Anti-tumor retamoebol and a method for preparing high purity thereof

Abstract

An antitumor tenacissimol and a high-purity preparation method thereof, three high-purity tenacissimols A, B and C with significant antitumor effects are prepared from tenacissima simultaneously through water decocting tenacissima, macroporous resin column chromatography, silica gel column chromatography and HPLC preparation and separation, and can be used for quality evaluation of tenacissima medicinal materials and preparation of antitumor drugs; the application has the advantages of simplicity, rapidness, high purity and low production cost.

Description

Technical Field

[0001] This invention relates to a technology in the field of biomedicine, specifically an antitumor herbal extract and its high-purity preparation method. Background Technology

[0002] Marsdenia tenacissima, also known as Tongguansan, Tongguangteng, Wuguteng, Naijiangteng, Tongtiansan, Longzhaocao, and Xianaiteng, is the dried stem of the plant Marsdenia tenacissima (Roxb.) Wight et Arn., belonging to the Asclepiadaceae family. A commonly used traditional Chinese medicine, it was first recorded in the *Diannan Bencao* (a medical text from Yunnan Province) and has a long history. It possesses various medicinal properties, including relieving cough and asthma, expectorating phlegm, promoting lactation, and clearing heat and toxins. It is widely used to treat cough with excessive phlegm, postpartum lactation insufficiency, rheumatic swelling and pain, and carbuncles. Modern research has found that Marsdenia tenacissima has significant anti-tumor effects and has been used to treat primary liver cancer, esophageal cancer, and gastric cancer, as well as lung cancer, with definite results. Summary of the Invention

[0003] This invention addresses the shortcomings of existing extraction techniques, which yield only saponins with low yields. It proposes a method for preparing tenacissimol, an antitumor compound, and its high-purity formulation. By separating the compounds from *Tengium styracifolium* through water decoction, macroporous resin chromatography, silica gel column chromatography, and HPLC, three high-purity tenacissimols (A, B, and C) with significant antitumor activity can be simultaneously prepared from *Tengium styracifolium*. This method can be used for quality evaluation of *Tengium styracifolium* and for the preparation of antitumor drugs. The method is simple, rapid, highly pure, and has low production costs.

[0004] This invention is achieved through the following technical solution:

[0005] This invention relates to a class of tenacissimol compounds, including tenacissimol A, tenacissimol B, and tenacissimol C, whose chemical structures are shown below:

[0006]

[0007] This invention relates to the application of the above-mentioned Tongguanteng alcohol compounds, using them for the preparation of antitumor drugs or for the identification of Tongguanteng medicinal materials.

[0008] This invention relates to a method for extracting the above-mentioned Marsdenia tenacissima compounds. The method involves decocting the dried stems of Marsdenia tenacissima (Roxb.) Wight et Arn. (a plant in the Asclepiadaceae family) in water, centrifuging the decoction, performing macroporous resin column chromatography, followed by silica gel column chromatography and HPLC preparative separation, and collecting three main chromatographic peaks to obtain purified Marsdenia tenacissima A, purified Marsdenia tenacissima B, and purified Marsdenia tenacissima C.

[0009] The macroporous resin column chromatography used D101 type macroporous resin and ethanol solution as eluent.

[0010] All silica gel column chromatography analyses were performed by TLC, and the main spot R was combined. f The flow rate was 0.2–0.5%, with a mixture of dichloromethane and methanol as the developing solvent.

[0011] The extraction method specifically includes: pulverizing *Tongguanteng* (a type of orchid), decocting it in water, and then performing D101 macroporous resin column chromatography. First, elute with 60% ethanol to remove impurities, then elute with 95% ethanol. Collect the 95% ethanol eluent, and recover the solvent under reduced pressure to obtain *Tongguanteng* ethanol extract. Perform silica gel column chromatography on the *Tongguanteng* ethanol extract, elute with dichloromethane, recover the dichloromethane, and discard the extract. Then, perform a dichloromethane-methanol gradient elution, collect the eluent, and perform TLC detection. Combine the main spots with the same (R²) f The fraction (0.2–0.5%) was removed, and the solvent was recovered under reduced pressure to obtain crude tongguantenol. The crude tongguantenol was then subjected to HPLC for preparative separation, and the three main peaks were collected and the solvent was recovered to obtain purified tongguantenol A, tongguantenol B, and tongguantenol C.

[0012] The water decoction method is described above, with a water-to-medicinal-material ratio of 10:1 to 20:1, a decoction time of 0.5 to 1 hour, and a decoction frequency of 1 to 2 times.

