A sesquiterpene coumarin compound isolated from ferula sinkiangensis and a preparation method and application thereof

Abstract

This invention belongs to the field of phytochemistry and pharmaceutical technology, specifically disclosing a method for preparing and purifying sesquiterpene coumarin compounds isolated from *Ferula samarkand*, including the structures of three sesquiterpene coumarin compounds isolated from *Ferula samarkand*, and the process for separating sesquiterpene coumarins. Addressing the current problem of unclear chemical composition and active ingredients of *Ferula samarkand*, this invention employs organic solvent extraction, concentration, and then utilizes two to three methods—normal-phase silica gel column chromatography, dextran gel chromatography, low-to-medium pressure rapid preparative chromatography, and semi-preparative high-performance liquid chromatography—for separation, ultimately yielding three sesquiterpene coumarin monomers. This solves the problem in existing technologies where the unclear chemical composition and active ingredients of *Ferula samarkand* make it difficult to separate and purify sesquiterpene coumarins.

Description

Technical Field

[0001] This invention relates to the fields of phytochemistry and pharmaceutical technology, specifically to a sesquiterpene coumarin compound isolated from Ferula samarkand, its preparation method, and its application. Background Technology

[0002] Coumarins are a diverse class of natural products, abundant in plants of the Apiaceae, Asteraceae, and Rutaceae families, and are a highly sought-after type of active compound in the field of natural product chemistry. With the rapid development of modern separation, purification, structural identification, and activity screening technologies, over 1000 natural coumarins have been discovered and reported. Their structural and bioactivity diversity provides an important material basis for innovative drug development.

[0003] As an important group within the Apiaceae family, *Ferula* is a high-quality natural resource of coumarins, with a research history dating back to 1935. This genus is rich in novel and highly active coumarins, especially sesquiterpene coumarins, which represent a core area of ​​chemical and pharmacological research in *Ferula*. Sesquiterpene coumarins are characterized by C-7 substitution and, based on differences in the sesquiterpene substituent skeleton, can be classified into three types: acyclic (chain), monocyclic, and bicyclic. They possess complex stereochemical structures and diverse structure-activity relationships, exhibiting broad and prominent biological activities in antitumor, antibacterial, insecticidal, and antioxidant activities, making them an important research subject for the natural source of antitumor drug candidates.

[0004] Samarkand asafoetida is a unique Ferula species resource endemic to Uzbekistan. Due to geographical distribution and limited research conditions, current research on this plant is relatively limited, and key issues such as its chemical composition, pharmacodynamic material basis, and mechanism of action remain unclear. Studies have shown that Samarkand asafoetida extract exhibits significant bioactivity in screening for anti-vitiligo and anti-tumor activities. Therefore, systematically studying its characteristic components, sesquiterpenoid coumarins, will not only enrich the chemical structure database of Ferula species and fill the research gaps in Samarkand asafoetida, but also provide a scientific basis for screening potential medicinal active ingredients, which is of great significance for promoting the high-value utilization of unique medicinal plant resources and the development of innovative drugs.

[0005] In summary, it is necessary to design a method for preparing and applying sesquiterpene coumarin compounds isolated from Samarkand Ferula aspera, in order to solve the problem that the lack of clarity regarding the chemical composition and pharmacological effects of Samarkand Ferula aspera under existing technologies makes it difficult to isolate and purify sesquiterpene coumarin compounds. Summary of the Invention

[0006] The purpose of this invention is to provide a sesquiterpene coumarin compound isolated from Ferula samarkand, its preparation method, and its application, in order to solve the problem that the lack of clarity regarding the chemical composition and pharmacologically active substances of Ferula samarkand under the existing technology makes it difficult to isolate and purify sesquiterpene coumarin compounds from Ferula samarkand.

[0007] To achieve the above objectives, the basic solution provided by this invention is: a sesquiterpene coumarin compound isolated from Ferula assamarkand, wherein the structural formula of the sesquiterpene coumarin compound is: Among them, compound formula (Ⅰ) is 7-[[(3'R,5'S,8'R,9'R,10'R)-decahydro-3',8'-dihydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; Compound formula (II) is 7-[[(5'S,8'R,9'R,10'R)-decahydro-3'-carbonyl-8'-hydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; The compound of formula (III) is 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one.

