Use of extracts of gemmiferous groundsel and active ingredients of chlorophyll in the treatment of organ fibrosis

Abstract

The application belongs to the technical field of medicine, and particularly relates to a use of an extract of Lysimachia christinae Hance and active ingredients chlorophyll in resisting organ fibrosis diseases. The application first discovers that chlorophyll (such as chlorophyll a and chlorophyll b), degradation intermediates (such as 7<1>-hydroxymethyl phaeophytin A, phaeophytin A, 3-hydroxyethyl phaeophytin B, 7<1>-hydroxymethyl phaeophytin A, 7<1>-hydroxymethyl-8<2>-hydroxyethyl phaeophytin A and phaeophytin A) and extracts (such as Lysimachia christinae Hance alcohol extract) containing the chlorophyll or degradation intermediates have good therapeutic effects on organ fibrosis diseases. They can inhibit TGF-beta signals and then inhibit fibrosis genes (fibronectin, alpha-SMA and Col1a1) expression, so as to realize the prevention and treatment of organ fibrosis diseases, and have high application potential in the development of drugs for preventing and treating organ fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of Lysimachia christinae extract and its active ingredient chlorophyll in the treatment of organ fibrosis. Background Technology

[0002] Organ fibrosis is a common pathological process in the progression of various chronic diseases to organ dysfunction. Characterized by excessive extracellular matrix deposition and tissue remodeling, it can occur in almost all organ systems, including the liver, lungs, kidneys, and heart. The main harm of fibrosis lies in its progressive and irreversible nature. Continuous fibrosis damages normal organ structure, leading to a reduction in parenchymal cells and impaired blood supply, ultimately resulting in organ failure. Epidemiological data show that organ fibrosis has a high prevalence in the general population. Taking the kidney as an example, renal fibrosis is a core pathological feature of all types of chronic kidney disease (CKD) and is closely associated with the progression and deterioration of CKD. Given the central role of fibrosis in organ damage and failure, developing effective drug treatment strategies targeting its pathogenesis is of significant clinical and public health value for delaying disease progression and reducing mortality.

[0003] Fibrosis is a common pathological process in the progression of diseases in multiple organs, involving the synergistic regulation of multiple signaling pathways. Among them, TGF-β signaling is recognized as a core pathway promoting fibrotic lesions in various organs, including the kidneys. Activation of TGF-β signaling begins with the sequential binding of extracellular TGF-β ligands (TGF-β1 / 2 / 3) to membrane receptors TGFBR2 and TGFBR1, which then phosphorylates and activates intracellular transcription factors Smad2 and Smad3. Activated Smad2 / 3 binds to Smad4 to form a complex, which translocates into the nucleus and regulates the expression of fibrosis-related target genes. Preclinical studies have shown that targeting TGF-β signaling can significantly improve fibrotic lesions in various organ tissues. Therefore, TGF-β signaling is an important target for the development of drugs against organ fibrosis.

[0004] Chlorophyll is the core pigment for photosynthesis in green plants and most algae, and it is also the main natural pigment source in commonly consumed vegetables. More than 50 types of chlorophyll molecules exist in nature, with chlorophyll a and chlorophyll b being the most abundant. The content of chlorophyll a in green plants is about three times that of chlorophyll b. Chemically, both chlorophyll a and b consist of a porphyrin ring chelating magnesium ions and a hydrophobic phytol side chain. The difference lies in the substituent on the 7th carbon atom (C7) of the porphyrin ring: chlorophyll a has a methyl group (-CH3), while chlorophyll b has an aldehyde group (-CHO). Due to the presence of the porphyrin ring, both chlorophyll a and b exhibit characteristic blue band (Soret band, 400–450 nm) and red band (Q band, 500–750 nm) absorption spectra. Under conditions such as light, heat, or leaf senescence, chlorophyll can degrade to generate various derivatives. Common degradation intermediates include pheophytin and phytol derivatives. Besides photosynthesis, chlorophyll has been reported to possess various beneficial health activities, including anti-tumor, detoxification, antioxidant, wound healing promotion, and antibacterial effects. However, to date, there have been no reports of chlorophyll inhibiting TGF-β signaling or its anti-organ fibrosis effects. Summary of the Invention

