Preparation method and application of a pair of phenylpropanoid coumarin compound enantiomers (±)-yakuchinone a

Abstract

The application relates to a preparation method of a pair of phenylpropanoid coumarin compound enantiomers (plus) - yahunuolignan A. Yahunugen is extracted by heating reflux with ethanol, ethanol is recovered under reduced pressure, an equal amount of diatomite is added, the solvent is volatilized, petroleum ether, dichloromethane, ethyl acetate and methanol are sequentially used for elution, recovery is carried out, each part is obtained; the ethyl acetate part is chromatographically separated, gradient elution is carried out, flow fractions are collected, detection analysis is carried out, the flow fractions are combined; purification, elution, collection of the chromatographic peak of the retention time, chiral resolution, collection of the flow fraction of the retention time are carried out, and (plus) - yahunuolignan A prepared by the above method is applied to the preparation of an anti-cervical cancer drug. The raw material is rich, the preparation method is easy to operate, (plus) - yahunuolignan A can be effectively prepared from yahunugen, the compound has anticancer activity, can be effectively used for preparing an anti-cervical cancer drug, a new use of yahunu medicinal materials is developed, and the application has practical application value.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the preparation method and application of the enantiomer (±)-Ahunu balsamin A, a phenylpropanoid coumarin compound. Background Technology

[0002] Cervical cancer (CESC) is characterized by the abnormal proliferation and malignant transformation of cervical cells. Studies have shown that natural active substances can serve as effective alternatives or sensitizers for chemotherapy drugs, demonstrating promising application prospects. Furthermore, traditional Chinese medicine (TCM), due to its wide availability, low cost, low toxicity, and multi-target synergistic effects, has become an important breakthrough point for the development of new anti-tumor drugs. Therefore, discovering anti-cervical cancer lead compounds from TCM and developing novel drugs with high selectivity and few side effects has significant academic value and clinical translational implications.

[0003] Yahunu is a plant belonging to the Menispermaceae family and the Menispermaceae family, specifically the Menispermaceae family. Cissampelos pareira L.var. hirsute The dried whole plant of *Symplocos spp.* possesses anti-inflammatory, analgesic, hemostatic, and tissue-regenerating effects. This invention is the first to discover a pair of enantiomers of phenylpropanoid coumarin compounds from plants of the *Symplocos spp.* Furthermore, these phenylpropanoid coumarin compounds exhibit rich and broad biological activities, such as immunomodulatory, anti-inflammatory, antimalarial, anti-cytotoxic, and hepatoprotective potential. The preparation method and biological activities of the phenylpropanoid coumarin compounds involved in this invention have not been publicly reported to date. Summary of the Invention

[0004] In view of the above situation and to overcome the defects of the prior art, the purpose of this invention is to provide a method for preparing (±)-Ahunu balsamin A, an enantiomer of phenylpropanoid coumarin, and its application. This method can effectively solve the problem of preparing (±)-Ahunu balsamin A from the root of Ahunu medicinal material and realizing its application in the preparation of anti-cervical cancer drugs.

[0005] The technical solution provided by this invention is a method for preparing the enantiomer (±)-Ahhunu balsamin A, a compound isolated from the root of Ahhunu, with the following molecular structural formula:

[0006] The preparation method is as follows: Using 30–50 kg of *Agonus edulis* as raw material, add 120–200 L of 70% ethanol (v / v) and extract three times by reflux at 92–95℃ for 1–1.5 hours each time. Recover the ethanol under reduced pressure to obtain 3.4–5.6 L of a suspension of the ethanol extract. Add an equal amount of diatomaceous earth, evaporate the solvent, and elute sequentially with 10.8–18 L each of petroleum ether, dichloromethane, ethyl acetate, and methanol. Recover the solvent to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, and methanol fraction. Separate the ethyl acetate fraction by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvents at v / v ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50. Each gradient uses 16.8–28.0 L of ethanol. L eluent was used, with a flow rate of 20–25 mL / min. Each fraction was collected in 900–1500 mL increments, resulting in 174 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4…Fr. 9; Component Fr.4 was separated by silica gel column chromatography, and eluted sequentially with a dichloromethane-methanol mixed solvent system at volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, using 450–750 mL of eluent for each gradient at a flow rate of 20–25 mL / min. Each fraction was collected in volumes of 75–125 mL, resulting in 68 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.4–0.7 mL / min, with each 4–7 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-auronin A. The ECD spectrum of this compound showed no significant Cotton effect (200–400 nm). Furthermore, the specific rotation value was zero, indicating that it was a racemic mixture; therefore, chiral separation was performed. [Column: Daicel chiral PAK AD-H (250 mm × 10 mm, 5...] m m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

[0007] The application of (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] prepared by the method of the present invention in the preparation of anti-cervical cancer drugs.