[0008] The beneficial effects of this invention are as follows: (1) This invention uses organic solvent extraction, extraction, and concentration, and then comprehensively utilizes two or three of the following methods for separation: normal phase silica gel column chromatography, dextran gel chromatography, medium-low pressure rapid preparative chromatography, and semi-preparative high performance liquid chromatography, to finally obtain three sesquiterpene coumarin monomer compounds; (2) The antitumor activity assay clearly shows that the sesquiterpene coumarin compounds isolated from Ferula samarkand have significant antitumor activity and inhibit human cervical cancer HeLa, colon cancer HT-29, and lung cancer A549 cells. Among them, compound (I) has an inhibitory effect on the IC50 of HeLa cells. 50 With a micrometer size of only 5.98 μm and outstanding activity, it provides a natural source for the preparation of antitumor drugs.

[0009] Option 2, a method for preparing sesquiterpene coumarin compounds isolated from Ferula assamarkand, comprising the following steps: S1: Extraction: First, the dried roots of Ferula samarkand were crushed at room temperature. Then, 95% ethanol was added to the crushed roots and the roots were soaked and extracted at room temperature. The extract was then concentrated under reduced pressure until there was no ethanol odor, thus obtaining crude extract of Ferula samarkand. S2: Extraction: The crude extract of Samarkand Ferula root was suspended in water to disperse it. Then, petroleum ether was added to the water for extraction. After standing and separating into layers, the organic phase was collected. The extraction was repeated until the organic phase was colorless. The collected organic phases were combined, and then the solvent was evaporated under reduced pressure to obtain the petroleum ether fraction extract. S3: Separation: The petroleum ether fraction obtained in S2 was separated using a normal-phase silica gel column chromatography method. Gradient elution was then performed using petroleum ether-dichloromethane as the solvent, and the eluent was collected. The fractions were then analyzed by silica gel thin-layer chromatography and combined to obtain 10 fractions Fr.A-Fr.J. Fraction Fr.G was separated by silica gel column chromatography using petroleum ether-ethyl acetate as the solvent for gradient elution. After analysis and combination, 9 subfractions were obtained. The subfraction Fr.GF was further separated by medium-low pressure rapid preparative chromatography using methanol-water as the solvent system for gradient elution, yielding 14 fractions. Fr.GF- 11. The compounds 7-[[(3'R, 5'S, 8'R, 9'R, 10'R)-decahydro-3', 8'-dihydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one and 7-[[(5'S, 8'R, 9'R, 10'R)-decahydro-3'-carbonyl-8'-hydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one were separated by semi-preparative high-performance liquid chromatography and isocratically eluted with acetonitrile-water mixture as eluent. S4: Fr.GF-13 was subjected to dextran gel chromatography, crudely separated with methanol as the mobile phase, and then separated by semi-preparative high performance liquid chromatography with acetonitrile-water mixture as eluent. Isocratic elution yielded compound 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one.

[0010] Option 3, an optimal choice from Option 2, involves soaking for 24 hours, extracting 5 times, and using a ratio of Samarkand fern root to ethanol of 1:6 in S1. This ensures the complete dissolution of sesquiterpene coumarins.

[0011] Option 4, a preferred embodiment of Option 2, involves the following steps in S3: the volume ratio of petroleum ether to dichloromethane is 100:0-0:100, the volume ratio of petroleum ether to ethyl acetate is 100:0-0:100, the volume ratio of methanol to water is 10:90-100:0, and the volume ratio of acetonitrile to water is 65:35. This stepwise elution from low to high polarity results in higher separation, is well-suited to the polarity characteristics of sesquiterpene coumarins, and effectively separates structurally similar compounds.

[0012] Option 5, which is an optimal choice for Option 2, involves using a reversed-phase column X-Select CSH in S3 for chromatographic separation. TM Premier BEH, Waters, 5μm, 10×250mm.

[0013] Option 6, which is a preferred option of Option 2, has an acetonitrile-water volume ratio of 60:40 in S4.

