阅读说明：本技术 大花独蒜兰丁二酸类化合物及其提取方法和应用 (Pleione bulbocodioides succinic acid compound and its extraction method and application ) 是由 吴沙沙 倪林 翟俊文 兰思仁 李威 吴美婷 彭东辉 秦思 韩茹 肖云 于 2021-09-18 设计创作，主要内容包括：本发明公开了大花独蒜兰丁二酸类化合物及其提取方法和其药物组合物与用途,其提取方法具体为大花独蒜兰的新鲜假鳞茎切碎,烘干至恒重,将干燥后的样品经粉碎机粉碎至粉末状,将粉末过四号筛,用75%甲醇回流提取2次,每次2 h,减压浓缩得浸膏。将浸膏溶解于24 L纯水中,选用大孔吸附树脂D101用乙醇-水系统进行洗脱,得到4个组分的浸膏,分别为DH-1、DH-2、DH-3、DH-4。其中60%乙醇部位的浸膏DH-3经过多次柱层析(硅胶、凝胶),最后用HPLC纯化,得到六个新丁二酸类化合物。本发明提取的得到新丁二酸类化合物可以用于制备治疗肝癌药物。(The invention discloses a major flower Pleione succinic acid compound, an extraction method thereof, a pharmaceutical composition thereof and an application thereof, wherein the extraction method specifically comprises the steps of cutting fresh pseudo bulbs of the major flower Pleione, drying to constant weight, crushing a dried sample into powder by a crusher, sieving the powder by a four-mesh sieve, performing reflux extraction for 2 times with 75% methanol for 2 hours each time, and performing reduced pressure concentration to obtain an extract. Dissolving the extract in 24L pure water, and eluting with macroporous adsorbent resin D101 with ethanol-water system to obtain extracts of 4 components, DH-1, DH-2, DH-3, and DH-4. Wherein the extract DH-3 of the 60 percent ethanol part is subjected to column chromatography (silica gel and gel) for multiple times, and finally purified by HPLC to obtain six neo-succinic acid compounds. The novel succinic acid compound extracted by the invention can be used for preparing a medicament for treating liver cancer.)

1. The giant typhonium giganteum and amaranth succinic acid compound is characterized in that: including compound 1, compound 2, compound 3, compound 4, compound 5, or compound 6, having the following structural formula:

、、、、、。

2. the method for extracting the giant pleione succinic acid compound as claimed in claim 1, which is characterized by comprising the following steps: the method comprises the following steps:

(1) cutting fresh pseudobulb of Pleione bulbocodioides, oven drying to constant weight, pulverizing into powder, sieving with a four-mesh sieve, reflux-extracting with 75wt% methanol for 2 times (each for 2 hr), and concentrating under reduced pressure to obtain extract;

(2) dissolving the extract obtained in the step (1) in pure water, and eluting with ethanol-water system by using macroporous adsorption resin D101 to obtain extracts of 4 components, namely DH-1, DH-2, DH-3 and DH-4;

(3) subjecting the extract DH-3 to silica gel column chromatography and gradient elution with dichloromethane-methanol system to obtain 7 fractions DH-3.1-DH-3.7, wherein the DH-3.3 is subjected to gradient elution with PRP512A macroporous adsorbent resin and ethanol-water system to obtain 10 fractions DH-3.3.1-DH-3.3.10;

(4) separating DH-3.3.3 of the step (3) by Sephadex LH-20 column chromatography, eluting with methanol to obtain 7 components of DH-3.3.3.1-DH-3.3.3.7, and purifying DH-3.3.3.3 by HPLC to obtain a compound 1 and a compound 2;

(5) and (3) purifying the DH-3.3.4 obtained in the step (3) by using Sephadex LH-20 column chromatography, eluting by using methanol to obtain 5 components of DH-3.3.4.1-DH-3.3.4.5, and purifying DH-3.3.4.2 by HPLC to obtain a compound 3, a compound 4, a compound 5 and a compound 6.

3. The use of the Pleione latifolia succinate of claim 1 in the preparation of a medicament for treating liver cancer.

Technical Field

The invention relates to six novel succinic acid compounds (1-6) in Pleione grandiflorum effective components and an extraction method thereof, and the six novel succinic acid compounds are applied to preparation of a medicament for treating liver cancer.

