Preparation method of aristolochic acid lactam components from houttuynia cordata

Abstract

This invention belongs to the field of traditional Chinese medicine pharmaceutical technology and relates to a method for preparing aristolochic acid lactam components from Houttuynia cordata. This invention establishes a method for rapidly enriching aristolochic acid lactam components from Houttuynia cordata using high-speed countercurrent chromatography. It enriches three lactam components—aristolochic acid lactam A II, aristolochic acid lactam FI, and aristolochic acid lactam B II—from Houttuynia cordata. The enrichment process of aristolochic acid lactam components in this invention is simple, rapid, and yields high amounts, making it suitable for industrial-scale preparation. It can be used for large-scale enrichment of aristolochic acid lactam compound reference standards in traditional Chinese medicine. Furthermore, it provides essential reference standards for the safety evaluation and quality control of Houttuynia cordata, a traditional Chinese medicine for clearing heat and detoxifying.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine pharmaceuticals and relates to a method for preparing aristolochic acid lactam components from Houttuynia cordata, and particularly to a method for enriching aristolochic acid lactam A II, aristolochic acid lactam FI, and aristolochic acid lactam B II from Houttuynia cordata. Background Technology

[0002] The existing technology discloses that Houttuynia cordata is the fresh whole herb or dried aerial parts of Houttuynia cordata, belonging to the Saururaceae family. It is mainly produced in central, southeastern, and southwestern provinces of my country. It has the effects of clearing heat and detoxifying, reducing swelling and draining pus, and promoting diuresis. In southwestern my country, it is commonly used as a vegetable or condiment. It is among the first batch of medicinal and edible herbs approved by the National Health Commission of China. Modern pharmacological studies have found that Houttuynia cordata possesses antibacterial, anti-inflammatory, antiviral, and anti-complement activities. It is widely used to treat enteritis, dysentery, nephritis edema, mastitis, and otitis media, suggesting that Houttuynia cordata has broad utilization value.

[0003] Studies have revealed that aristolochic acid compounds are widely found in plants of the Aristolochiaceae family and have been shown to have nephrotoxic, carcinogenic, and mutagenic effects. Aristolochic acid-producing compounds, the main metabolites of aristolochic acid compounds, are not only found in Aristolochiaceae plants but also in Houttuynia cordata. Previous studies have confirmed that aristolochic acid lactam I is more nephrotoxic than aristolochic acid I! Our preliminary research also found that several aristolochic acid lactam compounds in Houttuynia cordata have structures similar to aristolochic acid lactam I and also exhibit significant cytotoxicity to HK-2 renal tubular epithelial cells, suggesting that the preparation of control standards for these compounds will be of great significance for the quality control and safe and rational application of Houttuynia cordata.

[0004] Research and practice have shown that the content of aristolochic acid lactam compounds in Houttuynia cordata is very low, and the preparation of reference standards using traditional column chromatography is costly and yields low results. Therefore, the development of a method for the rapid enrichment of aristolochic acid lactam compounds from Houttuynia cordata has attracted the attention of those skilled in the art.

[0005] Based on the current state of the technology, the inventors of this application intend to provide a method for rapidly enriching aristolochic acid lactam components from houttuynia cordata. This application can help provide necessary research samples and reference standards for in-depth toxicological research, safety evaluation and quality control of aristolochic acid lactam components in traditional Chinese medicine. Summary of the Invention

[0006] The purpose of this invention is to provide a preparation process for aristolochic acid lactam components in Houttuynia cordata based on the current state of the technology, and particularly to a method for enriching aristolochic acid lactam A II, aristolochic acid lactam FI, and aristolochic acid lactam B II from Houttuynia cordata.

[0007] The structures of the three aristolochic acid lactam components described in this invention are shown in the following formulas:

[0008]

[0009] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0010] A method for preparing aristolochic acid A II, aristolochic acid FI, and aristolochic acid BII from houttuynia cordata includes the following steps:

[0011] (1) Extract Houttuynia cordata several times with organic solvents such as methanol or ethanol, combine the extracts, concentrate and recover the solvent, and evaporate to dryness to obtain an extract. The extraction method can be flash extraction, ultrasonic extraction or percolation extraction.

