semi-preparative HPLC method were as follows: a Welch Ultimate XB-C18 liquid chromatography column was used for isocratic elution separation, the mobile phase was acetonitrile / water with a volume ratio of 35:65, and the flow rate was 2.0 mL / min; the liquid chromatography peak Fr.4.12.8.8 was further separated by semi-preparative HPLC to obtain compound 3 (3.0 mg, t R =49.8min), and the semi-preparative HPLC conditions were as follows: a Welch Ultimate XB-C18 liquid chromatography column was selected, isocratic elution was used, the mobile phase was methanol / water with a volume ratio of 40:60, and the flow rate was 2.0mL / min.

[0055] Fraction Fr.4.12.9 was further separated and purified by semi-preparative HPLC to obtain compound 1 (7.0 mg, t R =66.1min), and the conditions for the semi-preparative HPLC method were as follows: a Welch Ultimate XB-C18 liquid chromatography column was selected, isocratic elution was used, the mobile phase was acetonitrile / water with a volume ratio of 36:64, and the flow rate was 2.0 mL / min.

[0056] (2) Structural identification of compounds 1-4

[0057] The structures of compounds 1-4 were identified using a variety of spectral techniques and ECD calculations. The results are shown below:

[0058] Compound 1: colorless cubic crystals; mp 189-190°C; [α]25D-56.4 (c 0.28, MeOH); UV (MeOH)λ max (logε)=207(4.16)nm; IR(KBr)ν max =3448,2925,2360,1767,1715,1636,1384,1148cm -1 ;ECD(MeOH)λ max (Δε)=201(-12.36),270(+16.44)nm; 1 H NMR and 13 C NMR data are shown in Table 1; HRESIMS [M+H] + m / z 291.1603 (calcd for C 17 H 23 O4 + ,291.1591), the molecular formula of the compound is determined to be C 17 H 22 O4.

[0059] By analyzing the 1 H NMR and 13 C NMR spectrum, 1 H- 1 The structure and absolute configuration of compound 1 were determined by H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, calculated ECD and X-ray single crystal diffraction pattern (CCDC 2302533). The absolute configuration of compound 1 is 1S, 3S, 4R, 5R, 7R.

[0060] Table 1 Compound 1 1 H(600MHz) and 13 C (150 MHz) NMR data

[0061]

[0062] Compound 2: colorless cubic crystals; mp 167-168°C; [α]25D+60.3 (c 0.10, MeOH); UV (MeOH)λ max (logε)=201(4.17)nm; IR(KBr)ν max =3423,2923,2871,1707,1656,1647,1636,1469,1422,1384,1009cm -1 ;ECD(MeOH)λ max (Δε)=200(+12.15),221(-16.46)nm; 1 H NMR and 13 C NMR data are shown in Table 2; HRESIMS [M+H] + m / z 225.1485 (calcd for C 13 H 21 O3 + ,225.1485), the molecular formula of the compound was determined to be C 13 H 20 O3.

[0063] By analyzing the 1 H NMR and 13 C NMR spectrum, 1 H- 1 The structure and absolute configuration of compound 2 were determined by H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, calculated ECD and X-ray single crystal diffraction pattern (CCDC 2302534). The absolute configuration of compound 2 is 1R, 4S, 5S, 7R, 10S.

[0064] Table 2 Compound 2 1 H(600MHz) and 13 C (150 MHz) NMR data

[0065]

[0066] Compound 3: colorless cubic crystals; mp 170-171°C; [α]25D-10.0 (c 0.12, MeOH); UV (MeOH)λ max (logε)=200(3.82),224(3.53),266(3.38)nm; IR(KBr)ν max =3422,2960,2920,1700,1453,1374,1284,1029,999cm -1 ;ECD(MeOH)λ max (Δε)=198(-2.16),228(+2.84),265(-0.44)nm; 1 H NMR and 13 CNMR data are shown in Table 3; HRESIMS [M+Na] + m / z 261.1458 (calcd for C 14 H 22 O3Na + ,261.1461), the molecular formula of the compound is determined to be C 14 H 22 O3.

[0067] By analyzing the 1 H NMR and 13 C NMR spectrum, 1 H- 1 The structure and absolute configuration of compound 3 were determined by H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, calculated ECD and X-ray single crystal diffraction pattern (CCDC 2302535). The absolute configuration of compound 3 is 1S, 4S, 5R, 7S, 10S.

[0068] Table 3 Compound 3 1 H(600MHz) and 13 C (150 MHz) NMR data

[0069]

[0070] Compound 4: colorless needle-like crystals; mp 184-185°C; [α]25D-18.0 (c 0.31, MeOH); UV (MeOH)λ max(logε)=211(4.35)nm; IR(KBr)ν max =3494,2937,2876,1698,1636,1305,1223cm -1 ;ECD(MeOH)λ max (Δε)=205(-2.53),265(+3.74)nm; 1 H NMR and 13 C NMR data are shown in the table below; HRESIMS [M+Na] + m / z 273.1461 (calcd for C 15 H 22 O3Na + ,273.1461), the molecular formula of the compound is determined to be C 15 H 22 O3.

