CN103833818B - The antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof - Google Patents

Abstract

The invention belongs to field of pharmaceutical chemistry technology, disclose the antitumor drug of a kind of Sasanguasaponin compound, its preparation method, application and preparation thereof.This Sasanguasaponin compound has structure shown in formula I.This Sasanguasaponin compound has certain restraining effect to liver cancer cell, breast cancer cell, melanoma cells, lung carcinoma cell, and effectively can suppress the growth of liver tumor, illustrate that this Sasanguasaponin compound has significant anti-tumor activity, can be applied to and prepare antitumor drug.

Description

Camellia oleifera saponin compound, its preparation method, application and antitumor drug prepared therefrom

technical field

The invention belongs to the technical field of medicinal chemistry, and in particular relates to a camellia oleifera saponin compound, its preparation method, application and antitumor drug prepared therefrom.

Background technique

Camellia oleifera (Camellia oleifera Abel), also known as tea seed tree, camellia oil tree, white flower tea, is an evergreen small tree of the family Theaceae (Theaceae) Camellia Linn, mainly distributed in tropical and subtropical regions. In my country, camellia oleifera is produced in the southwest and southeast regions, covering 17 provinces. Camellia oleifera is an important woody edible oil tree species, and it is called "the world's four largest woody oil tree species" together with olive, oil palm and coconut. Camellia oleifera oil is obtained by pressing oil from the seeds of camellia oleifera. The unsaturated fatty acid content of tea oil is as high as 90%, which is much higher than that of rapeseed oil, peanut oil and soybean oil; compared with olive oil, its vitamin E content is twice as high and has High nutritional value. Camellia oleifera also has high medicinal value. It is recorded in "Compendium of Materia Medica" that tea seeds, bitter cold and fragrant poison (saponin), are mainly used to treat dyspnea and cough, and remove disease scales; The skin has the effects of clearing heat and detoxification, regulating qi and relieving pain, promoting blood circulation and reducing swelling. It is mainly used to treat sore throat, stomach pain, toothache, bruises and burns.

The medicinal parts of camellia oleifera mainly include tea seed heart, camellia oil, camellia oleifera root, camellia oleifera root bark and tea oleifera. The chemical constituents isolated from Camellia oleifera include saponins, flavonoids, tannins, aromatic glycosides and alkaloids. Camellia oleifera saponin, also known as camellia oleifera saponin, is a kind of soap extracted from camellia oleifera seed cake. Medicine, aquatic products, building materials and many other fields. The study also found that Camellia oleifera saponin has the general properties of camellia plant saponins, bitter, spicy, and strong foaming properties; it also has a variety of good biological activities, which are manifested in many aspects, such as hemolysis, fish poisoning, bactericidal and antibacterial Active, anti-inflammatory, anti-hypertensive, anti-tumor, protects the cardiovascular system, kills insects, promotes plant growth, etc. Camellia oleifera saponins are mostly oleanane-type pentacyclic triterpenoid saponins, including aglycone, sugar, and organic acid. However, the complex structure of aglycone, many types of sugar parts and different combinations have resulted in a large number of saponins in camellia oleifera. , posed a challenge to the further purification and separation of camellia oleifera saponins. Therefore, although studies have found that the total saponin extract of camellia oleifera has anti-tumor activity, it is still unknown which monomer saponin plays the role, and further research is needed.

Contents of the invention

In view of this, the object of the present invention is to provide a camellia oleifera saponin compound, its preparation method, application and antitumor drug prepared therefrom. The camellia oleifera saponin compound has remarkable antitumor activity and can be applied to prepare antitumor drugs.

In order to realize the purpose of the invention of the present invention, the present invention adopts following technical scheme:

The invention provides a camellia oleifera saponin compound, which has the structure shown in formula I:

The present invention also provides a preparation method of camellia oleifera saponin compound, comprising the following steps:

Step 1: obtaining Camellia oleifera total saponins extract;

Step 2: Take the camellia oleifera total saponin extract obtained in step 1, and separate and purify it to obtain final product.

Preferably, in the preparation method provided by the present invention, step 1 is specifically:

After the camellia oleifera root is crushed, it is extracted to obtain an extract;

The resulting extract was concentrated to obtain a liquid extract;

Get the obtained liquid extract and mix with water, filter, and collect the filtrate;

The obtained filtrate is separated by macroporous resin, eluted with ethanol-water mixture, and the eluted fraction of ethanol-water mixture with a volume ratio of 70:30 to 95:5 is collected to obtain the total saponin extract of camellia oleifera.

In some embodiments of the present invention, the solution used for extraction in step 1 of the preparation method provided by the present invention is an ethanol-water mixture.

In some other embodiments of the present invention, in the ethanol-water mixture used for extraction in step 1 of the preparation method provided by the present invention, the volume percentage of ethanol is 30%-95%.

In some other embodiments of the present invention, in g/mL, the mass volume ratio of the pulverized camellia oleifera root in step 1 of the preparation method provided by the present invention to the solution used for extraction is 1:5-20.

In some other embodiments of the present invention, the preparation method provided by the present invention also includes a soaking process before the extraction in step 1, specifically taking the crushed camellia oleifera root in step 1 and mixing it with the solution used for extraction, at 30°C to 50°C Soak for 6h ~ 12h.

In some other embodiments of the present invention, the impregnation and extraction in step 1 of the preparation method provided by the present invention are specifically:

Mix the camellia oleifera root crushed in step 1 with the ethanol-water mixed solution, soak for 6h-12h at 30°C-50°C, heat to reflux, filter, and collect the filtrate to obtain the extract.

The extraction in step 1 of the preparation method provided by the present invention can be extracted by heating reflux once, filtered, and the filtrate is collected to obtain an extract; it can also be extracted by heating reflux 2 to 3 times, and the extract obtained by each heating reflux can be collected separately , combine the extracts obtained by heating to reflux each time, filter, collect the filtrate, and obtain the extract.

In some other embodiments of the present invention, the mass ratio of liquid extract to water in step 1 of the preparation method provided by the present invention is 1:1-10.

In some other embodiments of the present invention, step 1 of the preparation method provided by the present invention is specifically:

Take Camellia oleifera root and grind it; in g/mL, mix the crushed Camellia oleifera root with ethanol-water mixed solution at a mass volume ratio of 1:5-20, soak at 30°C-50°C for 6h-12h, heat and reflux for 1 After the first to third times, combine the extracts obtained by heating and reflux each time, filter, collect the filtrate, and obtain the extract. The resulting extract was concentrated to obtain a liquid extract. Mix the obtained liquid extract with water at a mass ratio of 1:1 to 10, filter through 100 mesh to 300 mesh, and collect the filtrate; after centrifuging the obtained filtrate, take the supernatant and separate it with D101 macroporous resin, and then use water and ethanol to volume The ethanol-water mixture with a percentage content of 30% to 60%, and the ethanol-water mixture with a volume percentage of 70% to 95% are eluted, and the ethanol with a volume percentage of 70% to 95% is collected. -The water mixture elutes the components to obtain the total saponin extract of camellia oleifera.