[0005] Addressing the problems of existing technologies, this invention, based on research into the anti-fibrotic effects of the traditional Chinese medicine *Lysimachia christinae*, discovered that its ethanol extract possesses significant TGF-β signaling inhibitory activity and anti-fibrotic efficacy. Component analysis identified its anti-fibrotic active components as chlorophyll (a and b) degradation intermediates. Further analysis using commercially available and spinach-derived chlorophyll revealed that chlorophyll a and b also exhibit significant TGF-β signaling inhibitory activity and anti-renal fibrosis efficacy. Therefore, this invention provides novel pharmaceutical uses for the ethanol extract of *Lysimachia christinae* and its derived active component chlorophyll (including its degradation intermediates), as well as its preparation method. Based on the common knowledge that chlorophyll in the plant kingdom shares a common chemical structure, this invention also indirectly demonstrates the anti-fibrotic pharmaceutical uses of chlorophyll from all possible plant pigments.

[0006] Use of chlorophyll or its degradation intermediates in the preparation of medicaments for the prevention and / or treatment of organ fibrosis.

[0007] The present invention also provides the use of extracts of traditional Chinese medicine containing chlorophyll or its degradation intermediates for the preparation of medicaments for the prevention and / or treatment of organ fibrosis.

[0008] Preferably, the chlorophyll is extracted from any chlorophyll-containing species, and the species is selected from at least one of algae, plants, cyanobacteria or protists; And / or, the chlorophyll is selected from at least one of chlorophyll a or chlorophyll b; And / or, the degradation intermediate is selected from at least one of the following compounds: 7¹-hydroxymethyl pheophytin A, pheophytin A, 3-hydroxyethyl pheophytin B, 7¹-hydroxymethyl pheophytin A, 7¹-hydroxymethyl-8²-hydroxyethyl pheophytin A, and pheophytin A.

[0009] Preferably, the organ fibrosis disease is selected from at least one of the following diseases: renal fibrosis, liver fibrosis, cardiac fibrosis, or pulmonary fibrosis.

[0010] Preferably, the drug inhibits TGF-β signaling activity, thereby inhibiting the expression of fibrosis genes, to prevent and / or treat organ fibrosis diseases.

[0011] Preferably, the herbal extract is an alcoholic extract of Lysimachia christinae.

[0012] Preferably, the preparation method of the Lysimachia christinae alcohol extract is as follows: Lysimachia christinae is percolated with 8-12 times the amount of 80-100% v / v ethanol aqueous solution, dried and concentrated to obtain the extract.

[0013] The present invention also provides a Lysimachia christinae extract, wherein the Lysimachia christinae extract is prepared by percolation extraction with 8-12 times the amount of 80-100% v / v ethanol aqueous solution, followed by drying and concentration.

[0014] Preferably, the method includes the following steps: percolating extracting Lysimachia christinae with 8-12 times the amount of 80-100% v / v ethanol aqueous solution, drying and concentrating to obtain the product.

[0015] The present invention also provides a pharmaceutical composition, which is a formulation made by adding pharmaceutically acceptable excipients to the above-mentioned Lysimachia christinae extract as the active ingredient.

[0016] This invention is the first to discover that chlorophyll and its degradation intermediates can inhibit TGF-β signaling (inhibiting Smad2 / 3 protein levels), thereby inhibiting the expression of fibrosis genes (fibronectin and Col1a1), thus achieving preventive and therapeutic effects against organ fibrosis. Specifically, this invention first experimentally discovered that the ethanol extract (95% ethanol) of the traditional Chinese medicine Lysimachia christinae has significant TGF-β signaling inhibitory activity. Furthermore, using the kidney as an example, it was found that the ethanol extract of Lysimachia christinae can significantly improve adenine-induced renal fibrosis in mice and protect damaged kidney function. Bioactivity-guided drug component analysis revealed that chlorophyll and its degradation intermediates are the key active components of Lysimachia christinae ethanol extract in inhibiting TGF-β signaling. To further demonstrate the anti-fibrotic drug activity of chlorophyll, this invention used high-purity (HPLC purity > 97%) chlorophyll isolated from vegetables (spinach) (a and b) to demonstrate that chlorophyll alone can also significantly improve adenine-induced renal fibrosis and damaged kidney function. The relevant case data of this invention demonstrate that the natural plant pigment chlorophyll has pharmaceutical uses in inhibiting TGF-β signaling and improving organ fibrosis, indicating that chlorophyll and its degradation intermediates have the potential to be used as therapeutic drugs for fibrotic diseases.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 Experimental results on the inhibition of TGF-β signaling and fibrosis gene expression by Lysimachia christinae extract in TCMK1 cells. A. CCK8 assay to test the effect of different concentrations of Lys treatment for 24 hours on TCMK1 cell viability. B. TCMK1 cells were treated with different concentrations of Lys and 5 ng / mL TGF-β1 for 24 hours. Western blot analysis of Smad2 / 3 and p-Smad2 / 3 expression (B), and RT-PCR and western blot analysis of fibrosis gene fibronectin and Col1a1 mRNA and protein expression (CE). p<0.001 vs. blank control group. # p<0.05, ## p<0.005, and ### p<0.001 vs. TGF-β1 group.

