A new compound, rhizochrysidin D, and a preparation method and application thereof

Abstract

This invention discloses a novel compound, cinnamon root leaf glycoside D, its preparation method, and its application. The preparation of cinnamon root leaf glycoside D includes: (1) extracting cinnamon root leaves by hot reflux with ethanol, followed by extraction with petroleum ether, ethyl acetate, and water-saturated n-butanol at a volume ratio of 1:1, and concentrating under reduced pressure to obtain an extract with three layers: a petroleum ether layer, an ethyl acetate layer, and a n-butanol layer; (2) mixing the ethyl acetate layer extract with silica gel, and then eluting it through a silica gel column in a gradient of dichloromethane-methanol = 1:0-0:1 by volume, observing the results on a thin-layer chromatography plate to obtain fractions A-K; fraction E is then eluted through an ODS column in a gradient of methanol-water = 1:9-9:1 by volume to obtain fractions E1-E9, of which E3 is separated by semi-preparative high-performance liquid chromatography to obtain cinnamon root leaf glycoside D. This compound exhibits good anti-inflammatory activity. It not only enriches the pharmacodynamic material basis of cinnamon root leaves but also enriches the treasure trove of natural chemical structures, providing new ideas for finding natural anti-inflammatory components.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a new compound, cinnabar root leaf glycoside D, its preparation method, and its application. Background Technology

[0002] Currently, the main drugs used to control and suppress inflammation include steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), and immunosuppressants, but they have serious adverse reactions. [1] Therefore, finding anti-inflammatory components from natural products is of great significance for anti-inflammatory drugs.

[0003] The genus *Ardisia* comprises approximately 500 species widely distributed in subtropical and tropical regions. [2,3] Modern genetic research considers *Ardisia* to be a genus within the subfamily Ardisiae of the family Myrtaceae. [4] The genus Ardisia is a rich source of novel and biologically active natural products. [5] In order to find new active natural products, the root of Aralia elata, a plant of the genus Aralia, was studied. Aralia elata is a commonly used natural medicine and is an evergreen shrub. [6] The fruit turns red when ripe; some varieties have white fruit, but this is relatively rare. [7,8] In China, the root of the red-fruited cinnabar plant is used as a traditional Chinese medicine called "cinnabar root". [9] It is widely used for a variety of diseases such as respiratory infections, toothache, joint pain, menstrual problems, and fertility regulation. [10-12] Previous phytochemical and pharmacological studies on *Ardisia crenata* have shown that it mainly contains triterpenoid saponins, isocoumarins, phytosterols, and benzoquinone compounds. [13,14] .

[0004] As a traditional natural medicinal plant, *Ardisia crenata* not only possesses extremely high medicinal value but also has promising market prospects. It is one of the main ingredients in the Kaihoujian spray, and its main component, bergenin, is included in the Chinese Pharmacopoeia as one of the main quality control standards for Kaihoujian spray. Further in-depth research on *Ardisia crenata* is of great significance for elucidating its pharmacodynamic material basis. Previous studies on *Ardisia crenata* mainly analyzed its roots, exploring their components, efficacy, and medicinal material basis. However, using only the roots of perennial plants for medicinal purposes resulted in significant resource waste. This study investigated the chemical composition and anti-inflammatory activity of the non-traditional medicinal part of *Ardisia crenata*—its leaves—aiming to expand the medicinal parts of *Ardisia crenata* and promote its rational resource utilization.

[0005] This invention separates and purifies the chemical components of a 70% ethanol extract of Cinnamomum cassia leaves using modern chromatography. A new lactone compound was discovered, its structure was identified using modern spectroscopic techniques, and its anti-inflammatory activity was screened, enriching the medicinal botanical content of Cinnamomum cassia root. Summary of the Invention

[0006] The purpose of this invention is to provide a novel compound, cinnamon root leaf glycoside D.

[0007] Another objective of this invention is to provide a new method for preparing the compound cinnamon root leaf glycoside D.

[0008] Another object of the present invention is to provide the application of the novel compound cinnamon root leaf glycoside D in the preparation of anti-inflammatory drugs.

