Application of biflavone compounds isochamaejasmine and genkwanol A in preparation of anticomplement drugs

Abstract

The invention belongs to the field of traditional Chinese medicine preparation, and relates to application of biflavone compounds isochamaejasmine and genkwanol A in preparation of anti-complement drugs. According to the present invention, the biflavone compounds such as the isochamaejasme and the genkwanol A are extracted from the ethyl acetate extraction part of the ethanol extract of the dried root of Stellera tianshanica plant Stellera tianshanica Pod. The in-vitro anti-complement activity evaluation experiment results prove that the biflavone compounds such as the isochamaejasme and the genkwanol A have strong inhibition effects on the classical pathway and the alternative pathway of the complement system, wherein the stellera tianshanica and the genkwanol A are extracted from the ethyl acetate extraction part of the ethanol extract of the dried root of Stellera tianshanica Pod. The biflavone compound isochamaejasmine and genkwanol A can be further used for preparing medicines for treating complement related diseases, and the complement related diseases comprise systemic lupus erythematosus, rheumatoid arthritis or acute respiratory distress syndrome.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine pharmaceuticals, and relates to isoluxin, a biflavonoid compound from *Pseudoeus tianshanensis*, and genkwa alcohol A, and their novel uses in the preparation of anticomplement drugs. Background Technology

[0002] Existing technologies reveal that overactivation of the complement system can trigger various serious diseases, including systemic lupus erythematosus, rheumatoid arthritis, and acute respiratory distress syndrome. Research on anticomplement drugs has been a hot topic and focus of global pharmaceutical research for many years. However, there is currently a lack of ideal treatments for these diseases, thus creating an urgent clinical need for novel, highly effective, low-toxicity, and specific complement inhibitors. The research and development of complement inhibitors from natural products has become an important research area that has received increasing attention in recent years due to its low cost and low toxicity. Domestic and international scholars have isolated a large number of monomeric compounds with complement system inhibitory effects from various natural products, including marine organisms, providing a broad prospect for the research and development of anticomplement drugs.

[0003] Tianshan fake wolfsbane ( Pedicularis verticilata *L.* (L.) belongs to the genus *L.* of the family Thymelaeaceae. This genus has a rich and long history of medicinal use, with records of its medicinal value appearing in ancient Chinese medical texts such as the *Shennong Bencao Jing* and *Bencao Gangmu*. *L.* (L.) is used medicinally for its root, which is neutral in nature, bitter and pungent in taste, and relatively toxic. It has diuretic, expectorant, antiseptic, and insecticidal effects. It is often used to treat symptoms such as qi and blood deficiency, fatigue and excessive sweating, exhaustion, and low blood pressure. Recent studies on the chemical composition and bioactivity of *L.* have revealed that its main chemical components include flavonoids, terpenes, coumarins, lignans, phenols, and volatile oils. Among these, biflavonoids and daphne diterpenoids are characteristic compounds of this genus, exhibiting various physiological activities such as antitumor, antioxidant, anti-HIV, anti-inflammatory, and insecticidal effects.

[0004] To date, there have been no reports of the biflavonoid compounds isoflavone and genistein A, which have complement-inhibiting effects, being found in this product. Summary of the Invention

[0005] The purpose of this invention is to provide new substances with anticomplement activity, specifically relating to isoflavone toxin and genkwa alcohol A, biflavonoid compounds isolated from *Pseudoesphala tianshanensis*.

[0006] A further objective of this invention is to provide the use of the biflavonoid compounds in the above-mentioned *Euphorbia tirucalli* in the preparation of anticomplement drugs.

[0007] This invention applies modern pharmacological screening methods to evaluate the anticomplement activity of isolated monomeric compounds, specifically from *Pseudoestus tianshanensis* (Tianshan pseudowolfsbane). Pedicularis verticilataTwo biflavonoids were isolated from the ethyl acetate extract of the dried root of L. and their activity on the classical complement pathway was confirmed.