[0008] This invention utilizes abundant raw materials and an easy-to-operate preparation method, which can effectively prepare (±)-cissacoumarinolignan A from the root of *Cissus chinensis*. This compound has anticancer activity and can be effectively used to prepare anti-cervical cancer drugs, opening up new uses for *Cissus chinensis* medicinal materials and having practical application value. Attached Figure Description

[0009] Figure 1 This is a molecular structural diagram of the present invention; Figure 2 This is the (±)-Ahunu balsamin A HR-ESI-MS image of the present invention; Figure 3 This invention (±)-Ahunu balsamin A 1 1H NMR spectrum (500MHz); Figure 4This invention (±)-Ahunu balsamin A 13 C NMR spectrum (125MHz); Figure 5 This invention (±)-Ahunu balsamin A 1 H- 1 HCOSY spectrum; Figure 6 The HSQC spectrum of (±)-Ahunu balsin A of this invention; Figure 7 The HMBC spectrum of (±)-Ahunu balsamin A of this invention; Figure 8 The NOESY spectrum of (±)-Ahunu balsamin A of this invention; Figure 9 The infrared spectrum of (±)-Ahunu balsamin A of the present invention; Figure 10 This is the UV spectrum of (±)-Ahunu balsamin A of the present invention; Figure 11 This is the ECD measurement diagram of (+)-Ahunu balsamin A of the present invention; Figure 12 This is the ECD measurement diagram of (–)-Ahunu balsamin A of the present invention; Figure 13 This is a resolution diagram of (±)-Ahunu balsamin A of the present invention. Detailed Implementation

[0010] The specific embodiments of the present invention will be described in detail below with reference to examples. Example

[0011] This invention discloses a method for preparing the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of a phenylpropanoid coumarin compound, which is isolated from cissacoumarinolignan root and has the following molecular structure:

[0012] The preparation method is as follows: 50 kg of dried Arundinaria root was used as raw material. 200 L of 70% ethanol was added, and the mixture was extracted three times under reflux at 95℃ for 1 hour each time. The ethanol was recovered under reduced pressure, yielding a 5.6 L suspension of the ethanol extract. 5.6 kg of diatomaceous earth was added, and the solvent was evaporated. The mixture was then eluted sequentially with 18 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 9 g of petroleum ether fraction, 588 g of dichloromethane fraction, 382 g of ethyl acetate fraction, and 1607 g of methanol fraction. The ethyl acetate fraction was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50. Each gradient was eluted with 28.0 L of ethanol. L eluent was used, and the flow rate was 25 mL / min. Each 1500 mL volume was considered one fraction, and 174 fractions were collected. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 750 mL of eluent for each gradient. The flow rate was 25 mL / min, and each 125 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.7 mL / min, with each 7 mL fraction collected. 73 fractions were collected, and each fraction was analyzed by silica gel TLC using GF… 254Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicelchiral PAK AD-H (250...]. mm × 10 mm, 5 m m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R =41.592 min fraction, yielding (–)-Ahunu balsamin A. Example

[0013] The preparation method of the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of a pair of phenylpropanoid coumarin compounds is as follows: 30 kg of dried cissacoumarino root is used as raw material, and 120 L of 70% ethanol is added for extraction by heating and reflux three times at a temperature of 92℃ for 1.5 hours each time. The ethanol is recovered under reduced pressure to obtain 3.4 L of ethanol extract suspension. 3.4 kg of diatomaceous earth is added, the solvent is evaporated, and the solution is eluted successively with 10.8 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvent is recovered to obtain the petroleum ether eluent fraction 5. 4g of the extract was extracted with 352g of dichloromethane, 229g of ethyl acetate, and 964g of methanol. The ethyl acetate extract was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50. Each gradient used 16.8 L of eluent at a flow rate of 20 mL / min. Each 900 mL fraction was collected, resulting in 174 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 450 mL of eluent for each gradient. The flow rate was 20 mL / min, and each 75 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.4 mL / min, with each 4 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAK AD-H (250...]. mm × 10 mm, 5 m m); mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R= 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A. Example