Preferably, in the preparation method provided by the present invention, step 2 is specifically:

Take the camellia oleifera total saponins extract obtained in step 1, separate it through a silica gel column under reduced pressure, elute it with a chloroform-methanol mixed solution, and collect the eluted components of the chloroform-methanol mixed solution with a volume ratio of 80:20 to 60:40 to obtain the first purification;

The purified product obtained for the first time was separated by a medium-pressure reversed-phase octadecylsilane bonded silica gel column, eluted with a methanol-water mixture, and a methanol-water mixture with a volume ratio of 70:30 to 90:10 was collected Elution component, get the second purification;

The purified product obtained for the second time was separated by a dynamic axial compression chromatographic column, eluted with a methanol-water mixture, and the eluted components of the methanol-water mixture with a volume ratio of 75:25 were collected to obtain the third purified product;

The purified product obtained for the third time was separated by a semi-preparative high-performance liquid chromatography column, eluted with a mixture of methanol-water-formic acid at a flow rate of 2mL/min, and the eluted components corresponding to a retention time of 52.5min were collected to obtain the product;

The semi-preparative high-performance liquid chromatography column is an octadecylsilane semi-preparative chromatographic column, and its model is 250mm×10mm, 5 μm;

The methanol-water-formic acid mixed solution is: a mixed solution composed of methanol and water in a volume ratio of 72:28 and mixed with formic acid accounting for 0.2% by mass of the resulting mixed solution.

In some other embodiments of the present invention, the particle size of the silica gel in the decompression silica gel chromatography column used in step 2 of the preparation method provided by the present invention is 60 mesh to 100 mesh.

In some other embodiments of the present invention, during the silica gel chromatography separation under reduced pressure in step 2 of the preparation method provided by the present invention, the chloroform-methanol mixed elution is a gradient elution, and the elution gradient is specifically: the volume ratio of chloroform and methanol is 90:10→80:20→70:30→60:40→50:50→40:60→30:70→20:80.

In some other embodiments of the present invention, the flow rate of the medium-pressure reversed-phase octadecylsilane-bonded silica gel chromatography column used in step 2 of the preparation method provided by the present invention is 25 mL/min.

In some other embodiments of the present invention, when the medium-pressure reversed-phase octadecylsilane bonded silica gel chromatography column is separated in step 2 of the preparation method provided by the present invention, the elution of the methanol-water mixture is gradient elution, and the elution The gradient is specifically: the volume ratio of methanol to water is 50:50→60:40→70:30→80:20→90:10→100:0.

In some other embodiments of the present invention, the dynamic axial compression chromatography column used in step 2 of the preparation method provided by the present invention is an octadecylsilane bonded silica gel chromatography column.

In some other embodiments of the present invention, when the dynamic axial compression chromatographic column is separated in step 2 of the preparation method provided by the present invention, the elution of the methanol-water mixture is a gradient elution, and the elution gradient is specifically: 60:40→ 75:25→80:20→90:10.

In some other embodiments of the present invention, the flow rate of the dynamic axial compression chromatographic column used in step 2 of the preparation method provided by the present invention is 150 mL/min.

In some other embodiments of the present invention, the semi-preparative high performance liquid chromatography column used in the preparation method provided by the present invention is an octadecylsilane bonded silica gel semi-preparative high performance liquid chromatography column.

Preferably, step 2 of the preparation method provided by the present invention also includes distillation and drying steps after the separation of the semi-preparative high-performance liquid chromatography column, specifically: taking the eluted components obtained from the separation of the semi-preparative high-performance liquid chromatography column for distillation and drying, that is have to.

In some other embodiments of the present invention, step 2 in the preparation method provided by the present invention is specifically:

Take the total saponin extract of camellia oleifera obtained in step 1, pass through a decompressed silica gel column, and carry out gradient elution with a chloroform-methanol system, the elution gradient is 90:10→80:20→70:30→60 in the volume ratio of chloroform and methanol :40→50:50→40:60→30:70→20:80, collected respectively, and combining the eluted components of the chloroform-methanol mixture with a volume ratio of 80:20, 70:30 and 60:40 to obtain the first primary purification;

The purified product obtained for the first time was separated by a medium-pressure reversed-phase octadecylsilane bonded silica gel column, and gradient elution was carried out with a methanol-water system, and the elution gradient was that the volume ratio of methanol and water was 50:50→60 :40→70:30→80:20→90:10→100:0, flow rate 25mL/min, collect separately, combine volume ratio of 70:30, 80:20, 90:10 methanol-water mixture for elution Components, to obtain the second purified product;

The purified product obtained for the second time is separated by a dynamic axial compression chromatographic column, and gradient elution is carried out with methanol-water mixtures of different volume ratios. The elution gradient is that the volume ratio of methanol and water is 60:40→75:25→ 80:20→90:10, the flow rate is 150mL/min, and the methanol-water mixture with a volume ratio of 75:25 is collected to obtain the third purified product;

The purified product obtained for the third time was separated by octadecylsilane bonded silica gel semi-preparative high performance liquid chromatography column, eluted with methanol-water-formic acid mixture, the flow rate was 2mL/min, and the collection retention time was 52.5min corresponding to The eluted components are distilled and dried to obtain a white amorphous powdery substance, namely;

Among them, the methanol-water-formic acid mixed solution is: a mixed solution composed of methanol and water in a volume ratio of 72:28 and mixed with formic acid accounting for 0.2% by mass of the resulting mixed solution.

The present invention also provides a kind of camellia oleifera saponin compound, the preparation method of this camellia oleifera saponin compound comprises the following steps:

Step 1: obtaining Camellia oleifera total saponins extract;

Step 2: Take the camellia oleifera total saponin extract obtained in step 1, and separate and purify it to obtain final product.

The present invention also provides an application of a camellia oleifera saponin compound in the preparation of antitumor drugs, the camellia oleifera saponin compound has a structure shown in formula I,

The preparation method of the camellia oleifera saponin compound comprises the following steps:

Step 1: obtaining Camellia oleifera total saponins extract;

Step 2: Take the camellia oleifera total saponin extract obtained in step 1, and separate and purify it to obtain final product.

In some embodiments of the present invention, the tumors targeted by the application provided by the present invention are liver tumors, melanoma, breast tumors or lung tumors.

The present invention also provides an antitumor drug, which includes the camellia oleifera saponin compound provided in the present invention and pharmaceutically acceptable excipients, and the camellia oleifera saponin compound has the structure shown in formula I,

The preparation method of the camellia oleifera saponin compound comprises the following steps:

Step 1: obtaining Camellia oleifera total saponins extract;

Step 2: Take the camellia oleifera total saponin extract obtained in step 1, and separate and purify it to obtain final product.

Preferably, the dosage form of an antitumor drug provided by the present invention is tablet, granule, capsule, injection, emulsion or suspension.

The invention provides a camellia oleifera saponin compound, its preparation method, application and antitumor drug prepared therefrom. The camellia oleifera saponin compound has the structure shown in formula I. The results of in vitro cell experiments confirmed that the camellia oleifera saponin compound has a certain inhibitory effect on liver cancer cells, breast cancer cells, melanoma cells, and lung cancer cells; The growth of liver tumors shows that the camellia oleifera saponin compound has significant antitumor activity and can be applied to the preparation of antitumor drugs.