[0008] The anticomplement-active biflavonoid compounds isolufenicol and genistein A of the present invention have the following structural formula:

[0009] The isolufenicol and genkwa alcohol A described in this invention are two biflavonoid compounds, and are prepared by the following method: The dried roots of *Pseudoestus tianshanensis* were pulverized and soaked three times in 95% ethanol at room temperature. The extracts were combined and concentrated until no alcohol odor remained. The extract was diluted with water and extracted three times sequentially with equal volumes of petroleum ether (60-90℃), ethyl acetate, and n-butanol to obtain petroleum ether, ethyl acetate, and n-butanol fractions. Activity testing of the three fractions showed that the ethyl acetate fraction had the strongest activity. The ethyl acetate fraction was then eluted sequentially with petroleum ether and ethyl acetate in gradient ratios of 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 9:1, 7:1, 5:1, 3:1, 2:1, and 1:1. After TLC analysis, the fractions were combined into seven groups. The resulting fractions were subjected to repeated silica gel column chromatography, Sephadex LH-20 chromatography, and preparative chromatography. Two biflavonoid compounds, isoygosin and genistein A, were isolated.

[0010] In this invention, isoygosin is a dark purple powder with the molecular formula C. 30 H 22 O 10 It is readily soluble in methanol, and under TLC ultraviolet light (254nm), it shows dark spots. When sprayed with a 10% concentrated sulfuric acid ethanol solution and heated, it turns yellow. 1 H-NMR (400 MHz, Methanol- d 4) Data: δ 7.14 (d, J = 8.6 Hz, 2H, H-2′″, 6′″), 6.83 (dd, J = 8.6, 1.6 Hz, 2H,H-2′, 6′), 6.72 (d, J = 8.0 Hz, 2H, H-3′″, 5′″), 6.62 (dd, J = 8.6, 1.5 Hz,2H, H-2′, 6′), 6.00 (d, J = 1.8 Hz, 1H, H-8″), 5.90 (d, J= 1.8 Hz, 1H, H-8), 5.82 (d, J = 1.8 Hz, 1H, H-6″), 5.58 (d, J = 4.8 Hz, 1H, H-2″), 3.48 (m, 1H,H-3), 3.25 (m, 1H, , H-3″); 13 C-NMR (100 MHz, Acetone- d 6) Data: δ 48.73 (C-3,.3″), 49.19 (C-3), 82.87 (C-2, 2″), 95.48 (C-6, 6″), 96.71 (C-8), 103.01 (C-10), 115.30 (C-3, 3′), 115.67 (C-3′″, 5′″), 128.57 (C-1′, 1′″), 129.72 (C-2′,6′), 129.72 (C-2′″, 6′″), 155.88 (C-4′″), 158.20 (C-4′), 164.26 (C-5, 5″),165.41 (C-9, 9″), 166.57 (C-7, 7″), 199.26 (C-4, 4″). Gentianol A: Pale yellow amorphous powder, molecular formula: C 30 H 22 O 10 HR-ESI-MS m / z : 543.12854 [M+H] +1 It is readily soluble in methanol, and under TLC ultraviolet light (254nm), it shows dark spots. When sprayed with a 10% concentrated sulfuric acid ethanol solution and heated, it turns yellow. 1 H-NMR (400 MHz, Methanol- d 4) Data: δ 7.09 (d, J = 8.5 Hz, 2H, H-10ʹʹ, H-14ʹʹ), 6.87 (d, J = 8.5 Hz, 2H, H-11ʹʹ, H-13ʹʹ), 6.70 (d, J = 8.6 Hz, 2H, H-2ʹ, H-6ʹ), 6.58 (d, J = 8.6 Hz, 2H,H-3ʹ, H-5ʹ)), 6.10 (s, 1H, H-6), 5.64 (d, J= 2.0 Hz, 1H, H-6ʹʹ), 5.62 (d, J = 2.0 Hz, 1H, H-8ʹʹ), 3.75 (td, J = 8.3, 5.3 Hz, 1H, H-3), 4.58 (d, J = 7.9Hz, 1H, H-2), 2.87 (dd, J = 16.1, 5.3 Hz, 1H, H-4), 2.50 (dd, J = 16.1, 8.6Hz, 1H, H-4); 13 C-NMR (100 MHz, Methanol- d 4) Data: δ 29.10 (C-4), 68.87 (C-3), 82.22 (C-2), 90.64 (C-6), 90.74 (C-8″), 92.58 (C-2″), 96.45 (C-3″), 96.45 (C-6″), 102.66 (C-4a), 103.85 (C-4″a), 104.71 (C-8), 115.64 (C-11″, 13″), 115.75(C-3′, 5′), 125.55 (C-9″), 128.57 (C-2′, 6′), 129.54 (C-10″, 14″), 130.68 (C-1′), 152.99 (C-8a), 157.73 (C-4′), 158.80 (C-5″), 159.09 (C-12″), 160.77 (C-7), 163.15 (C-5), 170.84 (C-7″), 174.16 (C-8″a), 197.51 (C-4″). The biflavonoids described above in this invention were tested for their anticomplement activity via the classical pathway in vitro. The results showed that these biflavonoids inhibited both the classical and alternative pathways of the complement system (as shown in Table 1). Table 1. Inhibitory effects of the compounds on the classical and alternative pathways of the complement system (Mean ± SD, n = 3) compound Compound Name <![CDATA[CH 50 (mg / ml)]]> <![CDATA[AP 50 (mg / ml)]]> 1 Wolf venom 0.04±0.04 0.07±0.03 2 Gentianol A 0.09±0.02 0.15±0.04 Positive control Heparin sodium 0.03±0.02 0.06±0.03 Among them, CH 50 This refers to the concentration of the test sample required to inhibit hemolysis by 50% via the classic route; AP 50 This refers to the concentration of the test sample required to inhibit hemolysis by 50% via the alternative pathway. The biflavonoid compounds isoyrus extract and genkwa alcohol A of this invention can be used to prepare anticomplement drugs.