[0013] The macroporous resin column chromatography used is of type D101, and the amount used is 1 to 2 times the mass of the drug material. The eluent for removing impurities is 60% ethanol; then elution is performed with 95% ethanol, and the eluent volume is 3 to 5 times the column volume (BV).

[0014] The silica gel column chromatography uses silica gel with a particle size of 200-300 mesh and a sample-to-silica gel ratio of 1:60-1:100 (sample to silica gel, w / w). The dichloromethane elution method involves eluting with dichloromethane for 4-6 BV, recovering the dichloromethane, and discarding the eluent. The gradient elution uses dichloromethane-methanol ratios of 39:1 and 19:1 (v / v), with each gradient eluting for 4-6 BV at a flow rate of 1 BV / h and a fraction volume of 0.25 BV. After collecting the eluent, TLC analysis is performed. Fractions with a main spot Rf of 0.2-0.5 are combined, and the solvent is recovered under reduced pressure to obtain crude guanshinol.

[0015] The HPLC preparative separation described above uses a C10 column. 18 The column (250*20mm, 5μm) was used with acetonitrile-water as the mobile phase and gradient elution for 30 min (7:13~9:1, v / v); the flow rate was 8.0 ml / min; DAD detection was performed, and the two main peaks with a maximum absorption wavelength of 240 nm (Tongguantengol A and Tongguantengol C) and one with a maximum absorption wavelength of 210 nm (Tongguantengol B) were collected sequentially. The solvent was recovered to obtain the purified Tongguantengol product. Attached Figure Description

[0016] Figure 1 To pass the test for Tengchun A 13 C-NMR spectrum (C5D5N, 150MHz);

[0017] Figure 2 To pass the test of Tengchun B 13 C-NMR spectrum (C5D5N, 150MHz);

[0018] Figure 3 For passing the test of Tengchun C 13 C-NMR spectrum (C5D5N, 150MHz). Detailed Implementation

[0019] Example 1

[0020] This embodiment is implemented under the following conditions and technical requirements:

[0021] 1. Add 1 kg of *Thunb. tung* to 15 L of water and decoct for 1 hour. Centrifuge the extract and perform macroporous resin (1 kg) column chromatography. Elute with 60% ethanol to remove 3 BV and impurities. Elute with 95% ethanol to remove 3 BV and recover the ethanol to obtain *Thunb. tung* alcohol extract (4.5 g).

[0022] 2. The above-mentioned *Tongguanteng* alcohol extract was subjected to silica gel column chromatography (270 g silica gel). Elution with dichloromethane for 6 BV was performed, and the dichloromethane was recovered while the extract was discarded. Elution was then performed using a dichloromethane-methanol gradient (6 BV per gradient), and the eluent was collected. TLC analysis was performed, and fractions with the main spot at 0.2–0.5 μm were combined. The solvent was recovered to obtain crude *Tongguanteng* alcohol (331 mg).

[0023] 3. The crude tongguantengol was separated by HPLC, and three main chromatographic peaks were collected to obtain 19 mg of purified tongguantengol A (purity 95.6%, HPLC method), 23 mg of purified tongguantengol B (purity 97.2%, HPLC method), and 15 mg of purified tongguantengol C (purity 96.7%, HPLC method).

[0024] The physical properties and spectral data of the obtained components are as follows:

[0025] Tongguan Tengchun A: White powder; 1 H, 13 C-NMR: See Table 1; HR-ESI-MS m / z: 429.2643 ([M+H] + C 26 H 37 O5, calculated value 429.2641).

[0026] Tongguan Tengchun B: White powder; 1 H, 13 C-NMR: See Table 1; HR-ESI-MS m / z: 349.2417 ([M+H] + C 21 H 33 O4, calculated value 349.2379).

[0027] Tongguan Tengchun C: White powder; 1 H, 13 C-NMR: See Table 1; HR-ESI-MS m / z: 347.2205 ([M+H] + C 21 H 31 O4, calculated value 347.2222).

[0028] Table 1. Passing through the vine alcohol 1 H-NMR (C5D5N, 600MHz), 13 C-NMR (C5D5N, 150MHz) data

[0029]

[0030]

[0031] Example 2

[0032] This embodiment is implemented under the following conditions and technical requirements:

[0033] 1. Add 1 kg of *Thunb. tongguanteng* (a type of herb) to 10 L of water and decoct twice, 0.5 h each time. Combine the two extracts. Centrifuge the extract and perform macroporous resin (2 kg) column chromatography. Elute with 60% ethanol for 5 BV to remove impurities; elute with 95% ethanol for 5 BV and recover the ethanol to obtain *Thunb. tongguanteng* alcohol extract (6.0 g).

