Lilium brownii extract, method for preparing the same and use thereof in preparing anti-inflammatory products

Abstract

This invention discloses a lily flower extract, its preparation method, and its application in the preparation of anti-inflammatory products. The preparation method of the lily flower extract includes the following steps: taking dried lily flowers, soaking them in water, reflux extraction, filtering, concentrating the filtrate, and drying to obtain the lily flower extract. The lily flower extract can be used to prepare anti-inflammatory products with excellent effects. The lily flower extract of this invention can significantly improve lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells and lipopolysaccharide-induced lung inflammatory damage. Its anti-inflammatory and lung inflammatory damage-improving effects can be used to prepare drugs for the prevention or treatment of inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extract technology, and particularly relates to a lily flower extract, its preparation method, and its application in the preparation of anti-inflammatory products. Background Technology

[0002] Lilies are important plants used for both medicinal and edible purposes, with a long history of cultivation and application. It is noteworthy that, for a long time, the non-medicinal parts of lilies—stems, leaves, and flowers—which produce a large quantity, have not been utilized in traditional medicinal practices and are mostly discarded as waste. This disposal method not only wastes resources but also contributes to environmental problems to some extent.

[0003] Traditional Chinese medicine (TCM) possesses the characteristic of comprehensive effects involving multiple components and targets, demonstrating unique advantages in the prevention and treatment of inflammatory diseases. Modern research has found that lily flowers are rich in flavonoids, saponins, and other chemical components, exhibiting certain biological activity and development potential. Ancient TCM texts also record lily flowers' ability to "moisten the lungs and clear heat," and its efficacy aligns somewhat with the TCM understanding of acute lung injury as "internal accumulation of phlegm and heat, and impaired lung function." In recent years, the concept of comprehensive utilization of TCM resources has received increasing attention. Lily flowers, as an important non-medicinal part, have considerable resources, but related research remains relatively weak.

[0004] With economic development and advancements in medical technology, the proportion of the aging population continues to increase. Furthermore, environmental pollution and increased stress leading to immune system abnormalities have resulted in the chronicity of inflammatory responses. Inflammatory responses can be caused by internal stimuli resulting from tissue damage, possessing certain physiological functions such as clearing damaged tissue and related factors, thereby repairing tissues and restoring their normal biological structure and defense mechanisms. However, they can also be caused by external stimuli such as bacterial or viral infections and trauma, in which case the body is prone to excessive inflammation, leading to inflammatory diseases. Imbalances in various intracellular inflammatory regulatory factors, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), as well as nitric oxide (NO), have been observed. Studies have shown that excessive inflammation exacerbates damage caused by infection or trauma. Endotoxins, such as lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria, are representative pro-inflammatory factors that can induce excessive inflammatory responses and generate related inflammatory factors. Therefore, substances that regulate cellular inflammatory factors can serve as preventative drugs and / or therapeutic agents for inflammatory diseases.

[0005] Currently, anti-inflammatory substances include nonsteroidal anti-inflammatory drugs such as benzyldamine, ibuprofen, flufenamic acid, and indomethacin; and steroidal drugs such as dexamethasone, prednisolone, hydrocortisone, and betamethasone. However, these drugs are not suitable for long-term use, which limits their application in the prevention and treatment of inflammatory diseases. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, this invention provides a lily flower extract, its preparation method, and its application in the preparation of anti-inflammatory products.

[0007] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing lily flower extract, comprising the following steps:

[0009] Take dried lily flowers, soak them in water, reflux to extract, filter, concentrate the filtrate, and dry to obtain the lily flower extract.

[0010] As a specific implementation plan, the method of soaking in water includes: adding 5-20 times the amount of water as the lily flower material and soaking for 20-40 minutes.

[0011] As a specific implementation plan, the reflux extraction method includes: heating and reflux extraction 1-3 times, each time for 0.5-4 hours. After each extraction, the residue is filtered, and 5-20 times the amount of water is added to the residue for further reflux extraction. Finally, the filtrates are combined.