Description of drawings

Fig. 1 shows the high-resolution mass spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 2 shows the proton nuclear magnetic resonance spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 3 shows the partially enlarged figure of the proton nuclear magnetic resonance spectrum of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 4 shows the partially enlarged figure of the proton nuclear magnetic resonance spectrum of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 5 shows the carbon nuclear magnetic resonance spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 6 shows the carbon nuclear magnetic resonance spectrogram local enlargement figure of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 7 shows the carbon nuclear magnetic resonance spectrogram local enlargement figure of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 8 shows the DEPT spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 9 shows the partial enlargement of the DEPT spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 10 shows the HSQC spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 11 shows the HMBC spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 12 shows the H-HCOSY spectrogram of the camellia oleifera saponin compound that embodiment 1 makes;

Fig. 13 shows the NOESY spectrogram of the camellia oleifera saponin compound prepared in Example 1.

detailed description

The invention discloses a camellia oleifera saponin compound, its preparation method, application and antitumor drug prepared therefrom. Those skilled in the art can refer to this content to obtain the camellia oleifera saponin compound and realize its application. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention Inside. The preparation method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method and application herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The reagents and raw materials used in the camellia oleifera saponin compound provided by the invention, its preparation method, application and the antitumor drug prepared therefrom can all be purchased from the market. The models and manufacturers of some instruments used in the embodiments of the present invention are as follows:

Chemical reagents: analytically pure, Sinopharm Chemical Reagent Co., Ltd.; electronic balance: Mettler-Toledo Instruments (Shanghai) Co., Ltd.; polarimeter 241PerkinElmer Inc., Waltham, MA, the United States; rotary evaporator: a wholly-owned factory of Tokyo Physical and Chemical Instruments; Thin-layer chromatography silica gel plate: HSGF254, produced by Yantai Zhifu Huangwu Silica Gel Development and Experimental Factory; various silica gels for column chromatography are produced by Qingdao Ocean Chemical Factory; NMR 500Hz: VarianInc., PaloAlto, CA, the United States; high-resolution mass spectrometry Q-TOF2: Micromass Company, UK; semi-preparative HPLC: LC-20AT, SPD-20A, Shimadzu Corporation, Japan; medium-pressure reversed-phase octadecylsilane preparative liquid chromatography: Büchi chromatography system, C- 650 pump, medium pressure column (460mm×26mmi.d., BüchiCorp., Flawil, Swiss); dynamic axial column chromatography: NP7000 pump (Newstyle), NU3000UV-VIS detector (Newstyle) and octadecylsilane column ( 650mm×100mm, 30μm, 1500g; Newstyle); octadecylsilane semi-preparative chromatographic column (C18 semi-preparative chromatographic column): 250mm×10mm, 5μm, American Kromsil Company.

In order to enable those skilled in the art to better understand the technical scheme of the present invention, the present invention will be further set forth below in conjunction with the examples:

Embodiment 1 has the preparation of the camellia saponin compound of structure shown in formula I

extract:

Take 2000g of dry Camellia oleifera root (collected from Qichun, Hubei in November 2011), crush it into sawdust with a grinder, add 20.0L of 70% ethanol aqueous solution, soak at 40°C for 10h, heat to reflux, and extract 3.0h, collect the extract, filter the extract, concentrate under reduced pressure to obtain 602g of liquid extract.

Macroporous resin enrichment:

Take the liquid extract obtained in the extraction step, add 6.02L of water, stir to dissolve, filter with a 100-mesh filter cloth, and centrifuge the filtrate at 12000r/min for 10min. After centrifugation, 5.3L of supernatant is obtained. Pore resin was further separated, and eluted successively with 10L water, 15L 30% ethanol aqueous solution, and 15L 70% ethanol aqueous solution, collected 70% ethanol aqueous solution eluted components, and concentrated under reduced pressure. After testing, the obtained concentrated solution contained camellia oleifera saponins, After drying in an oven at 100°C for 48 hours, 45 g of brown extract was obtained, which was named Camellia oleifera Total Saponins Extract.

Preparation of the first purified product:

The total saponin extract of camellia oleifera obtained by enrichment with macroporous resin was separated by decompression silica gel column (silica gel: 60-100 mesh), and gradient elution was carried out with chloroform-methanol system. The elution gradient was that the volume ratio of chloroform and methanol was 90:10→80:20→70:30→60:40→50:50→40:60→30:70→20:80, 2.0L for each gradient; collect each eluted fraction separately, and label. The obtained eluate was subjected to thin-layer plate analysis: thin-layer chromatography silica gel plate, BAW system, R _f value = 0.4-0.7. After testing, the following elution fractions contain similar components: 80:20 chloroform-methanol solution elution fraction, 70:30 chloroform-methanol solution elution fraction, 60:40 chloroform-methanol solution elution fraction point. Combine the 80:20 chloroform-methanol solution elution fraction, the 70:30 chloroform-methanol solution elution fraction and the 60:40 chloroform-methanol solution elution fraction, and evaporate to dryness under reduced pressure to obtain the first Subpurified 12.6 g.

Preparation of the second purification:

The purified product obtained for the first time was separated by medium-pressure reversed-phase octadecylsilane preparative liquid chromatography, and gradient elution was carried out with methanol-water system, and the elution gradient was that the volume ratio of methanol and water was 50:50→ 60:40→70:30→80:20→90:10→100:0, each gradient 1000mL, flow rate 25mL/min, detection wavelength 203nm. The eluted fractions were collected in sections to obtain six eluted fractions, which were marked one by one. Take the elution gradient corresponding to 60:40, 70:30, 80:20, and 90:10 for high-efficiency analysis liquid chromatography analysis. The results show that the elution gradient 70:30, 80:20, 90 : The components contained in the eluted components corresponding to 10 are similar (the HPLC time is 30min, and the detection peak is between 20min-28min), and the eluted components corresponding to the elution gradient are combined and concentrated under reduced pressure , to obtain 20 mL of the second purified product.

Preparation of the third purified product:

Take the purified product for the second time, and separate it by dynamic axial column chromatography (flow rate 150mL/min, detection wavelength 203nm), and use 60%, 75%, 80% and 90% methanol aqueous solution to elute successively in volume percentage, Each gradient is 3000mL and collected separately to obtain five eluted fractions, which are marked one by one. The obtained eluted components were analyzed by high performance analytical liquid chromatography (the detection peak was between 13.5min-15.0min), and 75% of the eluted components were collected (that is, the methanol-water mixture with a volume ratio of 75:25 eluted Components, HPLC detection peak at 14.5min), concentrated under reduced pressure to obtain 15mL of the third purified product.

Preparation of white amorphous powdery substance:

Take the third purified product and separate it on a C18 semi-preparative chromatographic column (250mm×10mm, 5μm) (flow rate 2mL/min, detection wavelength 203nm). : The mixed solution of 28 composition is then mixed with the mixed solution obtained by mixing 0.2% formic acid with the mass percentage of the obtained mixed solution) to elute at 52.5min. The obtained eluted fractions were concentrated under reduced pressure and dried to obtain 103 mg of a white amorphous powder.

Compound structure identification:

The resulting white amorphous powder was identified by TLC, acetic anhydride-concentrated sulfuric acid reaction, and Molish reaction. The identification results were: the color development result of thin-layer chromatography sulfuric acid ethanol was purple spots, and the reaction of acetic anhydride-concentrated sulfuric acid was positive. , Molish reaction was positive, suggesting that the substance may be a saponin compound. The high-resolution mass spectrogram of this substance is shown in Figure 1, the quasi-ion peak m/ _z1299.5965 [ _MH ]- in the mass _spectrogram , and the calculated value [MH]-, m /z1299.6010, basically the same, indicating that the molecular formula of the compound is C ₆₃ H ₉₆ O ₂₈ . Monosaccharides were obtained by complete acid hydrolysis with 2NTFA. The sugars were derivatized and analyzed by GC, and the presence of D-glucuronic acid, D-galactose, and D-xylose was detected.