[0011] The biflavonoid compounds isolus and genistein A of the present invention can be further used to prepare drugs for treating complement-related diseases, including systemic lupus erythematosus, rheumatoid arthritis, or acute respiratory distress syndrome. Attached Figure Description

[0012] Figure 1 Flowchart of extraction and separation of isolufenicol and genkwa alcohol A from the ethyl acetate extract of *Pseudoeus tianshanensis*. Detailed Implementation Example 1: Preparation of biflavonoid compounds Approximately 10.5 kg of dried roots of *Euphorbia tirucalli* were obtained. The roots were dried, pulverized, and extracted three times with 95% ethanol at room temperature, each extraction lasting 7 days. The solvent was recovered under reduced pressure, yielding a total extract of 2127.0 g. The extract was suspended in 4000 mL of distilled water at 50 °C and extracted three times consecutively with 5 L each of petroleum ether, ethyl acetate, and n-butanol. The extracts were then evaporated to dryness, yielding 192.5 g of the petroleum ether fraction, 853.8 g of the ethyl acetate fraction, and 335.5 g of the n-butanol fraction. The ethyl acetate fraction was mixed 1:1 with 100-200 mesh silica gel, dried, and ground into a uniform fine powder. It was then packed into a column using a wet method and eluted sequentially with a petroleum ether-ethyl acetate gradient. The elution ratios were 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 9:1, 7:1, 5:1, 3:1, 2:1, and 1:1, respectively. After TLC analysis, the fractions were combined into seven groups. Fraction 4 (21.8 g) was subjected to repeated column chromatography with petroleum ether-acetone (1:1), and then preparative chromatography was used to obtain the compound isuroxin (6.0 mg). Fraction 6 (168.4 g) was eluted with a normal-phase silica gel column using a gradient of dichloromethane:methanol (8:1) to obtain fraction 6f (70.5 mg), which was then eluted multiple times with an ODS column and Sephadex LH-20 to obtain compound genistein A (32.5 mg).

[0013] Example 2: In vitro anticomplement classical pathway test Take 0.1 ml of complement (guinea pig serum) and add barbiturate buffer (BBS) to prepare a 1:5 solution. Dilute this solution 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640 with BBS. Dissolve 0.1 ml each of 1:1000 hemolysin, each concentration of complement, and 2% sheep red blood cells (SRBC) in 0.3 ml of BBS. Mix well, incubate at 37 ℃ for 30 min, and then centrifuge at 5000 rpm and 4 ℃ for 10 min. Transfer 0.2 ml of the supernatant from each tube to a 96-well plate and measure the absorbance at 405 nm. A complete hemolysis group (0.1 ml of 2% SRBC dissolved in 0.5 ml of barbiturate buffer) was also included in the experiment. (ml triple-distilled water), using the absorbance of the blood vessel dissolved by triple-distilled water as the standard for complete hemolysis, calculate the hemolysis rate, plot the complement dilution as the X-axis and the percentage of hemolysis caused by complement at each dilution concentration as the Y-axis, select the lowest complement concentration that achieves a similar high hemolysis rate as the critical complement concentration required to ensure normal hemolysis of the system, take the critical concentration of complement and mix it with the test sample, pre-incubate in a water bath at 37 ℃ for 10 min, and then add an appropriate amount of BBS, hemolysin and 2% SRBC. After incubating each tube in a 37 ℃ water bath for 30 min, place it in a low-temperature high-speed centrifuge and centrifuge at 5000 rpm and 4 ℃ for 10 min. Then, collect 0.2 ml of the supernatant from each tube and plate it in a 96-well plate. Measure the absorbance at 405 nm. Simultaneously, a test sample control group, a complement group, and a complete hemolysis group were set up. The hemolysis rate was calculated by subtracting the corresponding absorbance value of the test sample control group from the absorbance value of the test sample. A graph was plotted with the test sample concentration as the X-axis and the hemolysis inhibition rate as the Y-axis to calculate the concentration CH of the test sample required to inhibit hemolysis by 50%. 50 The results of the in vitro anticomplement classical pathway assay are shown in Table 1.