[0012] As a specific implementation scheme, the filtrate is concentrated to a crude drug concentration of 0.5-2.0 g / mL.

[0013] Secondly, the present invention provides a lily flower extract, which is prepared by the aforementioned preparation method.

[0014] Thirdly, the present invention provides a composition comprising the aforementioned lily flower extract, and pharmaceutically acceptable excipients or food additives.

[0015] Fourthly, the present invention provides the use of the lily flower extract or the composition thereof in the preparation of anti-inflammatory products.

[0016] As a specific implementation scheme, the product can effectively reduce the production of nitric oxide, reduce the gene expression levels of cellular inflammatory factors and oxidation-related enzymes, and reduce the generation of ROS.

[0017] As a specific implementation plan, the product can inhibit lung inflammation and improve acute lung injury.

[0018] As a specific implementation scheme, the dosage form of the product includes any one of the following: powder, granule, pill, granule, microcapsule, fluid extract, decoction, capsule, tablet, oral liquid, syrup, mouthwash, spray, aerosol, and nasal rinse.

[0019] The product can be any one of the following: medicine, functional beverage, functional food, or health product.

[0020] Lily (Lilium spp.) is a plant used for both medicine and food. Its scales and leaves are the legally permitted medicinal / edible parts according to the pharmacopoeia. The flowers, stems, and leaves are mainly composed of phenols and flavonoids, while saponins and polysaccharides are concentrated in the scales and leaves, exhibiting an overall distribution characteristic of "high polysaccharide and saponin content in scales and leaves, high phenolic and flavonoid content in flowers and leaves, and moderate content in stems and leaves." The medicinal / edible lilies mentioned in existing technologies are usually the scales and leaves specified in the pharmacopoeia. These scales and leaves are mainly composed of starch (11%–19%) and sucrose (3.7%–10.4%), with low levels of phenols and flavonoids and high levels of polysaccharides and saponins. In contrast, lily flowers are mainly high in phenols and flavonoids, and low in starch and sugars. The main active ingredients of the two differ significantly.

[0021] Beneficial Effects: Compared with existing technologies, the lily flower used in this invention is a non-medicinal part of the traditional Chinese medicine lily, and its water extract has significant anti-inflammatory effects. This invention uses a lipopolysaccharide-induced inflammation model of RAW 264.7 cells to investigate the anti-inflammatory effect of lily flower. At the cellular level, it can effectively reduce nitric oxide production, decrease the gene expression levels of cellular inflammatory factors and oxidation-related enzymes, and reduce ROS production. In inflammatory disease models represented by the lipopolysaccharide-induced acute lung injury model, lily flower extract can prevent lung tissue damage caused by LPS, alleviate LPS-induced hemorrhage and edema, reduce serum levels of IL-6, IL-1β, TNF-α, MDA, and MPO, increase serum levels of T-SOD, CAT, and T-AOC, and decrease the gene expression levels of lung tissue inflammatory factors and oxidation-related enzymes. These results indicate that lily flower has preventive and / or therapeutic effects on inflammation and inflammatory diseases, and shows good promise in the development of functional drugs. Attached Figure Description

[0022] Figure 1Characterization of the components of the lily flower extract (Lily) of this invention. RP-HPLC-DAD(AB) of lily flower extract 1. HPLC-Q-TOF-MS / MS chromatogram (CD) of positive and negative ion fractions of lily flower extract 1.

[0023] Figure 2 This invention relates to the effects of lily flower extract (Lily) on LPS-induced RAW 264.7 cells. The experiments included: cell proliferation (A), effect on NO production (B), effect of Lily on the mRNA expression levels of IL-6 (C), IL-1β (D), TNF-α (E), and SOD1 (F), and its effect on ROS production (G). Compared with the LPS group, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05; ns, no significant difference; compared with the control group, #### p < 0.0001, ### p < 0.001, ## p < 0.01 and # p < 0.01.