[0012] (2) The extract obtained in step (1) is suspended in water and extracted four times with petroleum ether, dichloromethane or ethyl acetate. The extracts are combined, the solvent is concentrated and the extract is evaporated to dryness to obtain the extract.

[0013] (3) Separate the extract obtained in step (2) by normal phase silica gel column chromatography, using 5-7 column volumes of dichloromethane-methanol (120:1, 50:1, 10:1, 1:1) or dichloromethane-methanol (100:1, 50:1, 20:1, 10:1) gradient elution, and collect the eluent fraction of dichloromethane-methanol (50:1, 10:1) or dichloromethane-methanol (50:1, 20:1) according to the liquid chromatography-mass spectrometry (LCMS) analysis results to obtain the crude extract of aristolochic acid lactam components.

[0014] The crude extract obtained in step (3) was enriched by high-speed countercurrent chromatography. A hexane-ethyl acetate-methanol-water (1:2:1.55:1.45) or hexane-ethyl acetate-methanol-water (1:2:1.5:1.5) system was selected, with the upper phase (hexane-ethyl acetate) as the stationary phase and the lower phase (methanol-water) as the mobile phase. The mixture was partitioned at a flow rate of 2.0 mL / min and a main unit rotation speed of 850 r / min. The detection wavelength was 280 nm. The peak positions of the aristolochic acid lactam components were determined according to the characteristic LCMS spectrum. The eluent was collected, the solvent was concentrated and recovered, and the eluent was evaporated to dryness to obtain the aristolochic acid lactam component enrichment.

[0015] (4) The enriched product obtained in step (4) was further purified by high performance liquid chromatography. A C18 column (250×20mm, 5μm) was used, the column temperature was 25℃, and acetonitrile-water (50:50) or (53:47) was used for elution. The flow rate was 10mL / min. The main chromatographic peaks were collected to obtain aristolochic acid A II, aristolochic acid FI and aristolochic acid B II.

[0016] This invention establishes a method for rapidly enriching aristolochic acid lactam components from Houttuynia cordata using high-speed countercurrent chromatography. The method enriches three lactam components—aristolochic acid lactam A II, aristolochic acid lactam FI, and aristolochic acid lactam B II—from Houttuynia cordata, providing essential reference standards for the safety evaluation and quality control of this heat-clearing and detoxifying traditional Chinese medicine.

[0017] The present invention has the following beneficial effects:

[0018] The enrichment process for aristolochic acid lactam compounds provided by this invention is simple, rapid, and yields a high rate, making it suitable for industrial preparation and applicable to the large-scale enrichment of aristolochic acid lactam compound reference standards in traditional Chinese medicine. Attached Figure Description

[0019] Figure 1 The structures of aristolochic acid A II (AL AII), aristolochic acid FI (AL FI), and aristolochic acid B II (AL BII) are given.

[0020] Figure 2 The enrichment process flow diagrams are for Examples 1, 6, and 8.

[0021] Figure 3 The image shows the multi-stage mass spectra of aristolochic acid A II, aristolochic acid FI, and aristolochic acid B II in Example 5.

[0022] Figure 4 The proposed cleavage pathways for aristolochic acid A II, aristolochic acid FI, and aristolochic acid B II in Example 5.

[0023] Figure 5 The liquid chromatograms are of samples HH-PE1 to HH-PE11 in Example 6.

[0024] Figure 6 This is the high-speed countercurrent chromatogram of sample HH-PE-H from Example 8. Detailed Implementation

[0025] Example 1: Extraction and extraction of Houttuynia cordata

[0026] Take 5 kg of the lower part of Houttuynia cordata, crush it with a pulverizer, add 100 L of methanol, and perform flash extraction for 5 min at 100 V for one time. Combine the filtrates, concentrate and evaporate to dryness to obtain 1279.62 g of extract. Disperse the extract in 4 L of water and extract with petroleum ether four times to obtain 152.38 g of petroleum ether extract (e.g., ...). Figure 1 (As shown).