[0034] 2. The above-mentioned *Tongguanteng* alcohol extract was subjected to silica gel column chromatography (600g silica gel). Elution was performed with dichloromethane for 5 BV, and the dichloromethane was recovered, while the extract was discarded. Elution was then performed with a dichloromethane-methanol gradient (5 BV for each gradient), and the eluent was collected. TLC analysis was performed, and fractions with the main spot at 0.2–0.5 μm were combined. The solvent was recovered to obtain crude *Tongguanteng* alcohol (410 mg).

[0035] 3. The crude tongguantengol was separated by HPLC, and three main chromatographic peaks were collected to obtain 21 mg of purified tongguantengol A (purity 96.3%, HPLC method), 34 mg of purified tongguantengol B (purity 95.7%, HPLC method), and 21 mg of purified tongguantengol C (purity 97.0%, HPLC method).

[0036] Example 3

[0037] This embodiment is implemented under the following conditions and technical requirements:

[0038] 1. Add 1 kg of *Thunb. 20 L of water and decoct for 0.75 h. Centrifuge the extract and perform macroporous resin (1.5 kg) column chromatography. Elute with 60% ethanol for 4 BV to remove impurities. Elute with 95% ethanol for 4 BV and recover the ethanol to obtain *Thunb. 2 g* of ethanol extract.

[0039] 2. The above-mentioned Tongguanteng alcohol extract was subjected to silica gel column chromatography (400g silica gel). Elute with dichloromethane for 4 BV, recover the dichloromethane, and discard the extract. Elute with a dichloromethane-methanol gradient (4 BV for each gradient), and collect the eluent. Detect by TLC, combine fractions with the main spot at 0.2–0.5 μm, and recover the solvent to obtain crude Tongguanteng alcohol (376 mg).

[0040] 3. The crude tongguantengol was separated by HPLC, and three main chromatographic peaks were collected to obtain 18 mg of purified tongguantengol A (purity 96.3%, HPLC method), 22 mg of purified tongguantengol B (purity 95.7%, HPLC method), and 13 mg of purified tongguantengol C (purity 97.0%, HPLC method).

[0041] Example 4

[0042] This embodiment relates to the antitumor application of the aforementioned type of tranexamic acid, specifically its use in inhibiting the growth of A549 and Bel 7402 cells. The specific detection process is as follows:

[0043] Step 1) Human lung cancer cells A549 and human liver cancer cells Bel-7402 were seeded in RPMI 1640 medium containing 10% fetal bovine serum and cultured. The medium was changed and passaged every 24 hours. Cells in the logarithmic growth phase were harvested, digested into single cells with 0.25% trypsin, counted, and diluted with culture medium to a final concentration of 1×10⁻⁶ cells. 5 Single-cell suspension of 100 μL per well was seeded into 96-well culture plates and incubated overnight at 37°C in a 5% CO2 incubator. After the cells adhered, the experiment was performed.

[0044] Step 2) After culturing human lung cancer cells A549 and human liver cancer cells Bel-7402 for 24 hours, carefully aspirate the culture medium and add 100 μL of each concentration of drug solution (5 replicates per concentration). Add 100 μL of culture medium to the blank control group. Continue culturing in a 37℃, 5% CO2 incubator. After 48 hours, add 20 μL of 5 mg / mL MTT solution to each well and continue culturing in the incubator. After 4 hours, remove the cells, aspirate the supernatant from each well, add 100 μL of DMSO to each well, and shake at low speed on a horizontal shaker for 10 minutes (90 rpm, 37℃). After the purple crystals in each well have fully dissolved, measure the OD value of each well at a wavelength of 490 nm using a microplate reader.

[0045] The drug solutions of various concentrations refer to: 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μM.

[0046] The tumor cell growth inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - mean OD value of the treatment group / mean OD value of the control group) × 100%. Based on this, the half-maximal inhibitory concentration (IC50) was calculated using SPSS. 50 ).

[0047] The above experiment was repeated 3 times in parallel, and the average value was calculated. and standard deviation (SD). The specific results are shown in Table 1.

[0048] Table 1. Effects of Tongguan Tengli on the Inhibition of Tumor Cell Growth

[0049]

[0050] As shown in Table 1, all three components of the Tongguanteng alcohol group have significant cytotoxic activity and significantly inhibit the proliferation of tumor cells. Among them, the effect of Tongguanteng alcohol A is comparable to that of the positive control drug (5-fluorouracil).