The obtained white amorphous powdery substance is subjected to proton nuclear magnetic resonance spectrum detection, carbon nuclear magnetic resonance spectrum detection and two-dimensional nuclear magnetic resonance spectrum detection. The proton nuclear magnetic resonance spectrum of the white amorphous powdery substance is shown in Fig. 2, Fig. 3 and Fig. 4, wherein Fig. 3 and Fig. 4 are partial enlarged diagrams of the proton nuclear magnetic resonance spectrum of the substance. The carbon nuclear magnetic resonance spectrum of the white amorphous powdery substance is shown in Fig. 5, Fig. 6 and Fig. 7, and Fig. 6 and Fig. 7 are partial enlarged diagrams of the carbon nuclear magnetic resonance spectrum of the substance. The DEPT spectrum of the white amorphous powdery substance is shown in Figure 8 and Figure 9, wherein Figure 9 is a partial enlarged view of the DEPT spectrum of the substance. The HSQC spectrum of the white amorphous powder is shown in Figure 10. The HMBC spectrum of the white amorphous powder is shown in Figure 11. The H-HCOSY spectrum of the white amorphous powder is shown in Figure 12. The NOESY spectrum of the white amorphous powder is shown in FIG. 13 . See Table 1 for the NMR spectrum data of the white amorphous powdery substance.

The nuclear magnetic resonance data of table 1 white amorphous powder compound

position δ _{H δC} position δ _{H δC} 1 0.97m, 1.46m 38.3 22-O-Ang -- -- 2 1.94m, 2.24m 25.4 1" -- 168.3 3 4.11dd (11.0,4.5) 84.7 2" -- 129.1 4 -- 55.3 3″ 5.98dq(7.5,1.5) 137.3

5 1.46m 48.5 4″ 2.13d (7.5) 15.9 6 1.05m, 1.42m 20.4 5″ 1.99s 21.0 7 1.23m, 1.60m 32.5 GlcA -- -- 8 -- 41.8 1 4.81d (6.5) 104.0 9 1.84m 46.9 2 4.55m 78.3 10 -- 36.2 3 4.39m 85.1 11 1.88m, 1.99m 23.9 4 4.55m 70.7 12 5.49brs 123.7 5 4.44m 77.2 13 -- 143.0 6 -- nd 14 -- 40.4 Gal -- -- 15 1.63brs, 1.88brs 34.9 1 5.77d (7.5) 101.8 16 4.55brs 68.7 2 4.58m 84.1 17 -- 48.2 3 4.37m 75.1 18 3.18m 40.2 4 4.44m 71.0 19 1.50m, 3.18m 47.3 5 4.16m 77.1 20 -- 36.5 6 4.42m 62.0 twenty one 6.77d (10.5) 78.9 Xyl -- -- twenty two 6.39d (10.5) 73.8 1 5.12d (7.5) 107.8 twenty three 10.0brs 210.5 2 4.25m 76.4 twenty four 1.55s 11.2 3 4.11m 78.4 25 0.90s 16.0 4 4.37m 71.0 26 0.90s 17.0 5 3.56m 67.7 27 1.88s 27.7 -- 4.53m -- 28 3.48d (11.0) 63.8 Gal -- -- -- 3.71d (10.0) -- 1 5.93d (7.5) 103.0 29 1.18s 29.7 2 4.51m 73.9 30 1.42s 20.4 3 4.42m 75.5

21-O-Ang -- -- 4 4.53m 70.0 1' -- 167.8 5 4.53m 76.5 2' -- 129.1 6 4.55m 62.3 3' 6.05dq(7.5,1.5) 137.3 -- -- -- 4′ 2.17d (7.5) 16.0 -- -- -- 5' 2.09s 21.1 -- -- --

Note: The unit of chemical shift in the table is ppm, the unit of coupling constant (J) is Hz, and nd means the signal that exists but not detected.

Further structural analysis of the white amorphous powdery substance was carried out according to the obtained nuclear magnetic resonance spectrum and data:

The ¹³ CNMR spectrum of the white amorphous powdery substance shows a total of 63 carbon signals, and the compound contains 10 methyl carbons, 11 methylene carbons, 30 methine carbons and 12 quaternary carbon. Further analysis of the ¹³ CNMR spectrum, the spectrum shows four sugar end group carbon signals δ104.0, 101.8, 107.8 and 103.0, which are D-glucuronic acid, D-galactose, D-xylose and another D-galactose end group carbon. In addition, there is an aldehydic carbon signal at δ 210.5 in the downfield region. In the high field area of the ¹ HNMR spectrum of the white amorphous powder, there are six obvious triterpenoid saponin angle methyl hydrogen signals δ0.90(Me-25), 0.90(Me-26), 1.18(Me-29) , 1.42 (Me-30), 1.55 (Me-24) and 1.88 (Me-27); a oxymethylene δ3.48 (H-28, d, J=11.0Hz) and 3.71 (H-28, d, J=10.0Hz); four oxymethines δ4.11(1H,dd,J=11.0,4.5Hz,H-3),4.55(1H,brs,H-16),6.77(1H, d, J=10.5Hz, H-21) and 6.39 (1H, d, J=10.5Hz, H-22); an ene hydrogen proton δ5.49 (1H, brs, H-12) and an aldehyde hydrogen proton δ10.0 (1H, brs, H-23). In addition, the ¹ H NMR spectrum of the white amorphous powder also showed two groups of angelic acyl signals δ[6.05(1H,dq,J=7.5,1.5Hz,21-O-Ang-3'),2.17(3H ,d,J=7.5Hz,21-O-Ang-4') and 2.09(3H,s,21-O-Ang-5')]; δ[5.98(1H,dq,J=7.5,1.5Hz, 22-O-Ang-3″), 2.13 (3H, d, J=7.5Hz, 22-O-Ang-4″) and 1.99 (3H, s, 22-O-Ang-5″)].

In H-HCOSY, the methylene δ _H 1.63 (1H, brs) and 1.88 (1H, brs) adjacent to the oxymethine proton are related to δ _H 4.55 (1H, brs), indicating that they are in the ortho position And δ _H 4.55 is _H -16 signal, δ _H 1.63 and 1.88 are _H -15 signals; Correlation signals indicate that they are in ortho positions and δ _H 6.77 is H-21 signal and δ _H 6.39 is H-22 signal.

Through the HMBC spectrum analysis of the white amorphous powdery substance, it is known that the two angelica acyl groups are connected to the 21 and 22 positions of the compound, because in HMBC, δ _H 6.77 (1H, d, J=10.5Hz, H- 21) Correlates with δ _C 167.8 (21-O-Ang-1'), δ _H 6.39 (1H, d, J=10.5Hz, H-22) correlates with δ _C 168.3 (22-O-Ang-1'') relevant. Combined with ¹³ CNMR spectrum and ¹ HNMR spectrum data for comprehensive analysis, and with known aglycone 21β, 22α-O-diangeloyl-3β, 16α, 28-trihydroxyolean-12-en-23-al [Sci.Pharm1982,50,331] NMR Data comparison confirmed that the aglycone structure of the white amorphous powdery substance was 21β,22α-O-diangeloyl-16α,28-dihydroxyolean-12-en-23-aldehyde.