[0027] Example 2: Extraction and extraction of Houttuynia cordata

[0028] Take 19 kg of Houttuynia cordata, crush it with a pulverizer, soak it in 95% ethanol overnight, and then continuously percolate for 96 hours. Add 95% ethanol at a material-to-liquid ratio of 1:10 daily. Combine the filtrates, concentrate and evaporate to dryness. Extract the extract four times with petroleum ether to obtain 273.82 g of petroleum ether extract.

[0029] Example 3: Extraction and extraction of Houttuynia cordata

[0030] Take 5.05g of Houttuynia cordata, pulverize it with a grinder, add 100mL of methanol, sonicate for 30min for 30min, repeat 3 times, combine the filtrates, concentrate and evaporate to dryness to obtain 1.96g of methanol extract; disperse the extract in 10mL of water, extract with dichloromethane 4 times to obtain 0.12g of dichloromethane extract.

[0031] Example 4: Extraction and extraction of Houttuynia cordata

[0032] Take 5.03g of Houttuynia cordata, pulverize it with a grinder, add 100mL of methanol, sonicate for 30min for 30min, repeat 3 times, combine the filtrates, concentrate and evaporate to dryness to obtain 1.63g of methanol extract; disperse the extract in 10mL of water, extract with ethyl acetate 4 times to obtain 0.12g of ethyl acetate extract.

[0033] Example 5: UPLC-MS of characteristic peaks of the target analyte n Identification

[0034] Chromatographic separation was performed using a YMC-Triart C18 column (150 × 2.1 mm, 1.9 nm). The column temperature was 30℃, the flow rate was 0.3 mL / min, and elution was performed using a 0.1% formic acid-water-acetonitrile gradient (0–2 min, 10% acetonitrile; 2–12 min, 10–40% acetonitrile; 12–32 min, 40–50% acetonitrile; 32–45 min, 50%–100% acetonitrile; 45–55 min, 100% acetonitrile; 55–60 min, 100–10% acetonitrile; 60–65 min, 10% acetonitrile). The injection volume was 5 μL, and the wavelength was 277 nm. Primary mass spectrometry (MS) was performed using electrospray ionization (ESI) in positive ion mode with full scan detection. Secondary and tertiary mass spectrometry (MS) were performed using data-dependent scans. The mass range was m / z 100.0–1000.0. Capillary temperature 350℃, capillary voltage 35V, spray voltage 3.5KV, sheath flow rate 40psi, auxiliary flow rate 10psi.

[0035] Aristolochic acid A II, aristolochic acid FI, and aristolochic acid B II reference solutions were injected for detection, and primary, secondary, and tertiary mass spectra were obtained (e.g., ...). Figure 2(As shown). In positive ion mode, aristolochic acid A II, aristolochic acid FI, and aristolochic acid B II all form [M+H]. + Ions. Aristolochic acid A II and aristolochic acid FI are isomers; in secondary mass spectrometry, both lose CH3 to form m / z 251 [M+H-CH3]. + Ions, observed at low abundance in tertiary mass spectrometry at m / z 223 [M+H-CH3-CO] + The ion with higher abundance at m / z 195 is presumably [M+H-CH3-CO-HCN-H]. + Ions. Secondary mass spectrometry of aristolochic acid B II also first removes CH3 to form m / z 265 [M+H-CH3]. + The ion, with a tertiary mass spectrometry value of m / z 264 [M+H-CH3-H], was determined. + The ion suggests that the substituent on the left side forms a closed-ring structure of methylenedioxy (e.g., Figure 3 As shown). Based on the retention time (t) R The information on aristolochic acid and mass spectrometry fragments can be used to identify aristolochic acid A II, aristolochic acid FI, and aristolochic acid B II.