[0020] Figure 2 Experimental results showing the improvement of renal fibrosis in Ade mice by ethanol extract of Lysimachia christinae. A. Schematic diagram of animal experiment. B. Mouse body weight at the experimental endpoint. C and D. Blood urea nitrogen and creatinine. E. Appearance of kidney tissue. Note: In the Ade model, the kidneys showed weaker hemoglobin and stronger surface granularity, with improvement observed in all treatment groups. F and G. Sirius Red and HE staining. HK. RT-PCR, immunohistochemistry, and western blot analysis of fibrosis genes. fibronectin, Col1a1, α-SMA mRNA and protein expression. p<0.01 and p<0.001 Vs. Normal mice, # p<0.05, ## p<0.01, ### p<0.001 Vs. Ade. Scale bar for the top row of images in Figures E and F: 1 mm; scale bar for the bottom row of images in Figure F and the icons G and K: 50 μm.

[0021] Figure 3 Experimental results showing that chlorophyll is the main active component of Lys against TGF-β signaling. A. Schematic diagram of the bioactivity-guided Lys active component analysis strategy. B. Appearance of Lys extracts in solutions of different polarities and their inhibitory activity on Smad3 expression in TCMK1 cells. Extraction component numbers: 1 petroleum ether extract, 2 chloroform, 3 ethyl acetate, 4 n-butanol, 5 residual aqueous phase, 6 residual insoluble precipitate. C. Multi-wavelength three-dimensional absorption peak spectra of petroleum ether and chloroform extracts after HPLC separation, with dashed arrows indicating characteristic spectral absorption near 400 and 600 nm. D. Chlorophyll derivative components identified in petroleum ether and chloroform extracts by combining optical absorption spectroscopy and mass spectrometry. The upper figure shows the HPLC-MS optical absorption spectrum of the extracts (400-650 nm), and the lower figure shows the identified chlorophyll components, mainly demagnesiated and dephytolized derivatives. EF.TCMK1 cells were treated with different concentrations of chlorophyll a / b and TGF-β1 for 24 h, and the expression of Smad2 / 3, fibronectin and Col1a1 was analyzed by Western blot.

[0022] Figure 4 The following are the experimental results of spinach chlorophyll isolation and purification. A. Spinach chlorophyll isolation and purification process and the color appearance of the separated chlorophyll a / b (in methanol solution). B. Two-dimensional HPLC absorption spectra of spinach pigment mixture extracted with DCM and purified chlorophyll a / b. The component adjacent to the main absorption peak of chlorophyll a / b is the epimer of chlorophyll a / b, and Pheo-a (pheophytin a) is the absorption peak of pheophytin a. C. One-dimensional absorption spectrum of purified chlorophyll a / b. D. Mass spectrometry detection results of purified chlorophyll a / b.

[0023] Figure 5Experimental results showing that spinach-derived chlorophyll significantly improved renal fibrosis in Ade mice. A. Animal experimental flowchart. B. Animal weight change at the experimental endpoint. C. D. Serum urea nitrogen and creatinine. E. Kidney appearance. F. Sirius Red staining. G. HE staining. H. L. RT-PCR, immunohistochemistry, and western blot analysis of renal fibrosis genes. fibronectin, Col1a1, α-SMA mRNA and protein expression levels. p<0.001 Vs. normal mice, # p<0.05, ## p<0.01, ### p<0.001 Vs. Ade group. Scale bar for the top row of images in Figures E and F: 1 mm; scale bar for the bottom row of images in Figure F and the icons G and K: 50 μm. Detailed Implementation