[0014] The preparation method of the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of a pair of phenylpropanoid coumarin compounds is as follows: 40 kg of dried cissacoumarinolignan root was used as raw material. 160 L of 70% ethanol was added, and the mixture was extracted three times under reflux at 94℃ for 1.2 hours each time. The ethanol was recovered under reduced pressure, yielding a suspension of 4.5 L of ethanol extract. 4.5 kg of diatomaceous earth was added, and the solvent was evaporated. The mixture was then eluted sequentially with 14.4 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 7.2 g of the petroleum ether eluent and 7.2 g of the dichloromethane eluent. The chloromethane eluent was 470.4 g, the ethyl acetate eluent was 305.6 g, and the methanol eluent was 1285.6 g. The ethyl acetate extract was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50. Each gradient used 22.4 L of eluent at a flow rate of 23 mL / min. One fraction was collected in 1200 mL volumes, resulting in 174 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 600 mL of eluent for each gradient. The flow rate was 23 mL / min, and each 100 mL volume was considered one fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.6 mL / min, with each 6 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAK AD-H (250...]. mm × 10 mm, 5 m m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R =21.255 min fraction, yielding (+)-Ahunucurate A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A. Example

[0015] The preparation method of the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of a pair of phenylpropanoid coumarin compounds is as follows: 35 kg of dried cissacoumarinolignan root was used as raw material, and 140 L of 70% ethanol was added. The mixture was heated and refluxed three times at a temperature of 93℃ for 1.3 hours each time. The ethanol was recovered under reduced pressure, yielding a suspension of 3.9 L of ethanol extract. 3.9 kg of diatomaceous earth was added, and the solvent was evaporated. The mixture was then eluted successively with 12.6 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvent was recovered, yielding 6.3 g of the petroleum ether eluent and 6.3 g of the dichloromethane eluent. The chloromethane eluent was 411.6 g, the ethyl acetate eluent was 267.4 g, and the methanol eluent was 1124.9 g. The ethyl acetate extract was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50. Each gradient used 19.6 L of eluent at a flow rate of 22 mL / min. Each 1050 mL volume was considered one fraction, and 174 fractions were collected. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 525 mL of eluent for each gradient. The flow rate was 22 mL / min, and each 88 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.5 mL / min, with 73 fractions collected in 5 mL increments. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAK AD-H (250...]. mm × 10 mm, 5 m m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

[0016] Application of (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] prepared by the above method in the preparation of anti-cervical cancer drugs.

[0017] According to the methods given in the above embodiments, any amount of compound can be prepared as needed. The given embodiments are only used to illustrate specific implementation methods of the present invention, and are not intended to limit the scope of protection of the present invention. Any technical solutions that are essentially the same as those made by using equivalent substitutions or transformations are within the scope of protection of the present invention.