[0024] Figure 3 This invention demonstrates the protective effect of lily flower extract (Lily) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. (A) Body weight changes in mice during drug administration. (B) Measurement of lung dryness and wetness. (C) Histological evaluation of lung tissue in each experimental group after LPS stimulation. H&E staining (scale bar = 100 µm) was used to observe pathological changes in lung tissue. (D) Representative images of mouse lung tissue were observed and recorded. (E) Lung index was calculated. Compared with the LPS group, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05; ns, no significant difference; compared with the control group, #### p < 0.0001, ### p < 0.001, ## p < 0.01 and # p < 0.01.

[0025] Figure 4This invention investigates the protective effect of lily flower extract (Lily) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. The mRNA expression levels of IL-6 (A), IL-1β (B), TNF-α (C), SOD1 (D), and CAT (E) in lung tissue were measured. Compared with the LPS group, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05; ns, no significant difference; compared with the control group, #### p < 0.0001, ### p < 0.001, ## p < 0.01 and # p < 0.01.

[0026] Figure 5 This invention investigates the protective effect of lily flower extract (Lily) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Serum levels of T-SOD (A), CAT (B), T-AOC (C), MPO (D), and MDA (E), as well as (F) IL-6, IL-1β, and TNF-α, were measured. Compared with the LPS group, **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05; ns, no significant difference; compared with the control group, #### p < 0.0001, ### p < 0.001, ## p < 0.01 and # p < 0.01. Detailed Implementation

[0027] The technical solution of the present invention is illustrated below with detailed examples. However, it is worth noting that the scope of protection of the present invention is not limited to the specific embodiments described.

[0028] If specific techniques or conditions are not explicitly described in the examples, operations can be performed according to the techniques or conditions described in relevant literature in the field, or by following the relevant product instructions. Reagents or instruments, unless otherwise specified by manufacturer, are conventional products that can be purchased through legal channels.

[0029] The experimental methods described below follow general standards and, unless otherwise specified, employ standard experimental procedures. In the embodiments described, the test materials used are commercially available products, unless otherwise stated.

[0030] Example 1: Preparation of Lily Flower Extract 1

[0031] Take dried lily flowers, soak them in 10 times the amount of water for 30 minutes, reflux extract for 1 hour, and filter while hot; add 10 times the amount of water to the residue, reflux extract for 1 hour, filter while hot, combine the filtrates, concentrate to a crude drug concentration of 0.5 g / mL, and freeze-dry to obtain the lily flower extract 1.

[0032] Example 2 Preparation of Lily Flower Extract 2

[0033] Take dried lily flowers, soak them in 5 times the amount of water for 30 minutes, reflux extract for 0.5 hours, and filter while hot; add 5 times the amount of water to the residue, reflux extract for 0.5 hours, filter while hot, combine the filtrates, concentrate to a crude drug concentration of 1.5 g / mL, and freeze-dry to obtain the lily flower extract 2.

[0034] Example 3 Preparation of Lily Flower Extract 3

[0035] Take dried lily flowers, soak them in 20 times the amount of water for 30 minutes, reflux for 4 hours, and filter while hot; add 20 times the amount of water to the residue, reflux for 4 hours, filter while hot, combine the filtrates, concentrate to a crude drug concentration of 1.5 g / mL, and freeze-dry to obtain the lily flower extract 3.

[0036] Example 4: Chemical composition analysis and detection in lily flower extract 1

[0037] (1) Preparation of reversed-phase sample of lily flower extract 1

[0038] RP-HPLC-DAD characterization of lily flower extract 1 in Example 1: 58.58 mg of the above-mentioned lyophilized lily flower extract 1 was dissolved in 1 mL of 60% methanol, centrifuged, and the supernatant was filtered through a 0.22 μm microporous membrane as the reversed-phase chromatographic sample RP, and detected by a DAD detector.

[0039] (2) RP-HPLC-DAD high performance liquid chromatography conditions for lily flower extract 1

[0040] The chromatographic column was packed with octadecyl (C18) bonded silica gel, with a flow rate of 0.5–2.0 mL / min, a column temperature of 20–25 °C, and an injection volume of 5–10 μL. A DAD detector was used with a detection wavelength of 254 nm. The mobile phase was 0.1% formic acid water (A) – acetonitrile (B), and the gradient elution program is shown in Table 1.