[0036] Table 1 Mass Spectrometry Fragmentation Information of Aristolochic Acid Components in Houttuynia cordata

[0037]

[0038] Example 6: Normal-phase silica gel column chromatography separation of Houttuynia cordata extract

[0039] The petroleum ether extract from Example 1 was mixed with 2 kg of silica gel (200-300 mesh). Using dichloromethane-methanol as the elution system, eluted for 3 column volumes at a 120:1 ratio, 1 column volume at a 50:1 ratio, 1 column volume at a 10:1 ratio, and then 1 column volume at a 1:1 ratio. The eluted fractions were spotted onto a thin-layer chromatography (TLC) plate and preliminarily combined into 11 fractions (HH-PE1 to HH-PE11). Based on UPLC-MS... n Feature mapping tracks target components (such as...) Figure 4 As shown), HH-PE6 to HH-PE10 were collected and combined to obtain 26.14 g of HH-PE-H fraction, which was mainly eluted by dichloromethane-methanol (50:1, 10:1).

[0040] Example 7: Normal-phase silica gel column chromatography separation of Houttuynia cordata extract

[0041] The petroleum ether extract from Example 2 was mixed with 3.5 kg of silica gel (200-300 mesh). Using dichloromethane-methanol as the elution system, elution was performed at a ratio of 100:1 for 3 column volumes, 50:1 for 2 column volumes, 20:1 for 1 column volume, and 10:1 for another 1 column volume. Gradient elution was performed using a normal-phase silica gel column. The eluted fractions were initially combined into 8 fractions (HH-PE1 to HH-PE8) by thin-layer chromatography (TLC). The fractions were then analyzed according to UPLC-MS. n The target components were traced by the characteristic chromatogram. HH-PE5 to HH-PE7 were collected and combined to obtain 57.78 g of HH-PE-H fraction, which was mainly eluted by dichloromethane-methanol (50:1, 20:1).

[0042] Example 8: High-speed countercurrent chromatography enrichment and semi-preparative high-performance liquid chromatography purification

[0043] The HH-PE-H fraction from Example 6 was suspended in a small amount of the upper phase (n-hexane-ethyl acetate) and eluted by high-speed countercurrent chromatography. The elution system was n-hexane-ethyl acetate-methanol-water (1:2:1.5:1.5), with the upper phase as the stationary phase and the lower phase as the mobile phase. The flow rate was 2.0 mL / min, the rotation speed was 850 r / min, and the temperature was 25 °C. The sample loading was approximately 1000 mg each time. Data was collected using an N2000 online chromatography workstation. Based on the UV chromatographic peaks, 10 fractions HH-PE-H1 to HH-PE-H10 were collected and obtained (e.g., ...). Figure 5 As shown), based on the analysis results of UPLC-MS / MS (detection conditions are the same as in Example 5) (as shown), Figure 6 As shown), fractions HH-PE-H7 and HH-PE-H9 containing aristolochic acid lactams were collected respectively, and the solvent was recovered under reduced pressure to obtain 259.07 mg of enriched fraction HH-PE-H7 and 218.91 mg of enriched fraction HH-PE-H9 respectively.

[0044] The HH-PE-H7 fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with isocratic elution in acetonitrile-water (53:47) at a flow rate of 10 mL / min, a detection wavelength of 280 nm, and a column temperature of room temperature. Fractions were collected at 11.1–11.3 min and 12.3–12.7 min, and the solvent was recovered under reduced pressure to obtain aristolochic acid A II (5.35 mg) and aristolochic acid FI (7.08 mg). The HH-PE-H9 fraction was purified by reversed-phase HPLC with isocratic elution in acetonitrile-water (50:50) at a flow rate of 10 mL / min, a detection wavelength of 280 nm, and a column temperature of room temperature. Fractions were collected at 19.4–20.1 min, and the solvent was recovered under reduced pressure to obtain aristolochic acid B II (34.76 mg).