[0014] Option 7, an application of sesquiterpene coumarin compounds isolated from Ferula assamarkand, characterized in that the antitumor activity of sesquiterpene coumarin compounds (I), (II) and (III) isolated from Ferula assamarkand roots is determined, and the sesquiterpene coumarin compounds (I), (II) and (III) all have antitumor activity and can be used to prepare drugs against human cervical cancer cells, human colon cancer cells and lung cancer cells. Attached Figure Description

[0015] Figure 1 This invention relates to a sesquiterpene coumarin compound of formula (Ⅰ) isolated from Ferula aspera in Samarkand. 1 H NMR spectrum; Figure 2 This invention relates to a sesquiterpene coumarin compound of formula (Ⅰ) isolated from Ferula aspera in Samarkand. 13 C NMR spectrum; Figure 3 This invention relates to a sesquiterpene coumarin compound of formula (II) isolated from Ferula aspera in Samarkand. 1 H NMR spectrum; Figure 4 This invention relates to a sesquiterpene coumarin compound of formula (II) isolated from Ferula aspera in Samarkand. 13 C NMR spectrum; Figure 5 This invention relates to a sesquiterpene coumarin compound of formula (III) isolated from Ferula aspera in Samarkand. 1 H NMR spectrum; Figure 6 This invention relates to a sesquiterpene coumarin compound of formula (III) isolated from Ferula aspera in Samarkand.13 C NMR spectrum. Detailed Implementation

[0016] The present invention will be further described in detail below through specific embodiments: Example 1 A sesquiterpene coumarin compound isolated from Ferula aspera in Samarkand has the following structural formula: Among them, compound formula (Ⅰ) is 7-[[(3'R,5'S,8'R,9'R,10'R)-decahydro-3',8'-dihydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; Compound formula (II) is 7-[[(5'S,8'R,9'R,10'R)-decahydro-3'-carbonyl-8'-hydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; The compound of formula (III) is 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one.

[0017] Example 2 A method for preparing sesquiterpene coumarin compounds isolated from Ferula assamarkand includes the following steps: S1: Extraction: First, the dried roots of *Ferula samarkandi* were pulverized at room temperature. Then, 95% ethanol was added to the pulverized *Ferula samarkandi* roots and soaked at room temperature for 24 hours. The extraction was repeated 5 times, with the ratio of *Ferula samarkandi* roots to ethanol being 1:6. The extracts obtained from the 5 extractions were then combined and concentrated under reduced pressure until no ethanol odor was detected, yielding a crude extract of *Ferula samarkandi* roots. The crude extract of *Ferula samarkandi* roots was then suspended in water to disperse it. Petroleum ether was added to the water for extraction. After standing and separating the layers, the organic phase was collected. The extraction was repeated until the organic phase was colorless. The collected organic phases were then combined, and the solvent was evaporated under reduced pressure to obtain the petroleum ether fraction extract. S2: Separation: The petroleum ether fraction obtained in S1 was separated using a normal-phase silica gel column. Gradient elution was then performed using petroleum ether-dichloromethane (v / v) at a ratio of 100:0-0:100, and the eluent was collected. The fractions (collected from gradient elution) were then analyzed by silica gel thin-layer chromatography, and the fractions were combined to obtain 10 fractions Fr.A-Fr.J. Fraction Fr.G was separated by silica gel column chromatography using a petroleum ether-ethyl acetate (v / v) at a ratio of 100:0-0:100, and the fractions were combined to obtain 9 subfractions. Subfraction Fr.GF was further separated by medium-low pressure rapid preparative chromatography using a methanol-water (v / v) at a ratio of 10:90-100:0, yielding 14 fractions. Fr.GF-11 was then separated by semi-preparative high-performance liquid chromatography (reversed-phase column X-Select CSH). TM Separation was performed using a Premier BEH, Waters, 5 μm, 10 × 250 mm instrument. An acetonitrile-water mixture with a volume ratio of 65:35 was used as the eluent. Isocratic elution yielded compound (I) 7-[[(3'R, 5'S, 8'R, 9'R, 10'R)-decahydro-3', 8'-dihydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one and compound (II) 7-[[(5'S, 8'R, 9'R, 10'R)-decahydro-3'-carbonyl-8'-hydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; Compound (Ⅰ) is a white powder with the molecular formula C. 24 H 32 O5, with a skeleton type of bicyclic sesquiterpene coumarin; compound (II) is a white powder with the molecular formula C. 24 H 30 O5, with a skeleton type of bicyclic sesquiterpene coumarin; S3: Fr.GF-13 was coarsely separated by dextran gel chromatography using methanol as the mobile phase, followed by semi-preparative high-performance liquid chromatography (HPLC) using a reversed-phase X-Select CSH column. TM Premier BEH, Waters, 5μm, 10×250mm, using an acetonitrile-water mixture with a volume ratio of 60:40 as the eluent, was isocratically eluted to obtain compound (III) 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methylethylene-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; compound (III) was a colorless oil with the molecular formula C 24 H28 O4, with a skeleton type of bicyclic sesquiterpene coumarin.