[0018] The method of this invention is stable, reliable, and easy to operate. The obtained product (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] has anticancer activity and can be effectively used to prepare anti-cervical cancer drugs. Relevant information is as follows: I. Structural Identification of Compounds The compounds prepared by the method described in Examples 1-4 of this invention were subjected to UV, IR, and nuclear magnetic resonance spectroscopy. 1 H-NMR, 13 Identified by spectroscopic techniques such as C-NMR, DEPT, HSQC, HMBC, H-HCOSY, NOESY, and high-resolution mass spectrometry (HR-ESI-MS), all of them are (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] with the same structure. See the relevant spectra. Figure 2-13 As shown, the specific identification results are as follows: Yellowish-brown amorphous powder, readily soluble in methanol. HR-ESI-MS yielded quasi-molecular ion peaks. m / z 403.101 6 [M+H] + (Calculated value is 403.102 4), the inferred molecular formula is C. 20 H 18 O9, with an unsaturation degree of 12. IR spectral data of this compound show the presence of hydroxyl groups (3410 cm⁻¹). -1 ), carbonyl (1717 cm) -1 ), benzene ring (1625 cm) -1 (e.g., groups); UV spectroscopy shows characteristic absorption peaks of coumarin at 268 nm and 324 nm.) 1 1H NMR (500 MHz, CD3OD) yielded characteristic hydrogen proton signals for H-3 and H-4 on the coumarin nucleus. d H 6.19 (1H,d, J = 9.6 Hz, H-3), 8.15 (1H, d, J = 9.6 Hz, H-4); Aromatic proton signals of a set of ABX coupling systems d H 7.06 (1H, d, J = 2.0 Hz, H-2′), 6.87 (1H, d, J = 8.1 Hz, H-5′), 6.96 (1H, dd, J = 8.1, 2.0 Hz, H-6′); two hydroxymethyl signals d H 5.10 (1H, d,J = 8.2 Hz, H-7′), 4.10 (1H, m, H-8′); a single hydroxymethylene signal d H 3.85(1H, m, H-9′), 3.52 (1H, dd, J = 12.6, 3.9 Hz, H-9′) and two methoxy signals d H 3.88(3H, s, 3′-OCH3), 3.81 (3H, s, 6-OCH3). 13 C NMR (125 MHz, CD3OD) spectrum revealed a coumarin skeleton [1 carbonyl carbon]. d C 163.3 (C-2), two olefinic carbon signals d C 141.5 (C-4), 111.5 (C-3), one benzene ring d C 143.5 (C-7), 141.6 (C-9), 140.8 (C-5), 133.4 (C-6), 126.5 (C-8), 104.3 (C-10)]; a 1,3,4-trisubstituted phenyl[ d C 149.3 (C-3¢), 148.7 (C-4¢), 128.7 (C-1¢), 121.8 (C-6¢), 116.4 (C-5¢), 112.2 (C-2¢)]; one hydroxymethylene group d C 61.8 (C-9¢). From the methoxy hydrogen signal d H 3.88 (3H, s) and oxygen-containing aromatic carbon signals d C The HMBC correlation at 149.3 (C-3¢) suggests the presence of a 3-methoxy-4-hydroxyphenyl group in the structure. The above spectral data indicate that compound 1 is a phenylpropanoid coumarin. This compound has an unsaturation degree of 12, with the coumarin core and the 3-methoxy-4-hydroxyphenyl group occupying 11 unsaturations, leaving 1 unsaturation point, suggesting the presence of a ring in the structure. This, combined with the three oxygen-bound aliphatic carbon signals... d C 78.9 (C-7′), 79.7 (C-8′), 61.8 (C-9′), and H-7′ / H-8′, H-9′ / H-8′ 1 H- 1Based on H COSY correlation, it is inferred that a three-carbon sequence CH(O)CH(O)CH2OH exists in the structure, forming a dioxane ring structure. This is indicated by the methoxy hydrogen signal. d H 3.81 (3H, s) and coumarin nucleus d C The HMBC correlation at 133.4 (C-6) indicates that the 6-hydroxyl group is methylated, further suggesting the fusion of the dioxane with the coumarin core at the C-7 and C-8 positions. d H 5.10 (1H, d, J = 8.2 Hz, H-7′) and d C 128.7 (C-1′), 112.2 (C-2′), 121.8 (C-6′) and d H 3.85 (1H, m, H-9′) and d C 79.7 (C-8′) HMBC related, 3′-methoxy-4′-hydroxyphenyl linked at C-7′ position, hydroxymethyl linked at C-8′ position. (By...) d H 5.10 (1H, d, J = 8.2 Hz, H-7′) and d C The HMBC correlation of 126.5 (C-8) indicates that the dioxane linkage is 7-O-8′, 8-O-7′. The NOE correlation between H-7′ and H-9′ indicates that H-7′ and H-8′ are in the trans configuration. The NMR data of the compounds were analyzed and assigned based on HMBC and HSQC spectra (Table 1). The ECD spectra of the compounds showed no significant Cotton effect (200–400 nm) and a specific rotation of zero, suggesting that the compounds were a racemic mixture. Therefore, chiral resolution was performed [column: Daicel chiral PAK AD-H (250 mm × 10 mm, 5 μm); mobile phase: cyclohexane–isopropanol = 88:12, flow rate 2 ml / min], yielding equal amounts of compound 1a (…). t R =21.255 min) and compound 1b ( t R = 41.592 min). Compounds 1a and 1b exhibit opposite Cotton effects, and the measured ECD spectra are basically consistent with the calculated ECD spectra of isomers (7′R,8′R-1) and (7′S,8′S)-1, respectively (see Figure 3Thus, the absolute configurations of compounds 1a and 1b were determined to be 7′R,8′R and 7′S,8′S, respectively. Therefore, compounds 1a and 1b were identified as (+)-(7'R,8'R)-7′,8′-dihydro-7'-(3'-methoxy-4'-hydroxyphenyl)-8'(hydroxymethyl)-5-hydroxy -6-methoxy-2H-pyrano[2,3-f]-7,8-benzodioxin-2-one and (−)- (7'S,8'S)-7′,8′-dihydro-7'-(3'-methoxy- 4'-hydroxyphenyl)-8'(hydroxymethyl)-5-hydroxy-6-methoxy-2H-pyrano[2,3-f]-7,8-benzodioxin-2-one, and named (+)-cissacoumarinolignan A [(+)-cissacoumarinolignan A] and (−)-cissacoumarinolignan A [(−)-cissacoumarinolignan A].