[0024] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0025] Example 1: Chlorophyll or its degradation intermediates for the treatment of organ fibrosis. This embodiment provides chlorophyll or its degradation intermediate as an active ingredient for the treatment of organ fibrosis. The chlorophyll is selected from at least one of chlorophyll a or chlorophyll b. The degradation intermediate is selected from chlorophyll modified with pheophytin, phytol, and porphyrin ring side chains, specifically from at least one of the following compounds: 7¹-hydroxymethyl pheophytin A, pheophytin A, 3-hydroxyethyl pheophytin B, 7¹-hydroxymethyl pheophynic acid A, 7¹-hydroxymethyl-8²-hydroxyethyl pheophynic acid A, and pheophynic acid A.

[0026] The organ fibrosis disease is selected from at least one of the following diseases: renal fibrosis, liver fibrosis, cardiac fibrosis, or pulmonary fibrosis. Chlorophyll or its degradation intermediates inhibit TGF-β signaling (inhibiting Smad2 / 3 protein levels), thereby inhibiting the expression of fibrosis genes (fibronectin, α-SMA, and Col1a1), thus achieving the preventive and therapeutic effects on organ fibrosis diseases.

[0027] Example 2: The use of Lysimachia christinae alcohol extract for the treatment of organ fibrosis. This embodiment provides Lysimachia christinae alcohol extract as an active ingredient for the treatment of organ fibrosis. The preparation method of Lysimachia christinae alcohol extract (Lys) is as follows: 1 kg of dried Lysimachia christinae plant slices are mechanically ground into powder, and extracted by percolation with 10 L of 95% v / v ethanol. The extract is then subjected to rotary evaporation in a negative pressure water bath to remove the alcohol and excess water, yielding an oily paste.

[0028] In other embodiments, the amount and concentration of raw materials used in the extraction method can be adapted. For example, when the amount of ethanol aqueous solution is 8 times or 12 times that of Lysimachia christinae, or when the concentration of ethanol aqueous solution is 80% v / v or 100% v / v (anhydrous ethanol), the extracted extract has similar biological activity to the Lysimachia christinae ethanol extract obtained in Example 2.

[0029] The technical solution of the present invention will be further illustrated by the following experiments.

[0030] In Experiment 1, the ethanol extract of the traditional Chinese medicine Lysimachia christinae significantly inhibited TGF-β signaling and suppressed the expression of fibrosis genes in cultured cells. I. Experimental Methods 1. Preparation of Lysine indicum extract (Lys) for pharmaceutical purposes: The ethanol extract of Lysimachia christinae (Lys) was prepared according to the method described in Example 2. For cell experiments, Lys was diluted to 200 mg / mL with DMSO.

[0031] 2. Cell viability test: TCMK1 cells were seeded in 96-well plates and grown to 90% confluence. Different concentrations of Lys (80 μg / mL, 160 μg / mL, 320 μg / mL, and 640 μg / mL) were added for 24 hours. Then, the medium was replaced with serum-free medium containing 10% CCK8 reagent, and the cells were incubated for 1 hour. Absorbance was measured at 450 nm. Cell viability was converted to relative survival rate compared to the control group.

[0032] 3. Western blot test: TCMK1 cells were seeded in 6-well plates and grown to 80% confluence. Simultaneously, different concentrations of Lys (80 μg / mL, 160 μg / mL, 320 μg / mL, 640 μg / mL) and 5 ng / L TGF-β1 were added for 24 hours. Cells were collected, and proteins were extracted using RIPA lysis buffer. After determining the concentration, SDS-PAGE gel electrophoresis was performed at a loading rate of 20 μg. Cells were transferred to PVDF membranes, blocked with 2.5% bovine serum albumin (BSA) for 1 hour, and incubated overnight at 4°C with primary antibody. The next day, cells were washed with TBST, incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour, and then subjected to a chemiluminescence reaction using ECL substrate. The signal was detected using a chemiluminescence imaging system.