[0018] Table 1: Compounds of formulas (I)-(III) 1 H NMR and 13 C NMR data table (δ in ppm, J in Hz) Example 3 An application of sesquiterpene coumarin compounds isolated from Ferula samarkandi: The antitumor activity of sesquiterpene coumarin compounds (I), (II) and (III) isolated from Ferula samarkandi roots was determined. The results showed that sesquiterpene coumarin compounds (I), (II) and (III) isolated from Ferula samarkandi roots all have antitumor activity and can be used to prepare drugs against human cervical cancer cells (HeLa), human colon cancer cells (HT-29) and lung cancer cells (A549).

[0019] Therefore, the sesquiterpene coumarin compounds prepared in Experiment II were used in antitumor experiments, and the following results were obtained: MTT assay for cell viability: 1. After the revived RAW264.7 cells have grown to 80%-90% confluence, they are passaged and healthy cells are collected. 10 μL of cell suspension and 10 μL of trypan blue staining solution are mixed at a 1:1 volume ratio. After mixing, the cells are counted using a cell counter. 2. Adjust the cell concentration to 1×10⁻⁶. 4 The cells were seeded at a density of 100 μL per well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator until the cells adhered. 3. The next day, discard the original culture medium, add different concentrations of the test compound (set up 3 replicates), incubate for 1 hour, then add lipopolysaccharide (LPS) at a concentration of 1 μg / mL, and incubate for 16 hours in a constant temperature incubator at 37℃ and 5% CO2. 4. Discard the liquid in the wells, add 100 μL of 0.5 mg / mL MTT to each well, and continue incubation in an incubator at 37℃ and 5% CO2 for 3-4 hours before terminating the culture. 5. Remove the liquid from the wells, add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake for 10 min to fully dissolve the intracellular crystals, and measure the absorbance of each well at 490 nm using a microplate reader. Cell viability percentage (%) = (Compound OD - Blank OD / Control Group OD - Blank OD) × 100% Cell inhibition rate (%) = 1 - Cell viability % = [1 - (Compound OD - Blank OD / Control Group OD - Blank OD)] × 100% Initial screening was conducted under single-concentration conditions, such as a monomeric compound concentration of 50 μM and an extract concentration of 50 μg / μL, to test the activity of the samples. For samples exhibiting good activity under certain conditions, such as inhibition rates greater than 50%, 60%, and 70%, a subset of samples were selected for further testing of the dose-dependent relationship of activity. Graphpad Prism4 (a statistical analysis and graphing software specifically for biomedicine and pharmacology, primarily used for dose-response curve fitting and IC50 analysis) was used. 50 Software for calculating IC (including calculation, statistical testing, and scientific plotting) 50 The value was obtained by nonlinearly fitting the sample concentration to the sample activity, and duplicate wells (n≥3) were set for each sample in the test.

[0020] Table 2: Statistical Table of Antitumor Activities of Sesquiterpene Coumarin Compounds in Ferula Samarkandi In summary, this invention established an efficient and stable process for the separation and purification of sesquiterpene coumarins using *Ferula samarkandi* root as raw material. Through a multi-stage separation technique combining ethanol extraction, petroleum ether extraction, normal-phase silica gel column chromatography, medium-low pressure rapid preparative chromatography, dextran gel chromatography, and semi-preparative high-performance liquid chromatography, three novel sesquiterpene coumarin monomers were isolated from *Ferula samarkandi* root. The chemical structure and stereoconfiguration of each compound were identified by spectroscopic structure analysis. In vitro antitumor activity verification showed that the sesquiterpene coumarins isolated from *Ferula samarkandi* root exhibited significant inhibitory effects on the proliferation of human cervical cancer, human colon cancer, and human lung cancer cells, with compound (I) showing particularly outstanding activity.