[0019] Compound 1a is a yellowish-brown amorphous powder, readily soluble in methanol; a ]25 D+7.8 ( c 0.01, MeOH); ECD(MeOH) l max (Δ e 195 (+4.5), 220 (−2.3), 258 (+0.5), 282 (−0.2), 340 (+0.5). Compound 1b is a yellowish-brown amorphous powder, readily soluble in methanol; a ]25 D−7.5 ( c 0.01, MeOH); ECD(MeOH) l max (Δ e ) 195 (−5.2), 220 (+2.0), 258 (−0.2), 282 (+0.6), 340 (−0.8). The structure of (±)-Ahunu balsamin A and its HMBC and HH COSY correlation spectra are as follows:

[0020] Table 1. NMR (500 MHz, CD3OD) assignments for (±)-cissacoumarinolignan A

[0021] II. Cytotoxic Activity Assay 1. Experimental Materials The human cervical cancer cell line (HeLa) was provided by the Shanghai Institute of Biochemistry and Cell Biology, and the fetal bovine serum was purchased from Sigma.

[0022] 2. Cell Culture The test cells (human cervical cancer cells HeLa) were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture flasks were placed in a 37 °C, 5% CO2 saturated humidity incubator, and the culture medium was changed every 1-2 days. When the cells grew large enough to cover most of the bottom wall of the flask, they were digested with 0.25% trypsin and passaged.

[0023] 3. MTT method Logarithmic growth phase cells were cultured in 96-well plates, 100 μL per well (containing 4000 tumor cells), and incubated at 37 °C in a 5% CO2 incubator. The following day, the treatment groups were added with diluents containing different concentrations of the test compound, resulting in five dosage groups (40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM), with at least three parallel wells per group. The control group received the same volume of solvent as the treatment groups. Both were incubated at 37 °C in a 5% CO2 incubator. After 2 days, the culture medium was discarded, and 50 μL (1 mg / ml) MTT solution (prepared with culture medium) was added to each well. The plates were incubated at 37 °C for 4 hours, the supernatant was discarded, and 200 μL of DMSO was added to each well, followed by gentle shaking. The optical density (OD) was measured using a microplate reader at a detection wavelength of 490 nm. Cells treated with solvent control served as the control group. Etoposide was included as a positive compound in each experiment. The inhibitory rate of the drug on cells was calculated using the following formula. Based on the calculated inhibition rates at each concentration, the half-maximal inhibitory concentration (IC50) was obtained by processing the data using SPSS 26.0 software. 50 Repeat the test 3 times and take the average value as the final result.

[0024]

[0025] 4. Experimental Results The cytotoxic activity of (±)-ahunucurate A in human cervical cancer cells (HeLa) was tested using the MTT assay. The results showed that IC50... 50 The value is 35.17 μM.

[0026] Meanwhile, while conducting experiments on Example 1, the present invention also conducted the same experiments on other examples, and obtained the same or similar results, which will not be listed here one by one.

[0027] The applicant found in the literature on other phenylpropanoid coumarin compounds that their activities vary greatly due to differences in the position, number and type of the parent nucleus and the substituents attached to it. However, the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] showed excellent cytotoxic activity against human cervical cancer cells HeLa.

[0028] In summary, the method of this invention is stable, reliable, and easy to operate. The enantiomeric (±)-cissacoumarinolignan A prepared by this method has anticancer activity and can be effectively used to prepare anti-cervical cancer drugs. It has the prospect of preparing clinical anti-cervical cancer drugs, expands the medicinal value of cissacoumarinolignan, and provides technical support for the preparation of anti-cervical cancer drugs. It is a major innovation in the preparation of anti-cervical cancer drugs and has practical clinical and promotional application value.