[0033] 4. RT-PCR test: TCMK1 cells were treated as described in the Western blot assay above. Cells were collected, and total RNA was extracted using the Trizol method. After reverse transcription to synthesize cDNA, quantitative amplification was performed using SuperStar Universal SYBR Master Mix (Kangwei Century, Cat: CW3360H). The relative gene expression level was measured using a 2-1T / T ratio. -△△Ct The calculation was performed using the following method: The primer sequences used are as follows: fibronectin-F 5'-ATGTGGACCCCTCCTGATAGT-3' (SEQ ID NO. 1), fibronectin-R 5'-GCCCAGTGATTTCAGCAAAGG-3' (SEQ ID NO. 2); Col1a1-F: 5'-ATCCAACGAGATCGAGCTCA-3' (SEQ ID NO. 3), Col1a1-R 5'-AAGGGAGCCACATCGATGAT-3' (SEQ ID NO. 4); beta-actin-F 5'-AGAGGGAAATCGTGCGTGAC-3' (SEQ ID NO. 5), beta-actin 5'-CAATAGTGATGACCTGGCCGT-3' (SEQ ID NO. 6).

[0034] II. Experimental Results Treatment of renal tubular epithelial TCMK1 cells with different concentrations of Lys styrax ethanol extract (Lys) for 24 hours showed that cell viability was not affected when the Lys concentration was not higher than 320 μg / mL. Figure 1 A). Treatment of cells with 320, 160, and 80 μg / mL Lys dose-dependently inhibited the expression of Smad2 / 3 protein and simultaneously inhibited TGF-β1-induced Smad2 / 3 phosphorylation. Figure 1 B). Furthermore, Lys intervention can also dose-dependently inhibit the mRNA and protein expression of TGF-β1-induced fibrosis genes fibronectin and Col1a1 (B). Figure 1 CE).

[0035] Example 2: Lysimachia christinae alcohol extract improves adenine-induced renal fibrosis in mice. I. Experimental Methods 1. Preparation of Lysine indicum extract (Lys) for pharmaceutical purposes: The ethanol extract of Lysimachia christinae (Lys) was prepared according to the method described in Example 2. For in vivo experiments in mice, 1 kg of Lysimachia christinae ethanol extract (oily paste) was resuspended in corn oil to 333 mL. In subsequent steps, mice were administered doses of 10, 5, and 2.5 μL / g body weight by gavage, equivalent to doses of 30, 15, and 7.5 g (Lysimachia christinae slices) / kg body weight.

[0036] 2. Adenine (Ade)-induced CKD renal fibrosis mouse model and Lys intervention: Eight-week-old male C57BL / 6 mice were fed a diet containing 0.2% adenine for three weeks to induce renal fibrosis. Simultaneously, mice in the Lys intervention group were administered equal volumes of Lysimachia christinae extract (30, 15, and 7.5 g / kg / day) via gavage. Positive control mice were administered quercetin (Que) at 50 mg / kg / day via gavage, while normal and model mice were administered an equal volume of solvent (corn oil) via gavage. Mouse body weight was recorded at the start and end of the experiment, and blood and kidney tissue samples were collected for analysis of renal function and fibrosis-related indicators.

[0037] 3. Blood biochemical indicators: The serum creatinine and blood urea nitrogen levels of mice were analyzed using a fully automated biochemical analyzer.

[0038] 4. Pathological histological staining: After paraffin embedding and sectioning, mouse kidney tissue was stained with hematoxylin and eosin (HE) using an HE staining kit to assess structural changes in the kidney tissue, and fibrosis in the kidney tissue was assessed using a Sirius Red staining kit.

[0039] 5. Immunohistochemistry: After routine dewaxing and rehydration, kidney paraffin sections were boiled in 10 mM citric acid (pH 6.0) for 10 minutes for antigen retrieval, incubated with 3% H2O2 for 10 minutes to quench endogenous catalase, blocked with 2.5% BSA at room temperature for 1 hour, and incubated with primary antibody at 4°C overnight. The next day, after thorough washing, the primary antibody was identified using an HRP-conjugated anti-mouse / rabbit polymer universal kit (Zhongshan Jinqiao, Cat# PV-6000), and immunohistochemical signals were developed using DAB (3,3'-diaminobenzidine). After hematoxylin counterstaining of cell nuclei, sections were mounted with resin and observed and photographed under a white light microscope.

[0040] The remaining western blot and RT-PCR experimental procedures are as described in Experiment Example 1.