[0009] To achieve the objectives of this invention, the following technical solutions and steps are employed:

[0010] The present invention discloses a novel compound, cinnamon root leaf glycoside D, whose structural formula is shown in (Ⅰ):

[0011]

[0012] The method for preparing the novel compound cinnamon root leaf glycoside D according to the present invention includes the following steps:

[0013] (1) Extraction and extraction of Cinnabar root and leaves:

[0014] The roots and leaves of Cinnamomum cassia were dried, pulverized, and extracted with ethanol solution under reflux. The extract was then filtered and concentrated under reduced pressure until no ethanol odor was detected, yielding a crude extract. The concentrated crude extract was then extracted sequentially with petroleum ether, ethyl acetate, and water-saturated n-butanol at a volume ratio of 1:1. Each solvent was used for extraction several times. The extracts were combined and concentrated under reduced pressure to obtain an extract with three layers: a petroleum ether layer, an ethyl acetate layer, and a n-butanol layer.

[0015] (2) Separation of compounds from the ethyl acetate layer of Cinnamomum cassia root and leaves:

[0016] Weigh the ethyl acetate extract of cinnamon root obtained in step (1), mix it with 80-100 mesh silica gel, and then pass it through a silica gel column in a gradient of dichloromethane-methanol = 1:0-0:1 by volume. After recovering the solvent, spot the sample onto a GF column. 254 Thin-layer chromatography (TLC) was used to observe and combine fractions of the same composition to obtain fraction AK; fractions with clear spots on the TLC samples were selected for separation and purification by ODS, MCI, gel electrophoresis and / or semi-preparative high-performance liquid chromatography (SPLC).

[0017] The specific separation process is silica gel column separation: fraction E is passed through an ODS column and eluted with a methanol-water system of 1:9-9:1 by volume to obtain fractions E1-E9. Among them, E3 is separated by semi-preparative high performance liquid chromatography to obtain compound 4, namely cinnamon root leaf glycoside D.

[0018] The ethanol solution mentioned in step (1) of this invention is 60-80% ethanol, and the number of extractions is 2-4.

[0019] Preferably, the ethanol solution in step (1) of the present invention is 70% ethanol, and the number of extractions is 3.

[0020] The number of extractions in step (1) of this invention is 2-4 times.

[0021] Preferably, the number of extractions in step (1) of the present invention is 3 times.

[0022] The conditions for semi-preparative high performance liquid chromatography separation in step (2) of this invention are: methanol-water as the mobile phase and flow rate of 3 mL / min.

[0023] The methanol in the mobile phase of this invention is 40-45% methanol.

[0024] The methanol in the mobile phase of this invention is 42% methanol.

[0025] The application of cinnamon root leaf glycoside D described in this invention in the preparation of anti-inflammatory drugs.

[0026] Beneficial effects of this invention:

[0027] 1. The cinnabar root leaf glycoside D prepared by this invention is a new compound. The raw material used is cinnabar root and leaves, which are not traditional medicinal parts, making full use of resources and avoiding a great waste of data.

[0028] 2. The novel compound, cinnamon root leaf glycoside D, obtained in this invention, exhibits good anti-inflammatory activity and can be used to prepare anti-inflammatory drugs. In vitro anti-inflammatory experiments at the cellular level showed significant inhibition of both NO and inflammatory factors at concentrations greater than 5 μM / mL, indicating that this compound possesses good anti-inflammatory properties. This not only enriches the pharmacodynamic material basis of cinnamon root leaves but also expands the treasure trove of natural chemical structures, providing new insights for the search of natural anti-inflammatory components and offering new directions for future researchers. Attached Figure Description

[0029] Figure 1 The key HMBC of compound 4 and 1 H- 1 H COSY signal

[0030] Figure 2 CD spectrum of compound 4

[0031] Figure 3 Structure of compound 4

[0032] Figure 4 Compound 4 1 H-NMR spectrum

[0033] Figure 5 Compound 4 13C-NMR spectrum

[0034] Figure 6 DEPT 135° spectrum of compound 4

[0035] Figure 7 HSQC spectrum of compound 4

[0036] Figure 8 HMBC spectrum of compound 4

[0037] Figure 9 Compound 4 1 H- 1 H COSY spectrum

[0038] Figure 10 ORD spectrum of compound 4

[0039] Figure 11 UV spectrum of compound 4

[0040] Figure 12 High-resolution mass spectrum of compound 4

[0041] Figure 13 Effect of compound 4 on cell viability (Note: the horizontal axis represents compound concentration; compared with the control group, * indicates: P < 0.05)

[0042] Figure 14 Effect of compound 4 on the activity of LPS-induced cells (Note: Compared with the LPS-induced group, ** indicates P < 0.01, *** indicates P < 0.001; compared with the control group, ### (Indicates P < 0.001)