The HSQC spectrum of the white amorphous powder was analyzed, and the terminal carbon and hydrogen signals of the sugars connected to the saponin were assigned, as follows: δ _H 4.81 (1H, d, J=6.5Hz), 5.77 (1H , d, J=7.5Hz), 5.12 (1H, d, J=7.5Hz) and 5.93 (1H, d, J=7.5Hz), connected at δ _C 104.0 (glucuronic acid-C-1), 101.8 (Galactose-C-1), 107.8 (Xylose-C-1), 103.0 (Galactose'-C-1). In addition, about 12.6ppm of the 3-position of the compound aglycone shows that the sugar chain is connected to the 3-position of the aglycone, and in the HMBC spectrum, the glucuronic acid terminal group proton δ _H 4.81 is related to the 3-position δ _C of the aglycone 84.7, It was further confirmed that the sugar chain was connected to the 3 position of the aglycone. Further review of the literature found that the sugar chain of the compound was similar to the sugar chain data of camellenodiol reported in the literature [Bioorganic & medicinal chemistry letters 2010, 20, 7435-7439], thus inferring that the sugar chain of the compound was β-D-xylose-(1→2)-β -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronic acid. The connection order of the sugar chains can also be confirmed by analyzing the HMBC spectrum. In HMBC, the glucuronic acid anomeric proton δ _H 4.81 is related to the carbon δ _C 84.7 at the 3-position of the aglycone, and the galactose anomeric proton δ _H 5.77 is related to the glucuronic acid 3-position Carbon δ _C 85.1 is related, xylose end group proton δ _H 5.12 is related to galactose 2 carbon δ _C 84.1, galactose 2 hydrogen δ _H 4.58 is related to xylose end group carbon δ _C 107.8, another galactose end matrix The δ _H of 5.93 is related to the δ _C 78.3 of the 2-position carbon of glucuronic acid, and the δ _H 4.55 of the 2-position hydrogen of glucuronic acid is related to the δ _C 103.0 of the terminal carbon of the galactose. In addition, the anomeric proton coupling constants ³ J _H-1 and _H-2 of the four sugars are relatively large, indicating that the four sugars are all in the β-configuration.

In the NOESY spectrum, δ _H 6.77 (1H, d, J=10.5Hz, H-21) is related to δ _H 1.18 (3H, s, H-29), suggesting that H-21 is in the α configuration; δ _H 4.11 (1H ,dd,J=4.5,11.0Hz,H-3) is related to δ _H 10.0(1H,brs,H-23), suggesting that H-3 is in α configuration; δ _H 4.55(1H,brs,H-16) Correlation with δ _H 3.48 (1H, d, J=11.0Hz, H-28), 3.71 (1H, d, J=11.0Hz, H-28) suggests that H-16 is in the β configuration, and δ _H 6.39 (1H, d, J=10.5Hz, H-22) is correlated with δ _H 1.42 (3H, s, H-30), suggesting that H-22 is in the β configuration.

Therefore, this compound has the structure shown in formula I,

The structure of the compound was named 21β,22α-O-diangeloyl-16α,28-dihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)- β-D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide.

Embodiment 2 has the preparation of the camellia saponin compound of structure shown in formula I

extract:

Take 2000g of dry Camellia oleifera root (collected from Qichun, Hubei in November 2011), crush it into sawdust with a grinder, add 40.0L of 30% ethanol aqueous solution, soak at 30°C for 12h, heat to reflux, and extract three times , 0.5h each time, collect the extract, filter the extract, concentrate under reduced pressure, and obtain 628g of liquid extract.

Macroporous resin enrichment:

Take the liquid extract obtained in the extraction step, add 0.628L of water, stir to dissolve, filter with a 300-mesh filter cloth, and centrifuge the filtrate at 12000r/min for 10min. After centrifugation, 5.8L of supernatant is obtained. Pore resin was further separated, and eluted successively with 10L water, 15L 60% ethanol aqueous solution, and 10L 95% ethanol aqueous solution, collected 95% ethanol aqueous solution eluted components, and concentrated under reduced pressure. After testing, the obtained concentrated solution contained camellia oleifera saponins, After drying in an oven at 100°C for 48 hours, 55 g of brown extract was obtained, which was named Camellia oleifera Total Saponin Extract.

Preparation of the first purified product:

The total saponins extract of camellia oleifera obtained by enrichment with macroporous resin was separated by decompression silica gel column (silica gel: 60=100 mesh), and gradient elution was carried out with chloroform-methanol system. The elution gradient was that the volume ratio of chloroform and methanol was 90:10→80:20→70:30→60:40→50:50→40:60→30:70→20:80, 1.0L for each gradient; collect each eluted fraction separately, and label. The obtained eluate was subjected to thin-layer plate analysis: thin-layer chromatography silica gel plate, BAW system, R _f value = 0.4-0.7. After testing, the following elution fractions contain similar components: 80:20 chloroform-methanol solution elution fraction, 70:30 chloroform-methanol solution elution fraction, 60:40 chloroform-methanol solution elution fraction point. Combine the 80:20 chloroform-methanol solution elution fraction, the 70:30 chloroform-methanol solution elution fraction and the 60:40 chloroform-methanol solution elution fraction, and evaporate to dryness under reduced pressure to obtain the first Purified product 15.2g.

Preparation of the second purification:

The purified product obtained for the first time was separated by medium-pressure reversed-phase octadecylsilane preparative liquid chromatography, and gradient elution was carried out with methanol-water system, and the elution gradient was that the volume ratio of methanol and water was 50:50→ 60:40→70:30→80:20→90:10→100:0, each gradient 1000mL, flow rate 25mL/min, detection wavelength 203nm. The eluted fractions were collected in sections to obtain six eluted fractions, which were marked one by one. Take the elution gradient corresponding to 60:40, 70:30, 80:20, and 90:10 to carry out high-efficiency analysis liquid chromatography analysis, the results show that the elution gradient 70:30, 80:20, 90 : The components contained in the eluted components corresponding to 10 are similar (the HPLC time is 30min, and the detection peak is between 20min-28min), the eluted components corresponding to the elution gradient are combined, concentrated under reduced pressure, That is, 20 mL of the second purified product was obtained.

Preparation of the third purified product:

Take the purified product for the second time, and separate it by dynamic axial column chromatography (flow rate 150mL/min, detection wavelength 203nm), and use 60%, 75%, 80% and 90% methanol aqueous solution to elute successively in volume percentage, Each gradient is 3000mL and collected separately to obtain five eluted fractions, which are marked one by one. The obtained eluted components were analyzed by high performance analytical liquid chromatography (the detection peak was between 13.5min and 15.0min), and 75% of the eluted components were collected (that is, the methanol-water mixture with a volume ratio of 75:25 eluted Components, HPLC detection peak at 13.5min), concentrated under reduced pressure to obtain 15mL of the third purified product.

Preparation of white amorphous powdery substance:

Take the third purified product and separate it on a C18 semi-preparative chromatographic column (250mm×10mm, 5μm) (flow rate 2mL/min, detection wavelength 203nm). : The mixed solution of 28 composition is then mixed with the mixed solution obtained by mixing 0.2% formic acid with the mass percentage of the obtained mixed solution) for elution, and the chromatographic peak at 52.5min is collected, and the obtained eluted components are concentrated under reduced pressure and dried , to obtain 125 mg of white amorphous powder.