[0071] By analyzing the 1 H NMR and 13 C NMR spectrum, 1 H- 1 The structure and absolute configuration of compound 4 were determined by H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, calculated ECD and X-ray single crystal diffraction pattern (CCDC 2302536). The absolute configuration of compound 4 is 1R, 4S, 5S, 7R, 10S.

[0072] Table 4 Compound 4 1 H(600MHz) and 13 C (150 MHz) NMR data

[0073]

[0074] Specifically, the HRESIMS spectrum, HSQC spectrum, 1 H– 1 H COSY spectrum, HMBC spectrum and NOESY spectrum are shown in Figure 2. Figure 1-5 As shown, the HRESIMS spectrum, HSQC spectrum, 1 H– 1 H COSY spectrum, HMBC spectrum and NOESY spectrum are shown in Figure 2. Figure 6-10 As shown, the X-ray single crystal diffraction pattern of compound 1-4 is as follows Figure 11 shown.

[0075] Example 2

[0076] An in vitro cell inflammation model was established using LPS-induced RAW 264.7 cells to test the anti-inflammatory activity of compounds 1-4 synthesized in Example 1:

[0077] RAW 264.7 cells (1×10 5 ) were inoculated into 96-well culture plates and placed in an incubator at 37°C. After the cells adhered overnight, the drug-treated group was added with different concentrations (5μM, 10μM, 20μM, 40μM) of the test sample (compound 1-4) and incubated with the culture medium in a constant temperature incubator for 2 hours. The control group (using 20μM dexamethasone as a positive control drug) and the model group were added with blank culture medium with the same volume as the test sample and incubated in a constant temperature incubator for 2 hours. Subsequently, the cells were stimulated with LPS (1μg / mL) for 12 hours, and the NO content in the cell culture medium was determined by the Griess method. At the same time, the CCK-8 method was used to determine the cytotoxic effect of compounds 1-4 on the proliferation of RAW 264.7 cells.

[0078] The results are as follows Figure 12 As shown, compounds 1 (A) and 4 (B) inhibited LPS-induced NO production in RAW264.7 cells in a dose-dependent manner. The NO inhibitory activity of compound 1 at 40 μM was significantly superior to that of the positive drug dexamethasone (20 μM). At the same time, CCK-8 assay results showed that compounds 1-4 had no significant cytotoxicity.

[0079] Further research was conducted on the anti-inflammatory mechanism of compounds 1 and 4. Figure 13 As shown in Figures AD, compounds 1 and 4 significantly downregulated iNOs and COX-2 proteins that were upregulated after LPS induction. In addition, ELISA experiments showed that ( Figure 14 A and B), the release of TNF-α, IL-6 and PGE2 in cells after LPS stimulation was significantly inhibited by compounds 1 and 4. Therefore, the sesquiterpenoid compounds isolated by the present invention are potential anti-inflammatory drug leads and have the prospect of further development of anti-inflammatory drugs.

[0080] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for separating sesquiterpenoid compounds, characterized in that: The following steps are involved: (1) The dried roots of Piper melongena were extracted with ethanol, the combined extracts were concentrated to obtain an extract, the extract was extracted with petroleum ether, and the extracted components were subjected to silica gel column chromatography using a petroleum ether-ethyl acetate system for gradient elution to obtain 7 fractions Fr.1-Fr.7; (2) Fraction Fr.4 was purified by a normal phase silica gel column using a petroleum ether-ethyl acetate gradient elution system. The eluate was analyzed by thin layer chromatography and the same components were combined to obtain 21 fractions Fr.4.1-Fr.4.

21. (3) Fraction Fr.4.8 was purified by semi-preparative HPLC to obtain compound 4; (4) Fraction Fr.4.12 was purified by reverse phase silica gel column with a methanol-water gradient elution system to obtain 45 fractions Fr.4.12.1-Fr.4.12.45; Fraction Fr.4.12.9 was purified by a semi-preparative HPLC method to obtain compound 1; Compound 4 is: Compound 1 is 2. The method for separating sesquiterpenoids according to claim 1, wherein The blue pepper is a plant of the genus Lindera in the Lauraceae family (Lindera glauca (Siebold & Zucc.) Blume).

3. The method for separating sesquiterpenoids according to claim 1, wherein The ethanol used is industrial ethanol with a concentration of 80-95%, and the ethanol extraction method is a hot soaking method.

4. The method for separating sesquiterpenoids according to claim 1, wherein In step (1), the volume ratio of petroleum ether and ethyl acetate during elution is changed to 40:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1; in step (2), the volume ratio of petroleum ether and ethyl acetate during elution is changed to 12:1, 10:1, 8:1, 5:1, 3:1, and 0:1; and in step (4), the volume ratio of methanol and water during elution is changed to 20:80, 40:60, 60:40, 80:20, and 100:

0.

5. The method for separating sesquiterpenoids according to claim 1, wherein In step (3), the conditions for the semi-preparative HPLC method are: selecting a Welch Ultimate XB-C18 liquid chromatography column, isocratic elution separation, and acetonitrile / water as the mobile phase.

6. The method for separating sesquiterpenoids according to claim 1, wherein Fraction Fr.4.12.9 was purified by a semi-preparative HPLC method using a Welch Ultimate XB-C18 liquid chromatography column, isocratic elution, and acetonitrile / water as the mobile phase.

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