[0043] Figure 15 NO release from compound 4 (Note: Compared with the LPS-induced group, *** indicates P < 0.001, ns indicates P > 0.05; compared with the control group, ### (Indicates P < 0.001)

[0044] Figure 16 TNF-α release of compound 4 (compared with the LPS-induced group, ** indicates P < 0.01, *** indicates P < 0.001; compared with the control group, ### (Indicates P < 0.001)

[0045] Figure 17 IL-1β release of compound 4 (compared with the LPS-induced group, ** indicates P < 0.01, *** indicates P < 0.001; compared with the control group, ### (Indicates P < 0.001)

[0046] Figure 18 IL-4 release of compound 4 (compared with the LPS-induced group, ** indicates P < 0.01, *** indicates P < 0.001; compared with the control group, ### (Indicates P < 0.001)

[0047] Figure 19 IL-10 release of compound 4 (compared with the LPS-induced group, ** indicates P < 0.01, *** indicates P < 0.001; compared with the control group, ### (Indicates P < 0.001) Detailed Implementation

[0048] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. For those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0049] Example 1

[0050] (1) Extraction and extraction of Cinnamon root and leaves:

[0051] 4.9 kg of Cinnamomum cassia root and leaves were weighed, dried and pulverized, and extracted three times with 70% ethanol under hot reflux. The extracts were combined and concentrated under reduced pressure until no ethanol odor was detected to obtain crude extract. The concentrated crude extract aqueous solution was extracted with petroleum ether, ethyl acetate and water-saturated n-butanol in a 1:1 volume ratio, three times with each solvent. The extracts were combined and concentrated under reduced pressure to obtain extracts of three layers: 33.06 g of petroleum ether layer, 521.6 g of ethyl acetate layer and 408.2 g of n-butanol layer.

[0052] (2) Separation of compounds from the ethyl acetate layer of Cinnamomum cassia root and leaves:

[0053] 220.8 g of ethyl acetate extract of *Ardisia crenata* root was weighed, mixed with 80-100 mesh silica gel, and then passed through a silica gel column in a gradient of dichloromethane-methanol (v:v) 1:0-0:1. After solvent recovery, the sample was spotted onto a GF column. 254 Thin-layer chromatography (TLC) was used to observe and combine fractions of the same composition to obtain fraction AK; fractions with clear spots on the TLC samples were selected for separation and purification by ODS, MCI, gel electrophoresis and / or semi-preparative high-performance liquid chromatography (SPLC).

[0054] The specific separation process was as follows: fraction E was passed through an ODS column and eluted with a methanol-water gradient system (1:9-9:1) to obtain fractions E1-E9. Fraction E3 was then subjected to semi-preparative high-performance liquid chromatography (HPLC) with 42% methanol-water as the mobile phase at a flow rate of 3 mL / min to obtain compound 4 (t). R=24.6 min, 3.4 mg).

[0055] The structural formula of compound 4 is as follows:

[0056]

[0057] Example 2: Synthesis method of novel alkaloid derivatives

[0058] (1) Extraction and extraction of Cinnamon root and leaves:

[0059] 4.9 kg of Cinnamomum cassia root and leaves were weighed, dried and pulverized, and extracted four times with 60% ethanol under hot reflux. The extracts were combined and concentrated under reduced pressure until no ethanol odor was detected to obtain crude extract. The concentrated crude extract aqueous solution was extracted sequentially with petroleum ether, ethyl acetate and water-saturated n-butanol at a volume ratio of 1:1, with each solvent being extracted four times. The extracts were combined and concentrated under reduced pressure to obtain an extract of three layers: 32.22 g of petroleum ether layer, 512.7 g of ethyl acetate layer and 400.5 g of n-butanol layer.

[0060] (2) Separation of compounds from the ethyl acetate layer of Cinnamomum cassia root and leaves:

[0061] 220.1 g of ethyl acetate extract of *Ardisia crenata* root was weighed, mixed with 80-100 mesh silica gel, and then passed through a silica gel column in a gradient of dichloromethane-methanol (v:v) 1:0-0:1. After solvent recovery, the sample was spotted onto a GF column. 254 Thin-layer chromatography (TLC) was used to observe and combine fractions of the same composition to obtain fraction AK; fractions with clear spots on the TLC samples were selected for separation and purification by ODS, MCI, gel electrophoresis and / or semi-preparative high-performance liquid chromatography (SPLC).