Using the same compound structure identification method as in Example 1, the structure of the white amorphous powdery substance prepared in this example was identified, and the obtained high-resolution mass spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, DEPT spectrum, and HSQC spectrum were obtained. Figure, HMBC spectrogram, H-HCOSY spectrogram, NOESY spectrogram data are basically consistent with the relevant spectrogram data obtained in Example 1, and the results show that the white amorphous powdery substance is a compound having a structure shown in formula I,

Embodiment 3 has the preparation of the camellia saponin compound of structure shown in formula I

extract:

Take 2000g of dry Camellia oleifera root (collected from Qichun, Hubei), grind it into sawdust with a grinder, add 10.0L of 95% ethanol aqueous solution, soak at 50°C for 6h, heat to reflux, and extract twice, each After 1 hour, the extract was collected, filtered, and concentrated under reduced pressure to obtain 638 g of liquid extract.

Macroporous resin enrichment:

Take the liquid extract obtained in the extraction step, add 6.0L of water, stir to dissolve, filter with a 200-mesh filter cloth, and centrifuge the filtrate at 12000r/min for 10min. After centrifugation, 5.5L of supernatant is obtained. Pore resin was further separated, and eluted with 10.0L water, 15.0L 40% ethanol aqueous solution, and 15.0LL 80% ethanol aqueous solution successively, and the eluted components of 80% ethanol aqueous solution were collected and concentrated under reduced pressure. After testing, the obtained concentrated solution contained camellia oleifera saponin Compounds were dried in an oven at 100°C for 48 hours to obtain 52 g of brown extract, which was named Camellia oleifera Total Saponins Extract.

Preparation of the first purified product:

The total saponin extract of camellia oleifera obtained by enrichment with macroporous resin was separated by decompression silica gel column (silica gel: 60-100 mesh), and gradient elution was carried out with chloroform-methanol system. The elution gradient was that the volume ratio of chloroform and methanol was 90:10→80:20→70:30→60:40→50:50→40:60→30:70→20:80, 1.0L for each gradient; collect each eluted fraction separately, and label. The obtained eluate was subjected to thin-layer plate analysis: thin-layer chromatography silica gel plate, BAW system, R _f value = 0.4-0.7. After testing, the following elution fractions contain similar components: 80:20 chloroform-methanol solution elution fraction, 70:30 chloroform-methanol solution elution fraction, 60:40 chloroform-methanol solution elution fraction point. Combine the 80:20 chloroform-methanol solution elution fraction, the 70:30 chloroform-methanol solution elution fraction and the 60:40 chloroform-methanol solution elution fraction, and evaporate to dryness under reduced pressure to obtain the first Purified product 18.5g.

Preparation of the second purification:

The purified product obtained for the first time was separated by medium-pressure reversed-phase octadecylsilane preparative liquid chromatography, and gradient elution was carried out with methanol-water system, and the elution gradient was that the volume ratio of methanol and water was 50:50→ 60:40→70:30→80:20→90:10→100:0, each gradient 1000mL, flow rate 25mL/min, detection wavelength 203nm. The eluted fractions were collected in sections to obtain six eluted fractions, which were marked one by one. Take the elution gradient corresponding to 60:40, 70:30, 80:20, and 90:10 to carry out high-efficiency analysis liquid chromatography analysis, the results show that the elution gradient 70:30, 80:20, 90 : The components contained in the eluted components corresponding to 10 are similar (the HPLC time is 30min, and the detection peak is between 20min-28min), the eluted components corresponding to the elution gradient are combined, concentrated under reduced pressure, That is, 20 mL of the second purified product was obtained.

Preparation of the third purified product:

Take the purified product for the second time, and separate it by dynamic axial column chromatography (flow rate 150mL/min, detection wavelength 203nm), and use 60%, 75%, 80% and 90% methanol aqueous solution to elute successively in volume percentage, Each gradient is 3000mL and collected separately to obtain five eluted fractions, which are marked one by one. The obtained eluted components were analyzed by high performance analytical liquid chromatography (the detection peak was between 13.5min and 15.0min), and 75% of the eluted components were collected (that is, the methanol-water mixture with a volume ratio of 75:25 eluted Components, the HPLC detection peak was at 15.0min), and 15mL of the third purified product was obtained.

Preparation of white amorphous powdery substance:

Take the third purified product and separate it on a C18 semi-preparative chromatographic column (250mm×10mm, 5μm) (flow rate 2mL/min, detection wavelength 203nm). : The mixed solution of 28 composition is then mixed with the mixed solution obtained by mixing 0.2% formic acid with the mass percentage of the obtained mixed solution) for elution, and the chromatographic peak at 52.5min is collected, and the obtained eluted components are concentrated under reduced pressure and dried , to obtain 173 mg of white amorphous powder.

Using the same compound structure identification method as in Example 1, the structure of the white amorphous powdery substance prepared in this example was identified, and the obtained high-resolution mass spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, DEPT spectrum, and HSQC spectrum were obtained. Figure, HMBC spectrogram, H-HCOSY spectrogram, NOESY spectrogram data are basically consistent with the relevant spectrogram data obtained in Example 1, and the results show that the white amorphous powdery substance is a compound having a structure shown in formula I,

Embodiment 4 has the preparation of the camellia saponin compound of structure shown in formula I

extract:

Take 2000g of dry camellia oleifera root, crush it into sawdust with a pulverizer, add it into 20.0L ethanol aqueous solution for extraction, and concentrate the obtained extract to obtain 655g of liquid extract.

Macroporous resin enrichment:

Add 6.0L of water to the obtained liquid extract, stir until dissolved, and filter to obtain 5.8L of filtrate; separate the obtained filtrate through a macroporous resin, elute with ethanol-water mixture, and collect volume ratio of 70:30-95 : 5 ethanol-water mixed solution elution fraction, after detection, wherein contains camellia oleifera saponins compound, through oven 100 ℃, 48h drying, obtain brown extractum 55g, named as camellia oleifera total saponins extract.

Preparation of the first purified product:

Take the total saponins extract of camellia oleifera obtained by enrichment with macroporous resin, separate it on a decompressed silica gel column, and carry out gradient elution with chloroform-methanol system, each gradient is 1000mL, and the collection volume ratios are 80:20, 70:30, 60 : 40 chloroform-methanol mixture eluting components. The obtained three eluted fractions were subjected to thin-layer plate analysis: thin-layer chromatography silica gel plate, BAW system, R _f value = 0.4-0.7. After testing, the three eluted fractions contained similar components. The three eluted fractions were combined and evaporated to dryness under reduced pressure to obtain 15.9 g of the first purified product.

Preparation of the second purification:

The purified product obtained for the first time was separated by medium-pressure reversed-phase octadecylsilane preparative liquid chromatography, and gradient elution was carried out with methanol-water system, each gradient was 1000mL, and the collection volume ratio was 70:30, 80 :20, 90:10 methanol-water mixture eluted components. The obtained three eluted components were subjected to high-efficiency analytical liquid phase detection, and the components contained in the three eluted components were similar (the high-efficiency analytical liquid chromatography time was 30min, and the detection peak was between 20min-28min), and these three eluted components were combined The three eluted fractions were concentrated under reduced pressure to obtain 20 mL of the second purified product.

Preparation of the third purification:

The purified product was taken for the second time, separated by dynamic axial column chromatography, eluted with methanol-water system, and 450 mL of the eluted fraction of methanol-water mixture with a volume ratio of 75:25 was collected. The obtained eluted components were analyzed by high performance analytical liquid chromatography, and a detection peak appeared at 15.0 min. The eluted components were collected and concentrated under reduced pressure to obtain 15 mL of the third purified product.