[0041] Table 1. Elution gradient of RP-HPLC-DAD mobile phase

[0042]

[0043] (3) HPLC-Q-TOF-MS / M colorimetric conditions of lily flower extract 1

[0044] The lily flower extract of Example 1 was characterized by HPLC-Q-TOF-MS / MS. The sample preparation method was the same as that of Example 4.

[0045] An Agilent 6530 Q-TOF mass spectrometer with an ESI ion source was used. Positive and negative ion scanning modes were employed. The primary mass spectrometry scanning range was 50-3000 m / z, and the secondary mass spectrometry scanning range was also 50-3000 m / z. The capillary voltage was 3500 V, the dry gas (N2) flow rate was 9.0 L / min, the dry gas temperature was 325 °C, the nebulizer pressure was 35 psi, and the fragmentator voltage was 205 V. Data processing was performed using Agilent MassHunter Qualitative Analysis 10.0 software, with a maximum permissible error range of 10 ppm.

[0046] Experimental results: such as Figure 1 As shown.

[0047] Example 5: Anti-inflammatory effect of lily flower extract 1

[0048] 1. Effects of Lily on LPS-induced NO and inflammatory factors in RAW 264.7

[0049] RAW 264.7 cells were seeded in 96-well microplates. They were then pretreated with Lily (0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, calculated as the mass of the corresponding crude drug) at 37 ˚C for 1 h, followed by treatment with LPS (1.0 μg / mL) for 24 h. The accumulated nitrite concentration in the culture medium was determined according to the Griess reaction. Absorbance was monitored at 540 nm using a microplate reader.

[0050] RAW264.7 cells were seeded in 6-well plates. Cells were pretreated with Lily (0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, drug concentrations were calculated based on the corresponding crude drug mass) and cultured at 37˚C for 1 h, followed by treatment with LPS (1.0 μg / mL) for 24 h with or without Lily treatment. After culture, the culture medium was discarded, and cells were collected. Total RNA was extracted from RAW264.7 cells using an RNA separator and total RNA extraction reagent. The mRNA transcription levels of IL-6, IL-1β, TNF-α, and SOD1 in LPS-induced macrophages after Lily treatment were detected by real-time quantitative polymerase chain reaction (qPCR).

[0051] Experimental results: such as Figure 2 As shown in Figure B, the accumulation of the metabolite NO in the culture supernatant was measured using Griess' reagent to evaluate the effect of Lily on NO production in RAW 264.7 cells. Compared with the control group, LPS (p < 0.0001) significantly promoted NO production in macrophages. Compared with the control group and the LPS-treated group, Lily pretreatment inhibited NO production in RAW 264.7 cells, showing a clear concentration-dependent effect among different concentrations. Figure 2 As shown in CF, the mRNA expression of all genes related to inflammatory cytokines and oxidation-related enzymes was affected by Lily. Figure 2 As shown in Figure C, the expression level of IL-6 after LPS stimulation was significantly higher than that in the control group (p < 0.0001). Treatment with different concentrations of Lily effectively downregulated IL-6 expression in a significant concentration-dependent manner. Furthermore, Lily significantly inhibited the expression of IL-1β in LPS-treated macrophages within 24 hours. Figure 2 D), TNF-α Figure 2 E) and SOD1 ( Figure 2 The production of F). These results indicate that Lily can significantly reduce the expression of IL-6, IL-1β, TNF-α and SOD1 genes, thereby exerting an anti-inflammatory effect.

[0052] 2. Effect of Lily on LPS-induced intracellular ROS production in RAW 264.7 cells

[0053] Macrophages were seeded in 6-well plates. They were then incubated with Lily (0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL) for 24 h at 37°C for 1 h. Subsequently, they were treated with or without LPS (1 μg / mL) for 24 h and further incubated with DCFH-DA (10 μM) for 30 min. After incubation, cells were collected, and the mean DCFH-DA was determined by flow cytometry.