Background

Liver cancer is the 4 th most fatal malignant tumor worldwide, occupies the 3 rd position of the global cancer fatality rate, has high malignancy degree and high recurrence rate, is easy to transfer, and seriously threatens the health and the life of human beings. Although the chemical treatment of liver cancer has been greatly developed, no good medicine for eliminating liver cancer exists in clinic at present, and the problems of strong drug resistance, great toxic and side effects and the like exist. Therefore, the search for novel anti-liver cancer drugs with novel structures and small toxic and side effects is urgent. And the new compound separated from the plant is an important way for finding the lead compound.

Pleione grandiflorum is a plant of Pleione genus Pleione belonging to Orchidaceae, mainly produced in Yunnan province of China, and has abundant resources. The succinic acid compound is a specific component in orchidaceae plants, and has relatively strong anti-liver cancer activity.

Disclosure of Invention

The invention discovers that the Pleione majorana extract has the effect of treating the liver cancer for the first time, six new succinic acid compounds are separated from the effective part, and pharmacodynamic evaluation shows that the Pleione majorana extract has a good effect of treating the liver cancer.

The structural formula of the major flower Pleione succinate compound 1-6 disclosed by the invention is as follows:

、、、、、。

the invention also provides an extraction method of the compound, which comprises the following steps:

50 kg of fresh pseudobulb of Pleione bulbocodioides is cut into pieces, dried in an oven at 50 deg.C to constant weight (about 18 h), and the dried sample is pulverized into powder by a pulverizer to obtain 7 kg of sample. Sieving the powder with a sieve IV, extracting with 75% methanol under reflux for 2 times, each time for 2 hr, and concentrating under reduced pressure to obtain 2.45 kg of extract. Dissolving the extract in 24L pure water, and eluting with ethanol-water system using macroporous adsorbent resin D101 to obtain extracts of 4 components, DH-1 (87.01 g), DH-2 (308.31 g), DH-3 (156.44 g), and DH-4 (145.00 g). Since the target product is a medium-polar component, the medium-polar component DH-3 was selected and further separated and purified, and fraction DH-3 was separated and purified by silica gel column chromatography using a dichloromethane-methanol gradient (dichloromethane: methanol =20:1 to 1: 1) to obtain 7 fractions (DH-3.1 → DH-3.7). By silica gel thin layer chromatography, medium polar component DH-3.3 (17 g) was selected and further separated and purified, PRP512A macroporous adsorbent resin was used, and 10 fractions (DH-3.3.1 → DH-3.3.10) were obtained by eluting with ethanol-water system (ethanol: water =1: 9-0: 1). DH-3.3.3.3 (3.35 g) were chromatographed on a Sephadex LH-20 column eluting with methanol to give 7 fractions (DH-3.3.3.1 → DH-3.3.3.7). DH-3.3.3.3 (586.5 mg) was purified by semi-preparative HPLC (mobile phase: aqueous methanol, 55%) to give Compound 1 (28.0 mg) and 2 (8.2 mg). DH-3.3.4 (2.2 g) was purified by Sephadex LH-20 column chromatography eluting with methanol to give 5 fractions (DH-3.3.4.1-DH-3.3.4.5). DH-3.3.4.2 (366.4 mg) was purified by semi-preparative HPLC (mobile phase: aqueous MeOH, 55%) to give compounds 3 (4.1 mg), 4 (24.3 mg), 5 (12.1 mg) and 6 (4.4 mg).

The invention also provides a pharmaceutical composition which takes the compounds 1-6 of the invention as active ingredients. The pharmaceutical composition may be prepared according to methods well known in the art. The compounds of the invention may be formulated into any dosage form suitable for human or animal use by combining them with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants. The compounds of the present invention are generally present in the pharmaceutical compositions in an amount of 0.1 to 95%.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by enteral or parenteral routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oromucosal, ophthalmic, pulmonary and respiratory, dermal, vaginal, rectal, and the like. The dosage form for administration may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The liquid dosage forms can be solution (including true solution and colloidal solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion, liniment, etc.; the solid dosage form can be tablet (including common tablet, enteric coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, and enteric coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol, spray, etc., and the semisolid dosage form can be ointment, gel, paste, etc.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle drug delivery systems.

For tableting the compounds of the invention, a wide variety of excipients known in the art may be used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the humectant can be water, ethanol, isopropanol, etc.; the binder can be starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

To encapsulate the administration units, the active ingredient of the compounds of the invention can be mixed with diluents and glidants and the mixture can be placed directly into hard or soft capsules. Or the effective component of the compound of the invention can be prepared into granules or pellets with diluent, adhesive and disintegrating agent, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, glidants used to prepare the compound tablets of the present invention may also be used to prepare capsules of the compound of the present invention.