[0062] The specific separation process is as follows: fraction E is passed through an ODS column and eluted with a methanol-water system (1:9-9:1) gradient to obtain fractions E1-E9. Among them, E3 is subjected to semi-preparative high performance liquid chromatography with 40% methanol-water as the mobile phase and a flow rate of 3 mL / min to obtain compound 4 (3.4 mg).

[0063] Example 3: Synthesis method of novel alkaloid derivatives

[0064] (1) Extraction and extraction of Cinnamon root and leaves:

[0065] 4.9 kg of Cinnamomum cassia root and leaves were weighed, dried and pulverized, and extracted twice with 80% ethanol under hot reflux. The extracts were combined and concentrated under reduced pressure until no ethanol odor was detected to obtain crude extract. The concentrated crude extract aqueous solution was extracted with petroleum ether, ethyl acetate and water-saturated n-butanol in a 1:1 volume ratio, with each solvent being extracted twice. The extracts were combined and concentrated under reduced pressure to obtain an extract with three layers: 33.03 g of petroleum ether layer, 516.9 g of ethyl acetate layer and 404.7 g of n-butanol layer.

[0066] (2) Separation of compounds from the ethyl acetate layer of Cinnamomum cassia root and leaves:

[0067] 220.2 g of ethyl acetate extract of *Ardisia crenata* root was weighed, mixed with 80-100 mesh silica gel, and then passed through a silica gel column in a gradient of dichloromethane-methanol (v:v) 1:0-0:1. After solvent recovery, the sample was spotted onto a GF column. 254 Thin-layer chromatography (TLC) was used to observe and combine fractions of the same composition to obtain fraction AK; fractions with clear spots on the TLC samples were selected for separation and purification by ODS, MCI, gel electrophoresis and / or semi-preparative high-performance liquid chromatography (SPLC).

[0068] The specific separation process is as follows: fraction E is passed through an ODS column and eluted with a methanol-water system (1:9-9:1) gradient to obtain fractions E1-E9. Among them, E3 is subjected to semi-preparative high performance liquid chromatography with 45% methanol-water as the mobile phase and a flow rate of 3 mL / min to obtain compound 4 (3.4 mg).

[0069] Example 4

[0070] Application of cinnamon root leaf glycoside D in the preparation of anti-inflammatory drugs.

[0071] To verify the effectiveness of the invention, the inventive team conducted a series of experiments, as follows:

[0072] I. Extraction and Identification from Cinnabar Roots and Leaves

[0073] 1. Experimental medicinal materials

[0074] The medicinal material, Ardisia crenata Sims (batch number: 20200908), was collected from Lover's Valley in Guiyang City, Guizhou Province. It was identified by Professor Wei Shenghua as Ardisia crenata Sims, a plant of the genus Ardisia in the family Myrsinaceae. The specimen is preserved in the Laboratory of Traditional Chinese Medicine and Ethnic Medicine of Guizhou University of Traditional Chinese Medicine.

[0075] 2. Experimental instruments, reagents and materials

[0076] See Table 1.

[0077] Table 1. Partial list of experimental instruments, materials, and reagents

[0078]

[0079]

[0080] 3. Extraction and Separation

[0081] 3.1 Extraction and extraction of Cinnabar root and leaves

[0082] The roots and leaves of *Ardisia crenata* were dried and pulverized to obtain 4.9 kg. They were then extracted three times by hot reflux with 70% ethanol. The extracts were combined and the solvent was recovered until there was no ethanol odor. The concentrated crude extract aqueous solution was then extracted three times with petroleum ether, ethyl acetate, and water-saturated n-butanol in a 1:1 (v:v) ratio. The extracts were combined and concentrated under reduced pressure to obtain an extract with three layers: 33.06 g of petroleum ether layer, 521.6 g of ethyl acetate layer, and 408.2 g of n-butanol layer.

[0083] 3.2 Isolation of compounds from the ethyl acetate layer of Cinnamomum cassia roots and leaves

[0084] 220.8 g of ethyl acetate extract of *Ardisia crenata* root was weighed, mixed with 80-100 mesh silica gel, and then passed through a silica gel column in a gradient of dichloromethane-methanol (v:v) 1:0-0:1. After solvent recovery, the sample was spotted onto a GF column. 254 Thin-layer chromatography (TLC) was used to observe and combine fractions of the same composition to obtain fraction AK. Components with clearly defined spots on the TLC samples were selected for separation and purification using ODS, MCI, gel electrophoresis, and / or semi-preparative high-performance liquid chromatography (HPLC). The specific separation process followed a silica gel column separation procedure:

[0085] Fraction E was passed through an ODS column and eluted with a methanol-water gradient (1:9-9:1) to obtain fractions E1-E9. Fraction E3 was then subjected to semi-preparative high-performance liquid chromatography (HPLC) with 42% methanol-water as the mobile phase at a flow rate of 3 mL / min to obtain compound 4 (t). R =24.6 min, 3.4 mg).