[0045] Example 9: High-speed countercurrent chromatography enrichment and semi-preparative high-performance liquid chromatography purification

[0046] The HH-PE-H fraction from Example 7 was suspended in a small amount of the upper phase (n-hexane-ethyl acetate) and eluted by high-speed countercurrent chromatography. The elution system was n-hexane-ethyl acetate-methanol-water (1:2:1.55:1.45), with the upper phase as the stationary phase and the lower phase as the mobile phase. The flow rate was 2.0 mL / min, the rotation speed was 850 r / min, the temperature was 25 °C, and the sample loading was approximately 1000 mg each time. Data were collected using an N2000 online chromatography workstation. Based on the UV chromatographic peaks, 12 fractions HH-PE-H0 to HH-PE-H11 were collected. According to the UPLC-MS / MS analysis results (detection conditions were the same as in Example 5), fractions HH-PE-H6 and HH-PE-H9 containing aristolochic acid components were collected. The solvent was recovered under reduced pressure, and the enriched fractions HH-PE-H6 (449.93 mg) and HH-PE-H9 (519.84 mg) were obtained, respectively.

[0047] The HH-PE-H6 fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with isocratic elution in acetonitrile-water (53:47) at a flow rate of 10 mL / min, a detection wavelength of 280 nm, and a column temperature of room temperature. Fractions were collected at 11.0–11.4 min and 12.2–12.7 min, and the solvent was recovered under reduced pressure to obtain aristolochic acid A II (18.59 mg) and aristolochic acid FI (24.59 mg). The HH-PE-H9 fraction was purified by reversed-phase HPLC with isocratic elution in acetonitrile-water (50:50) at a flow rate of 10 mL / min, a detection wavelength of 280 nm, and a column temperature of room temperature. Fractions were collected at 19.2–19.8 min, and the solvent was recovered under reduced pressure to obtain aristolochic acid B II (165.06 mg).

[0048] The present invention has described the above embodiments. It will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing aristolochic lactam components in Houttuynia cordata, characterized in that, It comprises steps of: (1) mixing Houttuynia cordata with organic solvent in proportion, extracting several times, combining the extract, recovering the solvent, and obtaining extract; the organic solvent is selected from methanol or ethanol; (2) suspending the extract obtained in step (1) with water, extracting several times with organic solvent, combining the extract, recovering the solvent, and obtaining extract; the organic solvent is selected from petroleum ether, dichloromethane, or ethyl acetate; (3) subjecting the extract obtained in step (2) to normal phase silica gel column chromatography, eluting with dichloromethane-methanol mixed solvent, analyzing each tube eluate by liquid chromatography-mass spectrometry, collecting the eluate containing aristolochic acid components, recovering the solvent, and obtaining crude aristolochic acid components; the elution conditions of the normal phase silica gel column chromatography are: gradient elution of dichloromethane-methanol in the proportion of 120:1, 50:1, 10:1, 1:1 or gradient elution of dichloromethane-methanol in the proportion of 100:1, 50:1, 20:1, 10:1, collecting the elution part of dichloromethane-methanol 50:1, 10:1 or dichloromethane-methanol 50:1, 20:1; (4) subjecting the crude extract obtained in step (3) to high-speed counter-current chromatography separation, collecting the eluate containing aristolochic acid components according to the liquid chromatography-mass spectrometry analysis result of the eluate, and obtaining aristolochic acid component concentrate; the separation conditions of the high-speed counter-current chromatography are: using 1:2:1.55:1.45 or 1:2:1.5:1.5 mixed n-hexane-ethyl acetate-methanol-water system, wherein n-hexane-ethyl acetate is the stationary phase and methanol-water is the mobile phase; (5) further separating the concentrate obtained in step (4) by high performance liquid chromatography, collecting the chromatographic peak containing aristolochic acid components with acetonitrile-water mixed solvent as the mobile phase, recovering the solvent, and respectively obtaining aristolochic acid A II, aristolochic acid F I, and aristolochic acid B II.

2. The method of claim 1, wherein The extraction method of step (1) is selected from flash extraction, ultrasonic extraction, or percolation extraction.

3. The method of claim 1, wherein In the separation of high-speed counter-current chromatography of step (4), the chromatographic peak containing aristolochic acid components is determined according to the ultraviolet spectrum or mass spectrum of the effluent, and the corresponding effluent is collected and combined.

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