[0054] Experimental results: such as Figure 2 As shown in G, LPS treatment for 24 h significantly promoted ROS production. Conversely, Lily pretreatment significantly reduced LPS-induced reactive oxygen species (ROS) production in RAW 264.7 cells.

[0055] Example 6: Effect of Lily Flower Extract 1 on Improving Acute Lung Injury in Mice

[0056] Mice were randomly divided into four groups: a blank control group, a model group, a dexamethasone group (1 mg / kg), an LPS + Lily-L group (0.9 g / kg), an LPS + Lily-M group (1.8 g / kg), and an LPS + Lily-H group (3.6 g / kg), with six mice in each group. Control group mice were pretreated with saline orally for 13 days, while treatment group mice were given specified doses of Lily and Dex. On day 14, Lily and Dex were administered orally 1 hour before LPS treatment. Mice were then anesthetized with tribromoethanol (ip, 400 mg / kg). Model group mice received an intratracheal infusion of 10 µL / 10 g LPS (5 mg / kg), while normal control group mice received an intratracheal infusion of 10 µL / 10 g sterile saline. Mice were sacrificed 24 hours after LPS exposure, and samples were collected for subsequent experiments.

[0057] Experimental results: During the drug administration period, there was no significant difference in the body weight of mice. Figure 3 A); Compared with the control group, LPS-induced model group mice showed hemorrhage and congestion in lung tissue ( Figure 3 D) and pulmonary edema ( Figure 3 B); however, Lily, within a certain dose range (0.9, 1.8, and 3.6 mg / kg), and Dex (1 mg / kg) improved the degree of lung injury and edema under LPS exposure. Pathological lung tissue sections of model mice showed alveolar structure collapse, significant loss of lung tissue structure, widespread infiltration of inflammatory cells (neutrophils and monocytes), and thickening of alveolar walls (B). Figure 3 C). Lung injury score calculations indicated that Lily had a protective effect against lung lesions. Further evaluation of the effect of Lily on LPS-induced lung inflammation in ALI mice was conducted. Figure 4As shown in (AE), Lily can reduce the mRNA expression levels of lung tissue-related inflammatory factors and oxidases. Figure 5 (AF) indicates that Lily can inhibit the levels of T-SOD, CAT, T-AOC, MPO, MDA, IL-6, IL-1β and TNF-α in serum.

[0058] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for preparing lily flower extract, characterized in that, Includes the following steps: Take dried lily flowers, soak them in water, reflux to extract, filter, concentrate the filtrate, and dry to obtain the lily flower extract.

2. The method for preparing lily flower extract according to claim 1, characterized in that, The method of soaking in water includes: adding 5-20 times the amount of water as the medicinal material of lily flower and soaking for 20-40 minutes.

3. The method for preparing lily flower extract according to claim 1, characterized in that, The reflux extraction method includes: heating and reflux extraction 1-3 times, each time for 0.5-4 hours. After each extraction, filter, add 5-20 times the amount of water to the residue and continue reflux extraction, and finally combine the filtrates.

4. The method for preparing lily flower extract according to claim 1, characterized in that, The filtrate is concentrated to a crude drug concentration of 0.5-2.0 g / mL.

5. A lily flower extract, characterized in that, The lily flower extract is prepared by the preparation method according to any one of claims 1-4.

6. A composition, characterized in that, The composition comprises the lily flower extract of claim 5, and pharmaceutically acceptable excipients or food additives.

7. The use of the lily flower extract of claim 5, or the composition of claim 6, in the preparation of an anti-inflammatory product.

8. The application according to claim 7, characterized in that, The product can effectively reduce the production of nitric oxide, lower the gene expression levels of cellular inflammatory factors and oxidation-related enzymes, and reduce the generation of ROS.

9. The application according to claim 7, characterized in that, The product can suppress lung inflammation and improve acute lung injury.

10. The application according to claim 7, characterized in that, The dosage form of the product includes any one of the following: powder, granule, pill, granule, microcapsule, fluid extract, decoction, capsule, tablet, oral liquid, syrup, mouthwash, spray, aerosol, and nasal rinse. The product can be any one of the following: medicine, functional beverage, functional food, or health product.

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