[0086] 4. Structural analysis of compounds in the ethyl acetate layer of cinnabar root and leaves

[0087] 4.1 Identification of the structure of new compounds

[0088] Compound 4: Pale yellow solid, readily soluble in methanol; HR-ESI-MS, m / z: 529.1310 [M+Na] + ,and 13 C-NMR analysis suggests that the molecular formula of compound 4 is C 24 H 26 O 12 (The calculated value is 529.1316[M+Na]) + The unsaturation degree is calculated to be 12. 1 H-NMR gave a signal of 6 aromatic ring protons ( Figure 4 )δ H7.89 (2H,d,J=8.7,H-2”',6”'), 6.83 (2H,d,J=8.7,H-3”',5”'), 6.23 (1H,d,J=2.9,H-3’), 6.16 (1H,d,J=2.9,H-5’), suggesting the presence of two benzene ring units in the structure; and the existence of a terminal proton signal δ of the sugar. H 4.59 (1H,d,J=7.7,H-1”), suggesting that the structure contains sugar structural units; 13 C-NMR data showed carbon δ values ​​for 12 aromatic regions. C ( Figure 5 The values ​​of 103.4, 109.2, 116.2, 116.2, 122.0, 132.9, 133.0, 133.0, 138.5, 151.5, 155.9, and 163.7 confirm the existence of two benzene ring structures; the δ values ​​of the two ester carbonyl groups are also provided. C 167.9, 180.4 and 6 glycosyl data δ C 64.7, 71.6, 75.5, 76.1, 77.9, 107.5; the remaining 4 carbons, after DEPT 135° ( Figure 6 It was identified as having 3 methylene δ groups. C 28.2, 29.4, 83.0 and 1 methine δ C 36.4; via key 1 H- 1 H COSY signal ( Figure 9 The fragment was identified as a five-membered lactone ring. Compound 4 is presumed to be similar to compound 3, but lacks a phenolic hydroxyl group; key HMBC signals include H2-3, H2-4, and H-5 to C-2; H2-6 to C-4, C-5, C-1', C-5', and C-6'; H2-6" to C-7; and H2-2"' and 6"' to C-7, determining the linkage patterns of each group. See Table 2. Figure 1 .

[0089] through 1 H-NMR, HSQC ( Figure 7 ) and HMBC ( Figure 8 Analysis confirmed that its conformation was β-D-glucose; CD spectrum analysis confirmed that it was β-D-glucose. Figure 2Based on the spectral data, the 5-position of compound 4 was determined to be S configuration; therefore, the structure of compound 4 is fully characterized as ((2R,3S,4S,5R,6S)-(6-(2,4-dihydroxy-6-(((S)-5-oxotetrahydrofuran-2-yl)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-hydroxybenzoate, named Ardisicreolide D, with the structure shown below. Figure 3 ).

[0090] 4.2 Spectral data of compound 4:

[0091] HR-ESI-MS shows m / z: 529.1310 [M+Na] + ( Figure 12 Compound 4 has the molecular formula C. 24 H 26 O 12 (The calculated value is 529.1316[M+Na]) + Compound 4 has the highest UV absorption at 203 nm. Figure 11 ), UV(MeOH)λ(logε)203(4.56)nm; ( Figure 10 ).

[0092] Table 2 Ardisicreolide D 1 H and 13 C-NMR data (CD3OD)

[0093]

[0094]

[0095] 5. Summary

[0096] This study used a combination of modern chromatographic and spectroscopic techniques to isolate, purify, and identify a new compound from the ethyl acetate extract of the 70% ethanol extract of Ardisiccium chinense leaves. The compound was named (Ardisicreolide D) Ardisicreolide D(4), which is a lactone compound. Subsequent experiments will conduct preliminary screening of the pharmacological activity of the new compound in order to discover natural compounds with good activity, enrich the pharmacodynamic material basis of Ardisiccium chinense, and provide direction for future research.