Claims

1. A pair of enantiomers of phenylpropanoid coumarin compounds, (±)-cissacoumarinolignan A, which were isolated from cissacoumarinolignan and have the following molecular structure:

2. The method for preparing the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of the phenylpropanoid coumarin compound according to claim 1, characterized in that: Using 30–50 kg of dried Arjuna root as raw material, 120–200 L of 70% ethanol was added and extracted three times by heating and reflux at 92–95 °C for 1–1.5 hours each time. The ethanol was recovered under reduced pressure, yielding a suspension of 3.4–5.6 L of ethanol extract. An equal volume of diatomaceous earth was added, and the solvent was evaporated. The solution was then eluted sequentially with 10.8–18 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding the petroleum ether fraction, ethyl acetate fraction, dichloromethane fraction, and methanol fraction. The ethyl acetate fraction was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50, with each gradient using 16.8–28.0 L of ethanol. L eluent was used, with a flow rate of 20–25 mL / min. Each fraction was collected in 900–1500 mL increments, resulting in 174 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.4…Fr. 9; Component Fr.4 was separated by silica gel column chromatography, and eluted sequentially with a dichloromethane-methanol mixed solvent system at volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, using 450–750 mL of eluent for each gradient at a flow rate of 20–25 mL / min. Each fraction was collected in volumes of 75–125 mL, resulting in 68 fractions. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.4–0.7 mL / min, with each 4–7 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:

75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-aurinol A. The ECD spectrum of this compound showed no significant Cotton effect (200–400 nm). The specific rotation value is zero, indicating a racemic mixture. Chiral separation was then performed. [Column: Daicel chiral PAK AD-H (250 mm × 10 mm, 5...] μ m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

3. The method for preparing the enantiomer (±)-cissacoumarinolignan A of a pair of phenylpropanoid coumarin compounds according to claim 2, characterized in that: Using 50 kg of dried Arjuna root as raw material, 200 L of 70% ethanol was added and extracted three times by heating and reflux at 95℃ for 1 hour each time. Ethanol was recovered under reduced pressure, yielding a 5.6 L suspension of the ethanol extract. 5.6 kg of diatomaceous earth was added, and the solvent was evaporated. The solution was then eluted sequentially with 18 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 9 g of petroleum ether fraction, 588 g of dichloromethane fraction, 382 g of ethyl acetate fraction, and 1607 g of methanol fraction. The ethyl acetate fraction was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:

50. Each gradient used 28.0 L of ethanol. L eluent was used, and the flow rate was 25 mL / min. Each 1500 mL volume was considered one fraction, and 174 fractions were collected. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.

4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 750 mL of eluent for each gradient. The flow rate was 25 mL / min, and each 125 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.7 mL / min. 73 fractions were collected, and each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:

75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAK AD-H (250...]. mm × 10 mm, 5 μ m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R =21.255 min fraction, yielding (+)-Ahunucurate A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

4. A method for preparing the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of the phenylpropanoid coumarin compound according to claim 2, characterized in that: Using 30 kg of dried Arjuna root as raw material, 120 L of 70% ethanol was added and the mixture was extracted three times by heating and reflux at 92℃ for 1.5 hours each time. The ethanol was recovered under reduced pressure, yielding a 3.4 L suspension of the ethanol extract. 3.4 kg of diatomaceous earth was added, and the solvent was evaporated. The mixture was then eluted successively with 10.8 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 5.4 g of petroleum ether fraction, 352 g of dichloromethane fraction, 229 g of ethyl acetate fraction, and 96 g of methanol fraction. 4g; The ethyl acetate fraction was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:50, with 16.8 L of eluent for each gradient. The flow rate was 20 mL / min, and each 900 mL volume was considered one fraction. A total of 174 fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF. 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.

4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 450 mL of eluent for each gradient. The flow rate was 20 mL / min, and each 75 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.4 mL / min, with each 4 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:

75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAK AD-H (250...]. mm × 10 mm, 5 μ m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

5. The method for preparing the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of the phenylpropanoid coumarin compound according to claim 2, characterized in that: Using 40 kg of dried Arjuna root as raw material, 160 L of 70% ethanol was added and extracted three times by heating and reflux at 94 °C for 1.2 hours each time. The ethanol was recovered under reduced pressure, yielding a 4.5 L suspension of the ethanol extract. 4.5 kg of diatomaceous earth was added, and the solvent was evaporated. The extract was then eluted sequentially with 14.4 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 7.2 g of petroleum ether fraction, 470.4 g of dichloromethane fraction, 305.6 g of ethyl acetate fraction, and 1285.6 g of methanol fraction. The ethyl acetate fraction was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:

50. Each gradient used 22.4 L of ethanol. L eluent was used, the flow rate was 23 mL / min, and each 1200 mL volume was considered a fraction. 174 fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF. 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.