[0014] Example 3: In vitro anticomplement bypass pathway test Take 0.2 ml of complement (human serum) and add it to AP dilution buffer (barbital buffer, pH 7.4, containing 5 mg / mL). 2+8mM MEGTA) was prepared into a 1:5 solution, and then diluted 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640 solutions. 0.15 ml of complement, 0.15 ml of AP diluent, and 0.20 ml of 0.5% rabbit erythrocytes (RE) at each concentration were mixed, incubated at 37°C for 30 min, and then centrifuged at 5000 rpm and 4°C for 10 min. 0.2 ml of the supernatant from each tube was transferred to a 96-well plate, and the absorbance was measured at 405 nm. A complete hemolysis group was also included (0.20 ml of 0.5% RE dissolved in 0.3mM MEGTA). Using triple-distilled water (ml), the absorbance of the blood vessel dissolved by triple-distilled water was used as the standard for complete hemolysis. The hemolysis rate was calculated, and a graph was plotted with complement dilution as the X-axis and the percentage of hemolysis caused by complement at each dilution concentration as the Y-axis. The lowest complement concentration that achieved a similar high hemolysis rate was selected as the critical complement concentration required to ensure normal hemolysis of the system. The determined critical concentration of complement was mixed with the test sample, pre-incubated at 37°C for 10 min, and then 0.2 ml of 0.5% RE was added. After incubating each tube at 37°C for 30 min, it was placed in a low-temperature high-speed centrifuge at 5000 rpm and 4°C for 10 min. 0.2 ml of the supernatant from each tube was then transferred to a 96-well plate and centrifuged at 405 μm. The absorbance was measured at nm. The experiment included a test sample control group, a complement group, and a complete hemolysis group. The hemolysis rate was calculated by subtracting the corresponding absorbance value of the control group from the test sample absorbance value. A graph was plotted with the test sample concentration as the X-axis and the hemolysis inhibition rate as the Y-axis to calculate the required concentration of the test sample (AP) for 50% inhibition of hemolysis. 50 The results of the in vitro anticomplement bypass pathway test are shown in Table 1.

[0015] Table 1 shows the inhibitory effects of the compounds isorhynchonine and genkwa alcohol A of the present invention on the classical and alternative pathways of the complement system. (Mean ± SD, n=3).

[0016] The reagents used in the experiments of this invention are all known in the art and are commercially available.

Claims

1. The use of the biflavonoid compounds isolus and genkwa alcohol A, having the following structural formulas, in the preparation of anticomplement drugs, wherein the anticomplement drugs are used for the treatment of systemic lupus erythematosus, rheumatoid arthritis, or acute respiratory distress syndrome. The aforementioned biflavonoid compounds, isoygosin and genistein A, are characterized in that... Prepared by the following method: The dried roots of *Pseudoestus tianshanensis* were crushed and soaked three times at room temperature with 95% ethanol. The extracts were combined and concentrated until no alcohol odor remained. The extract was diluted with water and extracted three times sequentially with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain three fractions: petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction was then eluted sequentially with petroleum ether and ethyl acetate in the following ratios: 100:1, 80:1, 50:1, 30:1, 20:1, 15:1, 9:1, 7:1, 5:1, 3:1, 2:1, and 1:

1. After TLC analysis, the fractions were combined into seven fractions. The resulting fractions were then separated by repeated silica gel column chromatography, Sephadex LH-20 chromatography, and preparative chromatography to obtain two biflavonoid compounds, isoygosin and genistein A.

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