[0097] II. Study on the anti-inflammatory activity and mechanism of chemical components in the roots and leaves of Cinnamomum cassia

[0098] Cinnamon root is the main component of Kaihoujian spray, a compound preparation primarily used to treat pharyngitis, which forms the basis of this experiment. Previous research and literature review by our research group indicate that Cinnamon root can clear heat, soothe the throat, and resolve blood stasis, showing good efficacy in treating chronic pharyngitis. It is revered as a valuable medicine for throat ailments by the Miao people and is a commonly used traditional Chinese medicine in Guizhou for treating throat inflammation, confirming its excellent anti-inflammatory activity. Inflammation is a protective response of the body to stimuli, mainly involving immune cells, blood vessels, and various molecular mediators. This experiment used an LPS-induced RAW264.7 macrophage inflammation model in mice to measure major and common inflammatory factors such as NO, IL-1β, IL-4, IL-10, and TNF-α to evaluate the anti-inflammatory activity of the newly isolated compound from Cinnamon root leaves, enriching its pharmacodynamic material basis and providing a theoretical basis for rational drug use.

[0099] 1. Experimental instruments and reagents

[0100] See Table 3.

[0101] Table 3 Specifications and manufacturers of experimental instruments and reagents

[0102]

[0103]

[0104] 2. Experimental Methods

[0105] 2.1 Source of compounds and preparation of required solutions

[0106] 2.1.1 Compound Sources

[0107] The compound (4) used in the experiment was isolated and identified by me from the ethyl acetate fraction of the 70% ethanol extract of the leaves of *Ardisia crenata* at the Laboratory for the Application and Development of Special Functional Foods and Traditional Chinese Medicine and Ethnic Medicines of Guizhou University of Traditional Chinese Medicine.

[0108] 2.1.2 Preparation of reference solution

[0109] Accurately weigh the positive control dexamethasone (DMX) and add it to the culture medium to the required concentration.

[0110] 2.1.3 Sample solution preparation

[0111] Accurately weigh 1.0 mg of each of compound (4), dissolve them in DMSO (DMSO not exceeding 0.1%), add them to the culture medium and serially dilute to 160, 80, 40, 20, 10, 5 μM / mL to obtain the final product.

[0112] 2.2 Macrophage culture using RAW264.7

[0113] 2.2.1 Cell resuscitation

[0114] RAW264.7 cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. After centrifugation at 800 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 1.0 mL of preheated DMED medium. The cell suspension was then transferred to a culture dish and cultured in a 37°C incubator containing 5% CO2. The medium was changed the next day. When the cells reached 80-90% confluence in the culture dish, they could be passaged. The cell line from the time of thawing is recorded as the first generation. Cells from generations 3-8 are generally selected for experiments.

[0115] 2.2.2 Cell Count

[0116] Cell counting was performed using the hemocytometer method. Cells in the logarithmic growth phase were digested, thoroughly dispersed by pipetting, and diluted to an appropriate concentration. The cell suspension was then pipetted onto a hemocytometer along the edge of a coverslip, avoiding air bubbles. The hemocytometer was observed under a microscope, adjusting the field of view until the grid was clearly visible. The counting principle was "count the top, not the bottom; count the left, not the right." The cell concentration calculation formula is as follows:

[0117] Cell count / mL = (Number of cells in 8 large squares / 8) × 10 4 ×Dilution factor

[0118] 2.2.3 Cell passage

[0119] RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and streptomycin. When the cell adhesion density reached 80%, the medium was removed, and the cells were washed three times with sterile PBS. The cells were then digested with a suitable amount of trypsin preheated to 37°C for about 2-3 minutes. Under a microscope, when the cells became round and the gaps between them increased, the digestion solution was discarded. Fresh medium was added, and the cells were detached by pipetting. The cells were collected by centrifuging at 800 rpm / min for 5 minutes, and fresh medium was added to make a cell suspension. The cells were then transferred to new culture dishes at a certain ratio and cultured in an incubator containing 5% CO2 at 37°C for later use.

[0120] 2.2.4 Cell Culture

[0121] RAW264.7 cells were cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum (1000 U / mL penicillin and 1000 U / mL streptomycin) at 37°C with 5% CO2. Cell status was observed daily and the medium was changed as needed.

[0122] 2.2.5 Cell cryopreservation

[0123] When the cells reach 80-90% confluence, digest the cells with trypsin to prepare a cell suspension, centrifuge at 800 rpm for 5 min, then remove the supernatant, add a pre-prepared cryopreservation solution containing 7% DMSO (90% cell culture medium, 10% DMSO), suspend the cells, transfer the cell suspension to cryovials, label the cryovials, wrap them with cotton and place them at 4℃ for 10-15 min, then at -20℃ for 1-2 h, then place the cells in a cryopreservation box for short-term storage at -80℃, and then transfer them to liquid nitrogen for long-term storage.