In order to prepare the compound of the invention into injection, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator which are commonly used in the field can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol and glucose can be added as proppant for preparing lyophilized powder for injection.

In addition, colorants, preservatives, flavors, or other additives may also be added to the pharmaceutical preparation, if desired.

For the purpose of administration and enhancing the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The dosage of the pharmaceutical composition of the compound of the present invention to be administered may vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route and dosage form of administration, and the like. Generally, a suitable daily dosage range for a compound of the invention is from 0.001 to 150 mg/kg body weight, preferably from 0.1 to 100 mg/kg body weight, more preferably from 1 to 60 mg/kg body weight, most preferably from 2 to 30 mg/kg body weight. The above-described dosage may be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosage regimen including the use of other therapeutic means.

The compound or composition of the present invention can be administered alone or in combination with other therapeutic agents or symptomatic drugs, and when the compound of the present invention acts synergistically with other therapeutic agents, its dosage should be adjusted as appropriate.

The invention performs the cell experiment related to the liver cancer resistance on the new succinic acid compounds 1-6 of the Pleione mauritiana, and can better inhibit the growth of HepG2 liver cancer cells.

Drawings

FIG. 1 is a flow chart of the extraction method of the giant typhonium giganteum succinic acid compounds 1-6 of the invention.

Detailed Description

For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.

50 kg of fresh bulbodium giganteum of the Pleione majorana is cut up, dried in a 50 ℃ oven for 18 h, and the dried sample is crushed into powder by a crusher to obtain 7 kg of sample. Sieving the powder with a sieve IV, heating with 75% methanol in a water bath at 80 deg.C, condensing and refluxing for 2 hr each time, and concentrating under reduced pressure to obtain 2.45 kg of extract with yield of 35%. Dissolving the extract in 24L of pure water, performing gradient elution by using a macroporous adsorption resin D101 and an ethanol-water solvent (ethanol: water =0: 100-90: 10; volume ratio), collecting eluent, taking 500 mL as one component, and respectively concentrating under reduced pressure; the concentrated eluate was analyzed by silica gel Thin Layer Chromatography (TLC) to give 4 fractions of DH-1 (87.01 g), DH-2 (308.31 g), DH-3 (156.44 g) and DH-4 (145.00 g). According to the TLC analysis result, the medium polar component DH-3 is selected for further separation and purification.

Subjecting extract DH-3 (156.44 g) of 60% ethanol elution part to silica gel column chromatography, gradient eluting with dichloromethane-methanol solvent (dichloromethane: methanol =20: 1-1: 1), collecting each 500 mL fraction, concentrating under reduced pressure, and mixing to obtain 7 fractions (DH-3.1-DH-3.7); gradient-eluting DH-3.3 (17 g) with PRP512A resin and ethanol-water solvent (ethanol: water =10: 90-100: 0), collecting fractions in 250 mL conical flask, concentrating under reduced pressure, and mixing to obtain 10 fractions (DH-3.3.1-DH-3.3.10). TLC analysis, using sephadex LH-20 column chromatography of DH-3.3.3 (3.35 g), eluting with methanol, collecting fractions in 10 mL tubes, and mixing by TLC analysis to obtain 7 fractions (DH-3.3.3.1-DH-3.3.3.7). By High Performance Liquid Chromatography (HPLC), DH-3.3.3.3 (586.5 mg) with ultraviolet absorption of 279.4 nm and 222.6 nm target peak is selected for further separation and purification. DH-3.3.3.3 prepared by preparative liquid chromatography (column: C18, mobile phase: 55% methanol, set flow rate: 8.0 mL. min^-1Detection wavelength 230 nm) was purified to obtain compounds 1 (28.0 mg) and 2 (8.2 mg).

By TLC analysis, selectionDH-3.3.4 (2.2 g) was chromatographed on Sephadex LH-20 column, eluted with methanol, and fractions were collected in 10 mL tubes and combined by TLC to give 5 fractions (DH-3.3.4.1-DH-3.3.4.5). By HPLC analysis, DH-3.3.4.2 (366.4 mg) having UV absorption of 279.4 nm and 222.6 nm as the target peak was selected and subjected to preparative liquid chromatography (column: C18, mobile phase: 55% methanol, set flow rate: 8.0 mL. min^-1Detection wavelength 230 nm) to obtain compounds 3 (4.1 mg), 4 (24.3 mg), 5 (12.1 mg) and 6 (4.4 mg).