[0051] Example 5

[0052] This embodiment relates to a rapid and qualitative identification method for *Thunb. guanylate* based on the aforementioned type of *Thunb. guanylate* is pulverized, 2g is taken, and 50ml of ethyl acetate is added for ultrasonic extraction for 30min. Ethyl acetate is recovered under reduced pressure, 0.5ml of methanol is added, and the mixture is centrifuged. The supernatant is used as the test solution. The test solution and three *Thunb. guanylate solutions are spotted on the same silica gel G plate, developed with dichloromethane-methanol (19:1), and heated with 10% sulfuric acid-ethanol for color development. The sample shows spots of the same color at the same positions as the *Thunb. guanylate* solutions.

[0053] Compared with existing technologies, this method can simultaneously prepare three new compounds. Macroporous resin column chromatography removes most of the interference from water-soluble components, effectively enriching the tonkinensis alcohols and improving the yield. During silica gel column chromatography, dichloromethane elution is used first to further remove fatty acids, sterols, and fat-soluble pigments, enabling a simple and rapid preparation of tonkinensis alcohols. The obtained tonkinensis alcohols, as determined by HPLC, have a purity exceeding 95%. The organic solvents used throughout the preparation process, as well as the packing materials used in the separation, can be reused repeatedly. Furthermore, the simultaneous preparation of three tonkinensis alcohols fully utilizes raw materials and chemical reagents, significantly reducing preparation costs.

[0054] The above-described specific implementations can be partially adjusted by those skilled in the art in different ways without departing from the principles and purpose of the present invention. The scope of protection of the present invention is defined by the claims and is not limited to the above-described specific implementations. All implementation schemes within the scope of the claims are bound by the present invention.

Claims

1. A class of tranexamic acid compounds, characterized in that, Specifically, they are: Tongguantenol A, Tongguantenol B, and Tongguantenol C, whose chemical structures are shown below: 。 2. An application of the guanguan sucrose alcohol compound according to claim 1, characterized in that, Used in the preparation of antitumor drugs; The tumors mentioned are specifically: human lung cancer cells A549 and human liver cancer cells Bel-7402.

3. A method for extracting guanseng alcohol compounds according to claim 1, characterized in that, The dried stems of *Thunb. tongguanteng* were boiled in water, and the resulting decoction was centrifuged and subjected to macroporous resin column chromatography. After separation by silica gel column chromatography and HPLC, three main chromatographic peaks were collected to obtain purified *Thunb. tongguanteng* alcohol A, purified *Thunb. tongguanteng* alcohol B, and purified *Thunb. tongguanteng* alcohol C.

4. The method for extracting guanshanol compounds according to claim 3, characterized in that, The macroporous resin column chromatography refers to the following steps: after passing the aqueous decoction through a D101 macroporous resin column with a volume of 1-2 times that of the medicinal material, the decoction is eluted with 60% ethanol for 3-5 BV to remove water-soluble impurities; then eluted with 95% ethanol for 3-5 BV, the 95% ethanol eluent is collected, and the solvent is recovered under reduced pressure to obtain the tungsten extract.

5. The method for extracting guanseng alcohol compounds according to claim 4, characterized in that, The silica gel column chromatography described refers to the following steps: The extract of *Tongguanteng* alcohol is subjected to silica gel column chromatography, with the amount of silica gel being 60-100 times the sample weight; elution is performed with dichloromethane for 4-6 BV, the dichloromethane is recovered, and the extract is discarded; then, a dichloromethane-methanol gradient elution is performed at a volume ratio of 39:1-19:1, with each gradient elution for 4-6 BV, a flow rate of 1 BV / h, and 0.25 BV / split. The eluent is collected and then analyzed by TLC, using a dichloromethane-methanol volume ratio of 19:1 as the developing solvent. Using 10% sulfuric acid-ethanol as the colorimetric reagent, the color is developed by heating and then detected. Combined main spots with the same R value are considered. f 0.2-0.5 fractions were discharged, and the solvent was recovered under reduced pressure to obtain crude guanguan vine alcohol.

6. The method for extracting guanshanol compounds according to claim 5, characterized in that, The HPLC preparation and separation refers to the following: crude tongguantengol is prepared and separated by HPLC, that is, a 250*20mm, 5μm C18 column is used, and acetonitrile-water gradient elution with a volume ratio of 7:13-9:1 is used for 30 min; the flow rate is 8.0 ml / min; the detection wavelength is 240 nm and 210 nm, and the three main peaks are collected and the solvent is recovered to obtain purified tongguantengol A, tongguantengol B and tongguantengol C.

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