4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 600 mL of eluent for each gradient. The flow rate was 23 mL / min, and each 100 mL volume was considered one fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.6 mL / min, with each 6 mL fraction collected, resulting in 73 fractions. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:

75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAKAD-H (250...]. mm × 10 mm, 5 μ m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

6. The method for preparing the enantiomer (±)-cissacoumarinolignan A [(±)-cissacoumarinolignan A] of the phenylpropanoid coumarin compound according to claim 2, characterized in that: Using 35 kg of dried Arjuna root as raw material, 140 L of 70% ethanol was added and extracted three times by heating and reflux at 93℃ for 1.3 hours each time. Ethanol was recovered under reduced pressure, yielding a 3.9 L suspension of the ethanol extract. 3.9 kg of diatomaceous earth was added, and the solvent was evaporated. The extract was then eluted sequentially with 12.6 L each of petroleum ether, ethyl acetate, dichloromethane, and methanol. The solvents were recovered, yielding 6.3 g of petroleum ether fraction, 411.6 g of dichloromethane fraction, 267.4 g of ethyl acetate fraction, and 1124.9 g of methanol fraction. The ethyl acetate fraction was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvents at volume ratios of 100:0, 100:1, 60:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:30, and 100:

50. Each gradient used 19.6 L of ethanol. L eluent was used, and the flow rate was 22 mL / min. Each 1050 mL volume was considered a fraction, and 174 fractions were collected. Each fraction was analyzed by silica gel thin-layer chromatography using GF... 254 Thin-layer chromatography (TLC) plates were heated at 105 °C for 3–5 min using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. Based on the TLC results, fractions 1–3, 4–7, 8–30, 31–50, 51–74, 75–83, 84–141, 142–163, and 164–174 were combined to obtain components Fr.1…Fr.

4. …Fr.9; Component Fr.4 was separated by silica gel column chromatography, using a gradient elution system of dichloromethane-methanol mixed solvent with volume ratios of 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1, with 525 mL of eluent for each gradient. The flow rate was 22 mL / min, and each 88 mL volume was considered a fraction. Sixty-eight fractions were collected, and each fraction was analyzed by silica gel thin-layer chromatography using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent. The plates were heated at 105 °C for 3–5 min. Based on the TLC results, streams 1–33, 34–35, 36–42, 43–48, 50–61, and 62–68 were combined to obtain fractions Fr.4-1…Fr.4-6. Fractions Fr.4-6 were separated by Sephadex LH-20 column chromatography using methanol as eluent at a flow rate of 0.5 mL / min, with 73 fractions collected in 5 mL increments. Each fraction was analyzed by silica gel TLC using GF… 254 Thin-layer chromatography (TLC) was performed using petroleum ether-acetone (v / v) and dichloromethane-methanol (v / v) as developing solvents, respectively, and sulfuric acid-ethanol solution (v / v) as the colorimetric reagent (v / v). The mixture was heated at 105 °C for 3–5 min. Based on the TLC results, fractions 1–14, 15–19, 20–27, 28–43, and 44–73 were combined to obtain fractions Fr.4-6-1…Fr.4-6-3…Fr.4-6-5. Fraction Fr.4-6-3 was purified by preparative high-performance liquid chromatography (HPLC) using an acetonitrile-water mixed solvent system (v / v) of 25:

75. The chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the peak with a retention time of 25 min was collected to obtain the enantiomeric (±)-argentinin A, which was then chirally separated [column: Daicel chiral PAKAD-H (250...]. mm × 10 mm, 5 μ m); Mobile phase: cyclohexane–isopropanol = 88:12, flow rate: 2 ml / min)], collection retention time t R = 21.255 min fraction, yielding (+)-Ahunu balsamin A, collection retention time t R = 41.592 min fraction, yielding (–)-Ahunu balsamin A.

7. The use of the enantiomer (±)-cissacoumarinolignan A of any one of claims 1-6 in the preparation of an anti-cervical cancer drug.

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