Physicochemical and spectral data for compound 1 Pleioneside a are as follows:

2.99 (1H, d, J = 17.6 Hz, H-3a), 3.09 (IH, d, J = 17.6 Hz, H-3b), 1.67 (1H, m, H-5a), 1.73 (1H, m, H-5b), 1.78 (1H, m, H-6), 0.92 (1H, d, J = 6.4 Hz, H-7), 0.81 (1H, d, J = 6.4 Hz, H-8), 7.34 (2H, d, J = 8.8 Hz, H-2', 6'), 7.12 (2H, d, J = 8.8 Hz, H-3', 5'), 5.19 (1H, d, J = 12.0 Hz, H-7'a), 5.08 (1H, d, J = 12.0 Hz, H-7'b), 5.01 (1H, d, J = 7.6 Hz, H-1''), 3.60 (1H, m, H-2''), 3.78 (1H, m, H-3''), 4.99 (1H, t, J = 9.6 Hz, H-4''), 3.77 (1H, m, H-5''), 3.66 (1H, m, H-6''a), 3.57 (1H, m, H-6''b), 7.63 (2H, m, H-2''', 6'''), 7.42 (2H, m, H-3''', 5'''), 7.42 (1H, m, H-4'''), 7.77 (1H, d, J = 16.0 Hz, H-7'''), 6.60 (1H, d, J = 16.0 Hz, H-8'''), 4.95 (1H, d, J = 6.8 Hz, H-1''''), 3.22 (1H, t, J = 8.0 Hz, H-2''''), 3.40 (1H, m, H-3''''), 3.06 (1H, m, H-4''''), 3.35 (1H, m, H-5''''), 3.76 (1H, m, H-6''''a), 3.69 (1H, m, H-6''''b)。175.1 (C-1), 81.5 (C-2), 43.8 (C-3), 49.5 (C-5), 24.9 (C-6), 24.8 (C-7), 24.3 (C-8), 130.8 (C-1'), 131.4 (C-2', 6'), 117.8 (C-3', 5'), 159.2 (C-4'), 68.2 (C-7'), 102.2 (C-1''), 75.0 (C-2''), 75.7 (C-3''), 72.4 (C-4''), 76.2 (C-5''), 62.2 (C-'6'), 135.7 (C-1'''), 129.3 (C-2''', 6'''), 130.1 (C-3''', 5'''), 131.7 (C-4'''), 147.0 (C-7'''), 118.5 (C-8'''), 167.8 (C-9'''), 100.1 (1''''), 75.5 (2''''), 77.5 (3''''), 70.6 (4''''), 78.6 (5''''), 62.0 (6'''')。

physicochemical, spectral data for compound 2 pleionineside B are as follows:

2.99 (1H, d, J = 17.6 Hz, H-3a), 3.09 (IH, d, J = 17.6 Hz, H-3b), 1.67 (1H, m, H-5a), 1.73 (1H, m, H-5b), 1.78 (1H, m, H-6), 0.92 (1H, d, J = 6.4 Hz, H-7), 0.81 (1H, d, J = 6.4 Hz, H-8), 7.35 (2H, d, J = 8.8 Hz, H-2', 6'), 7.11 (2H, d, J = 8.8 Hz, H-3', 5'), 5.17 (1H, d, J = 12.0 Hz, H-7'a), 5.08 (1H, d, J = 12.0 Hz, H-7'b), 5.05 (1H, d, J = 8.0 Hz, H-1''), 3.68 (1H, m, H-2''), 5.20 (1H, m, H-3''), 3.66 (1H, m, H-4''), 3.60 (1H, m, H-5''), 3.92 (1H, dd, J = 2.0, 12.0 Hz, H-6''a), 3.76 (1H, dd, J = 5.2, 12.0 Hz, H-6''b), 7.64 (2H, m, H-2''', 6'''), 7.42 (2H, m, H-3''', 5'''), 7.42 (1H, m, H-4'''), 7.77 (1H, d, J = 16.0 Hz, H-7'''), 6.64 (1H, d, J = 16.0 Hz, H-8'''), 4.95 (1H, d, J = 8.0 Hz, H-1''''), 3.22 (1H, t, J = 8.0 Hz, H-2''''), 3.40 (1H, m, H-3''''), 3.07 (1H, m, H-4''''), 3.36 (1H, m, H-5''''), 3.76 (1H, m, H-6''''a), 3.70 (1H, m, H-6''''b)。175.2 (C-1), 81.6 (C-2), 44.0 (C-3), 49.6 (C-5), 24.9 (C-6), 24.8 (C-7), 24.3 (C-8), 130.8 (C-1'), 131.4 (C-2', 6'), 117.7 (C-3', 5'), 159.2 (C-4'), 68.2 (C-7'), 102.1 (C-1''), 75.0 (C-2''), 79.0 (C-3''), 69.5 (C-4''), 78.0 (C-5''), 62.1 (C-6''), 135.9 (C-1'''), 129.2 (C-2''', 6'''), 130.0 (C-3''', 5'''), 131.5 (C-4'''), 146.4 (C-7'''), 119.2 (C-8'''), 168.3 (C-9'''), 100.1 (1''''), 75.6 (2''''), 77.5 (3''''), 70.7 (4''''), 78.6 (5''''), 62.1 (6'''')。