[0041] II. Experimental Results In a mouse model of adenine (Ade)-induced renal fibrosis, patients were treated with gavage of Lys sphagnum extract (30, 15, and 7.5 g / kg / day) for 3 weeks. Figure 2 A) found that Lys could improve the damaged body weight of Ade mice, reduce blood urea nitrogen and creatinine, and restore the hemochromic appearance of the kidneys. Figure 2 BE), Sirius red and HE staining showed that Lys intervention reduced the area of ​​collagen staining in renal tissue and improved renal structural damage. Figure 2 F and G), RT-PCR, immunohistochemistry and western blot analysis showed that Lys can inhibit fibrosis-related genes (F and G), fibronectin, Col1a1, α-SMA mRNA and protein expression of () Figure 2 HL). The positive control drug, quercetin (Que, 50 mg / kg / day), had a similar effect. These results indicate that Lys has anti-renal fibrosis activity.

[0042] Experimental Example 3 revealed that chlorophyll is the key active ingredient in Lysimachia christinae extract that inhibits TGF-β signaling. I. Experimental Methods 1. Polar extraction of Lysimachia christinae alcohol extract: The ethanol extract of Lysimachia christinae (Lys) was prepared according to the method described in Example 2. 10 g of the ethanol extract (paste-like) from Lysimachia christinae slices was taken and resuspended in 40 mL of ultrapure water to form a suspension. Equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol were added sequentially for extraction. The extracted components were evaporated to dryness under negative pressure in a water bath, weighed, and dissolved in DMSO for subsequent cell experiments.

[0043] 2. Drug activity evaluation: TCMK1 cells were seeded in 6-well plates and grown to 90% confluence. They were then treated with the maximum non-toxic dose of each extract or chlorophyll a and chlorophyll b for 24 hours. Western blot was used to detect Smad3 protein expression.

[0044] 3. Component identification by ultra-high performance liquid chromatography-super-resolution mass spectrometry (UHPLC-URMS): Petroleum ether and chloroform extracts were redissolved in methanol, filtered through a 0.22 μm filter, and analyzed by UHPLC-URMS under the following conditions: Liquid chromatography was performed using an Ultimate 3000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA), equipped with a C8 column (1.7 μm, 2.1 mm × 100 mm, Waters). 5 μL of each extracted sample was injected for analysis, and the column temperature was set to 35 °C. The mobile phase consisted of water (phase A) and methanol (phase B), with a flow rate of 200 μL / min. The gradient elution program was set as follows: 0–5 min, 50%–100% B; 5–60 min, 100% B. After liquid chromatography analysis, qualitative mass spectrometry was performed using a high-resolution Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, USA) coupled with electrospray ionization to liquid chromatography. The analytical conditions were as follows: heating temperature 300℃; capillary temperature 350℃; capillary voltage 35V; spray voltage -3.0 kV in negative ion mode and 3.2 kV in positive ion mode; sheath gas (N2) flow rate 35 arb; auxiliary gas (N2) flow rate 10 arb. Primary mass spectrometry analysis of the samples was performed in both negative and positive ion modes (resolution R: 30000, mass-to-charge ratio scan range: 100–1500 Da), and secondary mass spectrometry was acquired using data-dependent scanning. Xcalibur 3.0 software was used for data acquisition and analysis.

[0045] II. Experimental Results Lys aqueous suspension was sequentially extracted with solvents of different polarities (petroleum ether, chloroform, ethyl acetate, and n-butanol), and the drug activity of different extract fractions was evaluated by the inhibition of Smad3 protein levels in TCMK1 cells. The experiment found that the main active ingredient of Lys was enriched in the petroleum ether and chloroform extract fractions. Figure 3 AB). High-performance liquid chromatography (HPLC) analysis showed that the petroleum ether and chloroform components contained multiple light-absorbing substances in the vicinity of 400 and 600 nm. Figure 3 C), based on UHPLC-URMS material quality and secondary fragmentation patterns, these substances were identified as chlorophyll a / b degradation intermediates (see Table 1). Figure 3 CD).

[0046] Table 1. Identified active ingredients In subsequent experimental studies, we also isolated these chlorophyll degradation intermediates, further verifying their inhibitory effect on organ fibrosis. Furthermore, since these active ingredients are chlorophyll degradation intermediates derived from chlorophyll molecules, this study also used commercially available chlorophyll a / b (MCE, catalog numbers HY-W127744 and HY-W127709) to treat TCMK1 cells. We found that chlorophyll a / b could dose-dependently inhibit Smad2 / 3 protein levels, while simultaneously inhibiting the expression of TGF-β1-induced fibrosis proteins fibronectin and Col1a1. Figure 3 These data indicate that the chlorophyll components in Lysimachia christinae (including chlorophyll and its degradation intermediates) have anti-TGF-β signaling and anti-in vitro cell fibrosis activity.