Preparation of white amorphous powdery substance:

Take the third purified product, and separate it with a semi-preparative HPLC column (250mm×10mm, 5μm) (flow rate 2mL/min, detection wavelength 203nm), and methanol-water-formic acid mixture (methanol and water by volume ratio The mixed solution composed of 72:28 is mixed with 0.2% formic acid in the mass percentage of the obtained mixed solution) for elution, and the chromatographic peak at 52.5 minutes is collected, and the obtained eluted components are concentrated under reduced pressure , and dried to obtain 152 mg of white amorphous powder.

Using the same compound structure identification method as in Example 1, the structure of the white amorphous powdery substance prepared in this example was identified, and the obtained high-resolution mass spectrum, hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, DEPT spectrum, and HSQC spectrum were obtained. Figure, HMBC spectrogram, H-HCOSY spectrogram, NOESY spectrogram data are basically consistent with the relevant spectrogram data obtained in Example 1, and the results show that the white amorphous powdery substance is a compound having a structure shown in formula I,

Example 5 In Vitro Cell Experiment

Experimental Materials:

Human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 were purchased from Shanghai Institute of Cells, Chinese Academy of Sciences. These four kinds of cells were cultured in RPMI1640 medium containing 10% inactivated NBS (plus 0.03% Glu, 0.06% Hepes, 0.2% NaHCO ₃ ) at 37°C, 5% CO ₂ , and passaged for 3 days. Cells in logarithmic growth phase were used for experiments. The camellia oleifera saponin compound used in the experiment is the camellia oleifera saponin compound with the structure shown in formula I prepared according to the preparation method provided in Example 1,

experimental method:

The conventional MTT method was used to determine the sensitivity of tumor cell lines to drugs. The specific experimental arrangements are as follows:

The experiment was divided into negative control group, experimental group and positive control group. The solution added to the test cells in the negative control group was complete medium, and 3 duplicate holes were set. The tested cells of the experimental group were added solutions containing the camellia oleifera saponin compound prepared in Example 1, the solvent was a mixed solution of DMSO and PBS with a volume ratio of 1:99, and the experiment was set at 5 concentrations, wherein the camellia oleifera saponin compound The concentrations were: 6.25 μg/mL, 9.375 μg/mL, 12.5 μg/mL, 18.25 μg/mL, 25.00 μg/mL, and each group had three replicate wells. A solution containing 5-FU (5-fluorouracil) was added to the test cells of the positive control group. The solvent was a mixed solution of DMSO and PBS with a volume ratio of 1:99. The concentration of 5-FU was 50 μg/mL. Three replicates were set. hole.

According to the experimental arrangement, the concentration of each cell was adjusted to 5×10 ⁴ /mL with complete medium, inoculated in 96-well culture plates, labeled one by one, 100 μL/well, and cultured overnight at 37°C and 5% CO ₂ . The experimental group was added with different concentrations of Camellia oleifera saponin compounds having the structure shown in Formula I prepared in Example 1 (final concentrations were: 6.25 μg/mL, 9.375 μg/mL, 12.5 μg/mL, 18.25 μg/mL, 25.00 μg /mL), 10 μL/well; the positive control group was added with 5-FU (50 μg/mL), 10 μL/well; the negative control group was added with an equal volume of complete medium, 10 μL/well; each group was set up with 3 replicate wells and cultured for 24 h . MTT (5 mg/mL) was added at 10 μl/well 4 hours before the termination of the culture, and DMSO 100 μL was added to each well after the culture was completed. After shaking on a shaker for 10 minutes, the absorbance A value at a wavelength of 490 nm was detected with a microplate reader.

Experimental data processing method:

The inhibitory effect of camellia oleifera saponins on different tumor cells was evaluated by IC ₅₀ value.

Calculate the tumor cell growth inhibition rate (InhibitionRate) according to the following formula:

Tumor cell growth inhibition rate (%)=[(A negative control group-A experimental group)/A negative control group]×100%;

Taking the concentration of camellia oleifera saponin compound as the abscissa, the tumor cell growth inhibition rate is plotted on the ordinate to obtain the inhibition rate-concentration curve, and then the concentration of the drug when the 50% inhibition rate is obtained, that is, the IC of the camellia oleifera saponin compound on the tested cells ₅₀ value (half inhibitory concentration), the IC ₅₀ value of the camellia oleifera saponin compound prepared in Example 1 on different test cells is shown in Table 2.

Table 2 IC ₅₀ values of camellia oleifera saponin compounds on four different tumor cells

test cells A549 B16 BEL-7402 MCF-7 _IC50 (μg/mL) 10.63±0.07 13.14±0.24 12.73±0.84 11.95±1.20

The experimental results showed that the positive control group 5-FU had a certain inhibitory effect on all the tested cells in this example at a concentration of 50 μg/mL. From the data of the experimental group, it can be seen that the Camellia oleifera saponin compound prepared in Example 1 has an _IC50 value of less than 15 μg/mL for these four tumor cells, indicating that the Camellia oleifera saponin compound has good antitumor activity, especially for A549 cells. The inhibitory effect is the most obvious, and its IC ₅₀ value is only 10.63μg/mL. The experimental results show that the camellia oleifera saponin compound with the structure shown in formula I prepared in Example 1 has a certain inhibitory effect on liver tumors, melanoma, breast tumors or lung tumors, and can be applied to the preparation of antitumor drugs.

The same experimental method was used to investigate the inhibitory effect of the camellia saponin compounds prepared in Examples 2 to 4 on human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 , obtained similar experimental results, illustrating that the Camellia oleifera saponin compound with the structure shown in Formula I prepared in Example 2 to Example 4 has a certain inhibitory effect on liver tumors, melanoma, breast tumors or lung tumors, and can be applied for the preparation of antineoplastic drugs.

Embodiment 6 in vivo experiment

Experimental Materials:

ICR mice, clean grade, male, weighing 18-22 g, were provided by Shanghai Slack Experimental Animal Co., Ltd. H ₂₂ and S ₁₈₀ liver cancer cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The camellia oleifera saponin compound used in the experiment is a compound with the structure shown in formula I prepared according to the preparation method provided in Example 1,

experimental method:

This example investigates the effect of the camellia oleifera saponin compound having the structure shown in formula I prepared in Example 1 on liver cancer. The subjects were H ₂₂ xenograft tumor model mice and S ₁₈₀ xenograft tumor model mice.