physicochemical and spectral data for compound 3 pleionineside C are as follows:

2.64 (1H, d, J = 16.0 Hz, H-3a), 2.94 (IH, d, J = 16.0 Hz, H-3b), 1.57 (1H, dd, J = 5.6, 14.0 Hz, H-5a), 1.66 (1H, dd, J = 5.6, 14.0 Hz, H-5b), 1.72 (1H, m, H-6), 0.91 (1H, d, J = 6.4 Hz, H-7), 0.79 (1H, d, J = 6.4 Hz, H-8), 7.26 (2H, d, J = 8.8 Hz, H-2', 6'), 7.09 (2H, d, J = 8.8 Hz, H-3', 5'), 4.99 (2H, m, H-7'), 7.28 (2H, m, H-2'', 6''), 7.09 (2H, m, H-3'', 5''), 4.99 (1H, m, H-7''), 5.05 (1H, d, J = 8.0 Hz, H-1'''), 3.67 (1H, m, H-2'''), 5.21 (1H, m, H-3'''), 3.68 (1H, m, H-4'''), 3.59 (1H, m, H-5'''), 3.91 (1H, dd, J = 2.0, 12.0 Hz, H-6'''a), 3.76 (1H, dd, J = 5.2, 12.0 Hz, H-6'''b), 4.90 (1H, d, J = 7.6 Hz, H-1''''), 3.48 (1H, m, H-2''''), 3.41 (1H, m, H-3''''), 3.44 (1H, m, H-4''''), 3.48 (1H, m, H-5''''), 3.88 (1H, dd, J = 2.0, 12.0 Hz, H-6''''a), 3.71 (1H, m, H-6''''b), 7.63 (2H, m, H-2''''', 6'''''), 7.42 (2H, m, H-3''''', 5'''''), 7.42 (1H, m, H-4'''''), 7.77 (1H, d, J = 16.0 Hz, H-7'''''), 6.64 (1H, d, J = 16.0 Hz, H-8''''')。176.0 (C-1), 76.6 (C-2), 46.2 (C-3), 49.2 (C-5), 25.0 (C-6), 24.7 (C-7), 23.9 (C-8), 131.1 (C-1'), 131.3 (C-2', 6'), 117.8 (C-3', 5'), 159.1 (C-4'), 67.9 (C-7'), 130.9 (C-1''), 131.1 (C-2'', 6''), 117.7 (C-3'', 5''), 159.0 (C-4''), 67.2 (C-7''), 102.0 (C-1'''), 73.2 (C-2'''), 78.9 (C-3'''), 69.4 (C-4'''), 77.9 (C-5'''), 62.1 (C-6'''), 102.2 (C-1''''), 74.8 (C-2''''), 77.8 (C-3''''), 71.2 (C-4''''), 78.0 (C-5''''), 62.4 (C-6''''), 135.8 (C-1'''), 129.2 (C-2''', 6'''), 130.0 (C-3''', 5'''), 131.5 (C-4'''), 146.5 (C-7'''), 119.1 (C-8'''), 168.4 (C-9''')。

physicochemical, spectral data for compound 4 pleionineside D are as follows:

2.60 (1H, d, J = 16.0 Hz, H-3a), 2.90 (IH, d, J = 16.0 Hz, H-3b), 1.57 (1H, dd, J = 6.0, 14.0 Hz, H-5a), 1.64 (1H, J = 6.0, 14.0 Hz, H-5b), 1.72 (1H, m, H-6), 0.92 (1H, d, J = 6.4 Hz, H-7), 0.79 (1H, d, J = 6.4 Hz, H-8), 7.35 (2H, d, J = 8.8 Hz, H-2', 6'), 7.11 (2H, d, J = 8.8 Hz, H-3', 5'), 5.12 (1H, s, H-7), 5.08 (1H, d, J = 8.0 Hz, H-1''), 3.71 (1H, m, H-2''), 5.20 (1H, m, H-3''), 3.67 (1H, m, H-4''), 3.60 (1H, m, H-5''), 3.92 (1H, dd, J = 2.0, 12.0 Hz, H-6''a), 3.76 (1H, dd, J = 5.2, 12.0 Hz, H-6''b), 7.63 (2H, m, H-2''', 6'''), 7.42 (2H, m, H-3''', 5'''), 7.42 (1H, m, H-4'''), 7.77 (1H, d, J = 16.0 Hz, H-7'''), 6.64 (1H, d, J = 16.0 Hz, H-8''')。176.4 (C-1), 76.6 (C-2), 45.8 (C-3), 49.8 (C-5),25.0 (C-6), 24.7 (C-7), 24.0 (C-8), 131.0 (C-1'), 131.2 (C-2', 6'), 117.7 (C-3', 5'), 159.0 (C-4'), 67.9 (C-7'), 102.0 (C-1''), 73.2 (C-2''), 78.8 (C-3''), 69.4 (C-4''), 77.8 (C-5''), 62.0 (C-6''), 135.7 (C-1'''), 129.2 (C-2''', 6'''), 130.0 (C-3''', 5'''), 131.6 (C-4'''), 146.5 (C-7'''), 118.9 (C-8'''), 168.5 (C-9''')。

physicochemical, spectral data for compound 5 pleionineside E are as follows:

2.62 (1H, d, J = 16.0 Hz, H-3a), 2.92 (IH, d, J = 16.0 Hz, H-3b), 1.57 (1H, dd, J = 5.6, 14.0 Hz, H-5a), 1.67 (1H, dd, J = 5.6, 14.0 Hz, H-5b), 1.71 (1H, m, H-6), 0.92 (1H, d, J = 6.4 Hz, H-7), 0.80 (1H, d, J = 6.4 Hz, H-8), 7.36 (2H, d, J = 8.8 Hz, H-2', 6'), 7.12 (2H, d, J = 8.8 Hz, H-3', 5'), 5.16 (1H, d, J = 12.0 Hz, H-7), 5.11 (1H, d, J = 12.0 Hz, H-7), 5.07 (1H, d, J = 8.0 Hz, H-1''), 3.69 (1H, m, H-2''), 5.19 (1H, m, H-3''), 3.66 (1H, m, H-4''), 3.60 (1H, m, H-5''), 3.91 (1H, dd, J = 2.0, 12.0 Hz, H-6''a), 3.75 (1H, dd, J = 5.2, 12.0 Hz, H-6''b), 7.64 (2H, m, H-2''', 6'''), 7.42 (2H, m, H-3''', 5'''), 7.42 (1H, m, H-4'''), 7.77 (1H, d, J = 16.0 Hz, H-7'''), 6.64 (1H, d, J = 16.0 Hz, H-8''')。176.0 (C-1), 76.6 (C-2), 45.9 (C-3), 49.1 (C-5), 25.1 (C-6), 24.7 (C-7), 23.9 (C-8), 131.0 (C-1'), 131.4 (C-2', 6'), 117.7 (C-3', 5'), 159.1 (C-4'), 67.9 (C-7'), 102.0 (C-1''), 73.2 (C-2''), 78.9 (C-3''), 69.4 (C-4''), 77.9 (C-5''), 62.1 (C-6''), 135.8 (C-1'''), 129.2 (C-2''', 6'''), 130.1 (C-3''', 5'''), 131.5 (C-4'''), 146.5 (C-7'''), 119.1 (C-8'''), 168.4 (C-9''')。

physicochemical and spectral data for compound 6 pleionineside F are as follows:

2.57 (1H, d, J = 15.6 Hz, H-3a), 2.86 (IH, d, J = 15.6 Hz, H-3b), 1.51 (1H, dd, J = 6.0, 14.0 Hz, H-5a), 1.59 (1H, dd, J = 6.0, 14.0 Hz, H-5b), 1.67 (1H, m, H-6), 0.88 (1H, d, J = 6.4 Hz, H-7), 0.74 (1H, d, J = 6.4 Hz, H-8), 7.18 (2H, d, J = 8.8 Hz, H-2', 6'), 7.03 (2H, d, J = 8.8 Hz, H-3', 5'), 4.89 (2H, m, H-7'), 7.22 (2H, m, H-2'', 6''), 7.06 (2H, m, H-3'', 5''), 4.94 (1H, m, H-7''), 4.90 (1H, m, H-1'''), 3.52 (1H, m, H-2'''), 3.44 (1H, m, H-3'''), 3.40 (1H, m, H-4'''), 3.75 (1H, m, H-5'''), 4.55 (1H, dd, J = 2.0, 12.0 Hz, H-6'''a), 4.38 (1H, dd, J = 7.2, 12.0 Hz, H-6'''b), 4.87 (1H, d, J = 7.6 Hz, H-1''''), 3.49 (1H, m, H-2''''), 3.43 (1H, m, H-3''''), 3.46 (1H, m, H-4''''), 3.47 (1H, m, H-5''''), 3.88 (1H, dd, J = 2.0, 12.0 Hz, H-6''''a), 3.70 (1H, m, H-6''''b), 7.61 (2H, m, H-2''''', 6'''''), 7.43 (2H, m, H-3''''', 5'''''), 7.43 (1H, m, H-4'''''), 7.69 (1H, d, J = 16.0 Hz, H-7'''''), 6.55 (1H, d, J = 16.0 Hz, H-8''''')。175.9 (C-1), 76.6 (C-2), 46.1 (C-3), 49.2 (C-5), 25.0 (C-6), 24.7 (C-7), 23.9 (C-8), 131.1 (C-1'), 131.2 (C-2', 6'), 117.8 (C-3', 5'), 159.1 (C-4'), 67.8 (C-7'), 130.9 (C-1''), 131.0 (C-2'', 6''), 117.7 (C-3'', 5''), 158.9 (C-4''), 67.1 (C-7''), 102.0 (C-1'''), 74.8 (C-2'''), 77.9 (C-3'''), 71.8 (C-4'''), 75.4 (C-5'''), 64.9 (C-6'''), 102.2 (C-1''''), 74.8 (C-2''''), 77.8 (C-3''''), 71.3 (C-4''''), 78.1 (C-5''''), 62.4 (C-6''''), 135.7 (C-1'''), 129.4 (C-2''', 6'''), 130.2 (C-3''', 5'''), 131.7 (C-4'''), 146.6 (C-7'''), 118.7 (C-8'''), 168.3 (C-9''')。

pharmacological experiments

Experimental material 1. test drugs: the monomer compound of the present invention. 2. Positive control: 5-Fluorouracil, supplied by Aladdin technologies, Inc. HPLC purity is more than or equal to 98 percent. 3. Cell: HepG2 liver cancer cell (China center for type culture preservation). 4. RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin).

Experimental example 1: inhibitory Activity of the Compound of the present invention on liver cancer cell

Cells were cultured in RRMI1640 medium containing 10% fetal bovine serum, 100U/mL penicillin and 100 mg/L at 37 ℃ in 5% CO₂Subculturing in a saturated humidity incubator, and selecting logarithmic growth cycle cells for experiments. Taking cells in logarithmic growth phase, digesting, fully blowing and beating into single cell suspension, counting, diluting into 1 × 10⁴cell/mL, inoculated in 96-well culture plate, and cultured at 37 ℃ for 24 h. Cells were treated with 10 μmol/L of compound 1-6 for 48 h. Then CCK8 was added and the cells were incubated for 2 h. Finally, the absorbance of each sample was measured at 450 nm by a microplate reader. Meanwhile, 5-fluorouracil was selected as a positive control.

Tumor cell growth inhibition (%) = (1-assay well assay/control well assay) × 100%

Inhibitory Activity of Compounds of Table 1 on liver cancer cells

Under the concentration of 10 mu M, the compounds 1-6 have certain inhibitory activity on HepG2 liver cancer cells, wherein the survival rate of the liver cancer cells under the action of the compounds 3 and 5 is lower, and the inhibitory rate is more than 50%, so that the compounds show better anti-liver cancer activity.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.