[0124] 2.3 Effect of the compound on RAW264.7 cell viability as determined by the CCK-8 assay

[0125] RAW264.7 cells in the logarithmic growth phase were harvested and the cell number was adjusted to 10-1. 6 Cells were seeded at a density of 100 μL / well in 96-well plates. Four replicates were set up for each well, including a blank control group, a normal control group, and experimental groups with different concentrations. 100 μL of PBS buffer was added around the perimeter of each well to prevent edge effects. After seeding, the 96-well plates were incubated at 37°C with 5% CO2. Once cells adhered, the supernatant was discarded, and different final concentrations of the sample were added. An equal volume of culture medium was added to the control wells. The plates were then incubated for 24 hours. The procedure was performed according to the CCK-8 kit instructions.

[0126] 2.4 Effect of the compound on LPS-induced RAW264.7 cell viability as determined by the CCK-8 assay

[0127] RAW264.7 cells in the logarithmic growth phase were harvested and the cell number was adjusted to 10-1. 6 Cells were seeded at a density of 100 μL / well in 96-well plates and incubated at 37°C with 5% CO2 until adherence. The supernatant was discarded. Three replicates were set up for each well: a blank control group, a normal control group, an LPS control group, a positive control group, and different concentration experimental groups. The blank group was treated with an equal volume of cell-free culture medium, and 100 μL of PBS buffer was added around the perimeter of each well to prevent edge effects. Different final concentrations of the sample were added to each well in the drug-treated groups. Cells were pretreated for 3 hours and then co-incubated with LPS stimulation for 24 hours. The procedure was performed according to the CCK-8 reagent kit instructions.

[0128] 2.5 Detection of NO content released from RAW264.7 cells using the Griess method

[0129] RAW264.7 cells in the logarithmic growth phase were harvested and the cell number was adjusted to 10-1. 5Cells were seeded at a density of 1 mL / well in 24-well plates, with three replicates for each group: normal control, LPS control, positive control, and experimental groups with different concentrations. The plates were incubated at 37°C with 5% CO2 for 24 hours, followed by pretreatment with 100 μL of different concentrations of sample for 3 hours, and then co-incubated with 10 μL of LPS for 24 hours. The cell culture medium was centrifuged (2000 rpm, 10 min), and the supernatant was transferred to a new centrifuge tube. The procedure was followed according to the NO kit instructions.

[0130] 2.6 ELISA was used to detect the levels of TNF-α, IL-1β, IL-4, and IL-10 released from RAW264.7 cells.

[0131] Take the supernatant from different groups in 2.5 and follow the instructions of the ELISA kit.

[0132] 3. Experimental Results

[0133] 3.1 Effect of compound (4) on RAW264.7 cell viability as determined by CCK-8 assay

[0134] The absorbance was measured, and data analysis was performed using ONE-AVOVA with GraphPad Prism 9.0.0. The results showed that compound (4) had no significant difference from the control group cells at a dose of less than 80 μM / mL (P > 0.05). It can be considered that the compound had no effect on the activity of RAW264.7 cells at this dose. Figure 13 As shown.

[0135] 3.2 Effect of compound 4 on LPS-induced RAW264.7 cell viability as determined by CCK-8 assay

[0136] By measuring absorbance and using GraphPad Prism 9.0.0 for ONE-AVOVA data analysis, the results showed that the cell activity of LPS-induced RAW264.7 cells was significantly different from that of the normal control group (P < 0.05); the cell activity of compound (4) was significantly different from that of LPS-induced RAW264.7 cells (P < 0.05). Since the cell activity of RAW264.7 cells significantly increased after LPS-induced inflammation, the activity of the drug-treated group decreased. Combined with the data from the dexamethasone positive control group, it is preliminarily speculated that compound (4) has certain anti-inflammatory activity. See Figure 14 .

[0137] 3.3 Griess method for detecting NO content released from RAW264.7 cells

[0138] The NO content in cell supernatant was measured using a NO kit, and data analysis was performed using GraphPad Prism 9.0.0 with ONE-AVOVA. The results showed that NO release was significantly increased in the LPS-induced RAW264.7 cell group compared to the normal control group; NO release was significantly decreased in the drug-treated group compared to the LPS-induced RAW264.7 cell group. (See...) Figure 15 .

[0139] 3.4 ELISA was used to detect the levels of TNF-α, IL-1β, IL-4, and IL-10 released from RAW264.7 cells.