Establishment of mouse H ₂₂ xenograft tumor model and mouse S ₁₈₀ xenograft tumor model and experimental arrangement:

Take H ₂₂ or S ₁₈₀ liver cancer (sarcoma) cell cryopreservation tubes, put them in a constant temperature water bath at 37°C to thaw, collect the cells by centrifugation, wash twice with PBS, resuspend the cells in PBS, and pass through the peritoneal cavity of ICR mice for 3 Above generation, the mice with obviously enlarged abdomen were taken, killed by dislocation, the abdomen was disinfected with alcohol, the milky white ascites was extracted with a disposable sterile syringe, injected into a sterilized centrifuge tube, counted by a cell counter, and the cell density was adjusted to 5× with normal saline. 10 ⁶ /mL, inoculate 0.2mL cell suspension into the right axilla of each mouse to establish the model. The next day, the mice were randomly divided into blank control group, experimental group 1, experimental group 2 and positive control group, 12 in each group, and administered. Experimental group 1 and experimental group 2 provided the camellia saponin compound with the structure shown in formula I prepared in embodiment 1, and the administration method was intraperitoneal injection, and the dosage of the two groups was: experimental group 1 was 1.5 mg/kg (i.e. 1.5 mg per 1 kg of body weight), the vehicle is 0.9% normal saline, once a day; the experimental group 2 is 3 mg/kg, the vehicle is 0.9% normal saline, once a day. The positive control group was provided with CTX (cyclophosphamide), the administration method was intraperitoneal injection, the administration dose was: 20mg/kg, the vehicle was 0.9% normal saline, once every other day. The blank control group was provided with a blank vehicle, namely 0.9% normal saline, injected intraperitoneally, with an injection volume of 0.2 mL, once a day. The mice in all groups were administered continuously for 10 days, the body weight of the mice was weighed every day, and the clinical manifestations of the mice were recorded. Two hours after the last administration, the tumor body was stripped off, the tumor weight was weighed, and the tumor inhibition rate was calculated.

Experimental data processing method:

The tumor inhibition rate was used to examine the inhibition of different groups of tumors, and the tumor inhibition rate was calculated according to the following formula:

Tumor inhibition rate (%)=[(average tumor weight of blank control group-average tumor weight of treatment group)/average tumor weight of blank control group]×100%

Table 3 shows the tumor weight and tumor inhibition rate of each group.

Table 3 Tumor weight and tumor inhibition rate of transplanted tumors in different groups

Note: Compared with blank control group, *P<0.05, **P<0.01

The experimental results showed that for the _H22 transplanted tumor model, compared with the blank control group, the tumor weights of the transplanted tumors in the positive control group, experimental group 1 and experimental group 2 were significantly smaller than those in the blank control group, and the difference was significant. It shows that the Camellia oleifera saponin compound with the structure shown in Formula I prepared in Example 1 can significantly inhibit the growth of _H22 liver cancer. For the _S180 transplanted tumor model, compared with the blank control group, the tumor weights of the transplanted tumors of the positive control group, experimental group 1 and experimental group 2 were significantly smaller than those of the blank control group, and the difference was significant, illustrating that Example 1 The prepared camellia saponin compound having the structure shown in formula I can significantly inhibit the growth of _S180 sarcoma. The experimental results also show that when the dosage of the camellia oleifera saponin compound with the structure shown in formula I prepared in embodiment 1 was 3.0mg/kg, the inhibitory rate to _H2 transplanted tumors reached 42.2%, suggesting that the camellia oleifera saponin compound Has significant anti-mouse H ₂₂ liver cancer growth effect; When the dosage of the Camellia oleifera saponin compound with the structure shown in Formula I prepared in Example 1 was 3.0 mg/kg, the inhibition rate of S ₁₈₀ transplanted tumors reached 32.1%, suggesting that the camellia oleifera saponin compound has a strong anti-growth effect on mouse _S180 liver cancer. The above results show that the camellia oleifera saponin compound having the structure shown in formula I prepared in Example 1 has good antitumor activity and can be applied to the preparation of antitumor drugs.

The same experimental method was used to investigate the inhibitory effect of the camellia oleifera saponin compounds prepared in Examples 2 to 4 on _H22 liver cancer and _S180 liver cancer in mice, and similar experimental results were obtained, indicating that the compounds obtained in Examples 2 to 4 Camellia oleifera saponins can effectively inhibit the growth of H ₂₂ liver cancer and S ₁₈₀ sarcoma. It shows that the camellia oleifera saponin compounds prepared in Examples 2 to 4 have good antitumor activity and can be applied to the preparation of antitumor drugs.

The preparation of embodiment 7 tablet

Get the Camellia oleifera saponins compound with the structure shown in I that Example 1 makes to add conventional adjuvant, make tablet according to conventional method.

The preparation of embodiment 8 granules

The Camellia oleifera saponin compound with the structure shown in I obtained in Example 2 was added with conventional adjuvants, and granules were prepared according to conventional methods.

The preparation of embodiment 9 capsules

The Camellia oleifera saponin compound with the structure shown in I obtained in Example 3 was added with conventional adjuvants, and capsules were made according to conventional methods.

The preparation of embodiment 10 injection

The Camellia oleifera saponin compound with the structure shown in I obtained in Example 4 was added with conventional adjuvants, and injections were made according to conventional methods.

The preparation of embodiment 11 emulsion

The Camellia oleifera saponin compound with the structure shown in I obtained in Example 1 was added with conventional adjuvants, and an emulsion was prepared according to a conventional method.

The preparation of embodiment 12 suspension

The Camellia oleifera saponin compound with the structure shown in I obtained in Example 1 was added with conventional adjuvants, and a suspension was prepared according to a conventional method.

The above are only preferred implementations of the present invention. It should be noted that the above preferred implementations should not be regarded as limiting the present invention, and the scope of protection of the present invention should be based on the scope defined in the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (7)

1. a camellia oleifera saponin compound is characterized in that, has the structure shown in formula I: 2. a preparation method of camellia oleifera saponin compound as claimed in claim 1, is characterized in that, comprises the following steps: Step 1: obtaining Camellia oleifera total saponins extract; Step 2: Take the total saponin extract of camellia oleifera, separate it through a silica gel column under reduced pressure, elute with a chloroform-methanol mixture, and collect the eluted components of the chloroform-methanol mixture with a volume ratio of 80:20 to 60:40, Get the first purification; The purified product for the first time was separated by a medium-pressure reversed-phase octadecylsilane bonded silica gel column, eluted with a methanol-water mixture, and a methanol-water mixture with a volume ratio of 70:30 to 90:10 was collected. Liquid eluting components, to obtain the second purification; The second purified product was separated by a dynamic axial compression chromatographic column, eluted with methanol-water mixture, and the eluted fraction of methanol-water mixed solution with a volume ratio of 75:25 was collected to obtain the third purified product ; The third purified product was separated by a semi-preparative high-performance liquid chromatography column, eluted with methanol-water-formic acid mixture, the flow rate was 2mL/min, and the eluted components corresponding to the retention time of 52.5min were collected to obtain ; The methanol-water-formic acid mixed solution is: a mixed solution composed of methanol and water in a volume ratio of 78:22 and mixed with formic acid accounting for 0.2% by mass of the mixed solution. 3. the preparation method according to claim 2, is characterized in that, step 1 is specially: After the camellia oleifera root is crushed, it is extracted to obtain an extract; The extract is taken and concentrated to obtain a liquid extract; Get the liquid extract and mix it with water, filter, and collect the filtrate; The filtrate is separated by macroporous resin, eluted with ethanol-water mixture, and the eluted components of ethanol-water mixture with a volume ratio of 70:30 to 95:5 are collected to obtain the total saponin extract of camellia oleifera. 4. use of camellia oleifera saponin compound as claimed in claim 1 in the preparation of antitumor drugs. 5. The application according to claim 4, characterized in that the tumor is liver tumor, melanoma, breast tumor or lung tumor. 6. An antitumor drug, characterized in that it comprises the camellia oleifera saponin compound as claimed in claim 1 and pharmaceutically acceptable auxiliary materials. 7. The antitumor drug according to claim 6, characterized in that its dosage form is tablet, granule, capsule, injection, emulsion or suspension.