[0047] Experimental Example 4: Chlorophyll can improve adenine-induced renal fibrosis I. Experimental Methods 1. Isolation and purification of spinach chlorophyll: The separation of spinach chlorophyll was performed using the well-established reverse-phase chromatography method (Petrovic, Sanja et al., (2012). Savremene tehnologije. 1. 16-24, https: / / www.researchgate.net / publication / 258696540), with appropriate methodological adjustments made based on specific circumstances. The specific procedures were as follows: Fresh spinach stems were removed, leaves were washed, freeze-dried, ground into powder, and extracted with dichloromethane at a ratio of 1:10 (m / v) using ultrasonic extraction. The powder was filtered, dried under negative pressure in a 40℃ water bath, and reconstituted with a small amount of methanol. The sample was purified using a medium-low pressure chromatography system (equipped with a preparative C8 column, Santai, Cat# SW-5822-120-SP). The mobile phase consisted of water (A) and methanol (B), with a gradient elution program set as follows: 0-5 min, 50%-100% B; 5-60 min, 100% B. The absorption spectrum and color of the eluent at 400-600 nm were continuously monitored. Fractions exhibiting characteristic chlorophyll absorption peaks at 400-600 nm were collected, evaporated to dryness, and weighed. Chlorophyll A and B exhibit characteristic bright green and blue-green colors, respectively, and also possess characteristic absorption spectra. Chlorophyll A has characteristic absorption peaks at 431 nm and 665 nm, while chlorophyll B has characteristic absorption peaks at 463 nm and 652 nm. This method can separate high-purity chlorophyll a and chlorophyll b (see...). Figure 4 In animal experiments, chlorophyll a and chlorophyll b were collected and administered together.

[0048] 2. Ade-induced renal fibrosis mouse model and chlorophyll intervention: Eight-week-old male C57BL / 6 mice were fed a diet containing 0.2% adenine and simultaneously administered 100 and 200 mg / kg / day of chlorophyll (a mixture of chlorophyll a and chlorophyll b) by gavage. Positive control mice were administered 50 mg / kg / day of quercetin (Que) by gavage, while normal and model control mice were administered a solvent (corn oil) by gavage. Three weeks after modeling and drug treatment, serum and kidney tissue were collected from the animals, and renal function and fibrosis-related indicators were analyzed according to the method described in Experiment Example 2.

[0049] II. Experimental Results To demonstrate whether chlorophyll from non-Moneywort-derived plants possesses the same anti-renal fibrosis effect, this experiment established a method for the large-scale isolation of chlorophyll a / b (Chl-a / b) from spinach, which is rich in chlorophyll. After freeze-drying, grinding, dichloromethane (DCM) extraction, and separation using a preparative C8 column, high-purity chlorophyll a / b was obtained. The isolated chlorophyll a was blue-green, and chlorophyll b was bright green. Figure 4 A). Two-dimensional absorption spectroscopy analysis showed that, compared to the unseparated spinach pigment mixture, column chromatography could achieve complete separation of high-purity chlorophyll a and b components, with HPLC purity > 97% (it should be noted that the peak-side substances of the main components of chlorophyll a and b are epimers of chlorophyll a and b) (see... Figure 4 B). One-dimensional absorption spectral analysis of the main component peaks showed that chlorophyll a had absorption peaks of 431 and 665 nm in the blue and red bands, respectively, with a peak ratio of approximately 1.2; chlorophyll b had absorption peaks of 463 and 652 nm in the blue and red bands, respectively, with a peak ratio of approximately 2.8 (see...). Figure 4 C). Mass spectrometry analysis of secondary fragments further confirmed that the separated substances were chlorophyll a and b, with chlorophyll a ion having a mass spectrometry value of 893.5 [M+H]. + The chlorophyll b ion spectra are 615.2, 555.2, 481.2; the chlorophyll b ion spectra are 692.2, 597.2, 569.2, 524.1 (of which 907.5 [M+H]). + Poor stability and missing) (see) Figure 4 D). These data are consistent with chlorophyll a / b spectral and mass spectrometry data reported in the literature (Photosynth Res 130, 335–346 (2016). PMID: 27113221 and Petrovic, Sanja et al., (2012). Savremene tehnologije. 1. 16–24, https: / / www.researchgate.net / publication / 258696540).