[0021] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A sesquiterpene coumarin compound isolated from Ferula assamarkand, characterized in that, The structural formula of the sesquiterpene coumarin compound is: Among them, compound formula (Ⅰ) is 7-[[(3'R,5'S,8'R,9'R,10'R)-decahydro-3',8'-dihydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; Compound formula (II) is 7-[[(5'S,8'R,9'R,10'R)-decahydro-3'-carbonyl-8'-hydroxy-4',4',8',10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one; The compound of formula (III) is 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one.

2. A method for preparing sesquiterpene coumarin compounds isolated from Ferula assamarkand, comprising the following steps: S1: Extraction: First, the dried roots of Ferula samarkand were crushed at room temperature. Then, 95% ethanol was added to the crushed roots and the roots were soaked and extracted at room temperature. The extract was then concentrated under reduced pressure until there was no ethanol odor, thus obtaining crude extract of Ferula samarkand. S2: Extraction: The crude extract of Samarkand Ferula root was suspended in water to disperse it. Then, petroleum ether was added to the water for extraction. After standing and separating into layers, the organic phase was collected. The extraction was repeated until the organic phase was colorless. The collected organic phases were combined, and then the solvent was evaporated under reduced pressure to obtain the petroleum ether fraction extract. S3: Separation: The petroleum ether fraction obtained in S2 was separated using a normal-phase silica gel column chromatography method. Gradient elution was then performed using petroleum ether-dichloromethane as the solvent, and the eluent was collected. The fractions were then analyzed by silica gel thin-layer chromatography and combined to obtain 10 fractions Fr.A-Fr.J. Fraction Fr.G was separated by silica gel column chromatography using petroleum ether-ethyl acetate as the solvent for gradient elution. After analysis and combination, 9 subfractions were obtained. The subfraction Fr.GF was further separated by medium-low pressure rapid preparative chromatography using methanol-water as the solvent system for gradient elution, yielding 14 fractions. Fr.GF- 11. The compounds 7-[[(3'R, 5'S, 8'R, 9'R, 10'R)-decahydro-3', 8'-dihydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one and 7-[[(5'S, 8'R, 9'R, 10'R)-decahydro-3'-carbonyl-8'-hydroxy-4', 4', 8', 10'-tetramethyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one were separated by semi-preparative high-performance liquid chromatography and isocratically eluted with acetonitrile-water mixture as eluent. S4: Fr.GF-13 was subjected to dextran gel chromatography, crudely separated with methanol as the mobile phase, and then separated by semi-preparative high performance liquid chromatography with acetonitrile-water mixture as eluent. Isocratic elution yielded compound 7-[[(3'R,5'S,9'R,10'R)-1',2',3',4',5',8',9',10'-octahydro-3'-hydroxy-4',4',10'-trimethyl-8'-methyl-9'-naphthyl]methoxy]-2H-1-benzopyran-2-one.

3. The method for preparing sesquiterpene coumarin compounds isolated from Ferula aspera in Samarkand according to claim 2, characterized in that, In S1, the soaking time was 24 hours, the number of extractions was 5, and the ratio of Samarkand fern root to ethanol was 1:

6.

4. The method for preparing sesquiterpene coumarin compounds isolated from Ferula samarkand according to claim 2, characterized in that, In S3, the volume ratio of petroleum ether to dichloromethane is 100:0-0:100, the volume ratio of petroleum ether to ethyl acetate is 100:0-0:100, the volume ratio of methanol to water is 10:90-100:0, and the volume ratio of acetonitrile to water is 65:

35.

5. The method for preparing sesquiterpene coumarin compounds isolated from Ferula aspera in Samarkand according to claim 2, characterized in that, In S3, chromatographic separation is performed using a reversed-phase column X-Select CSH. TM Premier BEH, Waters, 5μm, 10×250mm.

6. The method for preparing sesquiterpene coumarin compounds isolated from Ferula samarkand according to claim 2, characterized in that, In S4, the volume ratio of acetonitrile to water is 60:

40.

7. The application of a sesquiterpene coumarin compound isolated from Ferula samarkand, characterized in that, The antitumor activity of sesquiterpene coumarin compounds (Ⅰ), (Ⅱ) and (Ⅲ) isolated from the root of Ferula samarkandi all exhibited antitumor activity and could be used to prepare drugs against human cervical cancer cells, human colon cancer cells and lung cancer cells.

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