[0140] The NO content in cell supernatant was measured using an ELISA kit. Data analysis was performed using ONE-AVOVA with GraphPad Prism 9.0.0. Results showed that compared with the normal control group, the release of TNF-α, IL-1β, IL-4, and IL-10 was significantly increased in the LPS-induced RAW264.7 cell group; compared with the LPS-induced RAW264.7 cell group, the release of TNF-α, IL-1β, IL-4, and IL-10 was significantly decreased in the drug-treated group. (See Table 4.) Figures 16-19 .

[0141] Table 4. Release levels of inflammatory factors

[0142]

[0143] 4. Summary

[0144] By culturing normally growing mouse RAW264.7 macrophages after administering different concentrations of sample drugs, and using CC... The K-8 reagent kit was used to measure absorbance using an ELISA reader to calculate the relative cell activity. The optimal concentration for drug administration, defined as 80 μM / mL, was determined to have no significant inhibitory effect on cell activity. To preliminarily screen the anti-inflammatory effects of the test compounds on LPS-induced mouse RAW264.7 macrophages, LPS-induced RAW264.7 macrophages were cultured after being treated with different concentrations of the sample, and the relative cell activity was calculated. The results showed that LPS-induced inflammation further activated the cells, significantly increasing cell activity compared to the control group. The LPS-induced RAW264.7 macrophages in the treated group showed significantly decreased cell activity compared to the untreated group, suggesting a preliminary anti-inflammatory effect. To confirm the anti-inflammatory activity of the compounds, an ELISA kit was used to measure NO and inflammatory factors TNF-α, IL-1β, IL-4, and IL-10 in the cell supernatant. The results showed that compound 4 significantly inhibited NO and inflammatory factors at concentrations greater than 5 μM / mL, which is speculated to be related to the five-membered lactone ring, a common structural unit in the compound.

[0145] References:

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Claims

1. A novel compound, cinnamon root leaf glycoside D, characterized in that, The structural formula is shown in (Ⅰ): 。 2. A method for preparing the novel compound cinnamon root leaf glycoside D as described in claim 1, characterized in that, Includes the following steps: (1) Extraction and extraction of Cinnamon root and leaves: The roots and leaves of Cinnamomum cassia were dried, pulverized, and extracted with ethanol solution under reflux. The extract was then filtered and concentrated under reduced pressure until no ethanol odor was detected, yielding a crude extract. The concentrated crude extract was then extracted sequentially with petroleum ether, ethyl acetate, and water-saturated n-butanol at a volume ratio of 1:

1. Each solvent was used for extraction several times. The extracts were combined and concentrated under reduced pressure to obtain an extract with three layers: a petroleum ether layer, an ethyl acetate layer, and a n-butanol layer. (2) Separation of compounds from the ethyl acetate layer of Cinnamomum cassia root and leaves: Weigh the ethyl acetate extract of cinnamon root obtained in step (1), mix it with 80-100 mesh silica gel, and then pass it through a silica gel column in a gradient of dichloromethane-methanol = 1:0-0:1 by volume. After recovering the solvent, spot the sample. Thin-layer plate observation combined with the same components yielded fraction AK; fraction E was passed through an ODS column and eluted with a methanol-water gradient of 1:9-9:1 by volume to obtain fractions E1-E9, of which E3 was separated by semi-preparative high performance liquid chromatography to obtain compound 4, namely cinnamon root leaf glycoside D.

3. The preparation method according to claim 2, characterized in that, The ethanol solution mentioned in step (1) is 60-80% ethanol, and the number of extractions is 2-4.

4. The preparation method according to claim 3, characterized in that, The ethanol solution mentioned in step (1) is 70% ethanol, and the number of extractions is 3.

5. The preparation method according to claim 2, characterized in that, The number of extractions in step (1) is 2-4.

6. The preparation method according to claim 5, characterized in that, The number of extractions in step (1) is 3.

7. The preparation method according to claim 2, characterized in that, The conditions for semi-preparative high performance liquid chromatography separation in step (2) are: methanol-water as the mobile phase and flow rate of 3 mL / min.

8. The preparation method according to claim 7, characterized in that, The methanol is 40-45% methanol.

9. The preparation method according to claim 8, characterized in that, The methanol in question is 42% methanol.

10. The use of cinnamon root leaf glycoside D as described in claim 1 or cinnamon root leaf glycoside D prepared by any one of claims 2-9 in the preparation of anti-inflammatory drugs.

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