[0050] Ade mice were treated with spinach-derived chlorophyll a / b at doses of 100 and 200 mg / kg / day for 3 weeks, respectively. Figure 5 A), chlorophyll administration significantly alleviated the weight loss phenotype in Ade mice and reduced serum urea nitrogen and creatinine (A). Figure 5 BD). Chlorophyll treatment restored the hemochromatographic appearance of the kidneys in Ade mice. Pathological staining (Sirius red and HE) showed that chlorophyll intervention inhibited Adey-induced collagen deposition in kidney tissue and reduced renal structural disorder. Figure 5 EG). Furthermore, RT-PCR, immunohistochemistry, and western blot experiments confirmed that chlorophyll treatment significantly inhibited the fibrosis gene (EG). fibronectin, Col1a1, α- SMA mRNA and protein expression of () Figure 5 HL). The positive control drug quercetin (Que, 50 mg / kg / day) showed similar antifibrotic efficacy.

[0051] As can be seen from the above examples and experimental cases, chlorophyll (such as chlorophyll a and chlorophyll b), its degradation intermediates (such as 7¹-hydroxymethyl pheophytin A, pheophytin A, 3-hydroxyethyl pheophytin B, 7¹-hydroxymethyl pheophytin A, 7¹-hydroxymethyl-8²-hydroxyethyl pheophytin A, pheophytin A), and extracts containing these chlorophyll or degradation intermediates (such as Lysimachia christinae alcohol extract) have good therapeutic effects on organ fibrosis. Therefore, they have the potential to be developed into drugs for the prevention and treatment of organ fibrosis.

Claims

1. Use of chlorophyll or its degradation intermediates in the preparation of medicaments for the prevention and / or treatment of organ fibrosis.

2. Use of extracts of traditional Chinese medicine containing chlorophyll or its degradation intermediates in the preparation of medicaments for the prevention and / or treatment of organ fibrosis.

3. The use according to claim 1 or 2, characterized in that: The chlorophyll is extracted from any chlorophyll-containing species, which is selected from at least one of algae, plants, cyanobacteria, or protists. And / or, the chlorophyll is selected from at least one of chlorophyll a or chlorophyll b; And / or, the degradation intermediate is selected from at least one of the following compounds: 7¹-hydroxymethyl pheophytin A, pheophytin A, 3-hydroxyethyl pheophytin B, 7¹-hydroxymethyl pheophytin A, 7¹-hydroxymethyl-8²-hydroxyethyl pheophytin A, and pheophytin A.

4. The use according to claim 1 or 2, characterized in that: The organ fibrosis disease is selected from at least one of the following diseases: renal fibrosis, liver fibrosis, cardiac fibrosis, or pulmonary fibrosis.

5. The use according to claim 1 or 2, characterized in that: The drug inhibits TGF-β signaling activity, thereby suppressing the expression of fibrosis genes and achieving the prevention and / or treatment of organ fibrosis diseases.

6. The use according to claim 2, characterized in that: The herbal extract is an alcoholic extract of Lysimachia christinae.

7. The use according to claim 6, characterized in that: The preparation method of the Lysimachia christinae alcohol extract is as follows: Lysimachia christinae is percolated with 8-12 times the amount of 80-100% v / v ethanol aqueous solution, dried and concentrated to obtain the extract.

8. A Lysimachia christinae extract, characterized in that: The preparation method of the Lysimachia christinae extract is as follows: Lysimachia christinae is percolated with 8-12 times the amount of 80-100% v / v ethanol aqueous solution, dried and concentrated to obtain the extract.

9. The method for preparing the Lysimachia christinae extract according to claim 8, characterized in that, The process includes the following steps: extracting Lysimachia christinae by percolation with 8-12 times the volume of 80-100% v / v ethanol aqueous solution, drying and concentrating to obtain the final product.

10. A pharmaceutical composition, characterized in that: It is a formulation made with the Lysimachia christinae extract as the active ingredient as described in claim 8, and pharmaceutically acceptable excipients.

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