CN118290503A - Flavone glycoside compounds in golden camellia, preparation method thereof and application thereof in preparation of antitumor drugs - Google Patents

Abstract

The present invention belongs to the technical field of natural drug chemistry, specifically a flavonoid glycoside compound in Camellia chrysantha and a preparation method thereof and use thereof in preparing anti-tumor drugs. The method of separating flavonoid glycoside compounds from Camellia chrysantha has practicality and feasibility of industrial production, and provides a new approach for the comprehensive development and utilization of Camellia chrysantha resources; at the same time, the separated flavonoid glycoside compounds have good biological activity and are expected to be applied to the fields of medicine, health products and cosmetics. Therefore, the present invention has important practical application value and market prospects.

Description

Flavonoid glycoside compounds in Camellia chrysantha and their preparation method and use in the preparation of anti-tumor drugs

Technical Field

The invention belongs to the technical field of natural drug chemistry, and specifically relates to a flavonoid glycoside compound in golden camellia, a preparation method thereof, and use thereof in preparing anti-tumor drugs.

Background technique

Yellow Camellia is an evergreen shrub or small tree of the Camellia genus of the Theaceae family. It is considered to be a relic of the original camellia of the Quaternary glacial period, and is known as a "living fossil of plants" along with Ginkgo biloba and Metasequoia glyptostroboides. Currently, there are 42 species and 5 varieties of Yellow Camellia, which are mainly distributed in Guangxi, China and northern Vietnam. In addition to its ornamental value, Yellow Camellia also has high medicinal value. In 2010, it was listed in the "Medicine and Food of the Same Origin" catalog by the former Ministry of Health of China, becoming a new resource food. The "Chinese Zhuang Pharmacy" and "Guangxi Chinese Medicinal Materials Standards" record that Yellow Camellia is "slightly bitter, astringent, flat, and can clear away heat, detoxify, promote body fluid, and promote diuresis and reduce swelling."

Flavonoids are a class of compounds that are widely found in various parts of plants and are based on benzene rings and connected by three central carbon atoms. Their parent nucleus is 2-phenylchromone. Current studies have shown that flavonoids have pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor. Discovering flavonoids with novel structures and strong pharmacological effects from plants is of great significance for a deeper understanding of their biological functions and the development of new drugs and health products.

Summary of the invention

The present invention aims to solve the above technical problems and provides a flavonoid glycoside compound in Camellia chrysantha and a preparation method thereof and use thereof in preparing anti-tumor drugs.

The technical solution of the present invention is:

A flavonoid glycoside compound in Camellia chrysantha, the structure of which is shown in Formula I and/or Formula II:

Named as:

3,5,7,4′-tetrahydroxyflavonol-3-O-β- _D -glucopyranosyl-(1→3)-[β- _D -xylopyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-coumaryl-β- _D -glucopyranoside (Formula I); 3,5,7,4′-tetrahydroxyflavonol-3-O-α- _L -arabinopyranosyl-(1→3)-[β- _D -xylopyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-coumaryl-β- _D -glucopyranoside (Formula II).

The method for preparing the flavonoid glycoside compounds in Camellia chrysantha comprises the following steps:

1) Take Camellia chrysantha flowers, dry them, extract them three times with a 70% ethanol aqueous solution at 60° C. under hot reflux, combine the extracts, and concentrate under reduced pressure to remove ethanol to obtain Camellia chrysantha ethanol extract;

2) adding water to the Camellia chrysantha alcohol extract of step 1) and stirring until a suspension is formed;

3) extracting the suspension obtained in step 2) with an equal volume of ethyl acetate three times, filtering the extracted suspension, and concentrating under reduced pressure to remove part of the water to obtain a Camellia chrysantha aqueous extract;

4) using a macroporous resin to adsorb the water extract of Camellia chrysantha obtained in step 3), and desorbing the extract with ethanol aqueous solutions of different volume fractions to obtain fractions Fr.1 to Fr.12 containing flavonoid glycosides;

5) The fraction Fr.5 obtained in step 4) is adsorbed on a small pore resin and desorbed with methanol aqueous solution of different volume fractions to obtain secondary fractions Fr.5.1 to Fr.5.7 containing flavonoid glycoside compounds;

6) The secondary fraction Fr.5.2 of step 5) is adsorbed on a hydrophobic filler and desorbed with methanol aqueous solution of different volume fractions to obtain sub-fractions Fr.5.2.1 to Fr.5.2.9 containing flavonoid glycoside compounds;

7) The sub-fraction Fr.5.2.1 or Fr.5.2.2 of step 6) is separated and purified by semi-preparative high performance liquid chromatography to obtain the flavonoid glycoside compound.

Furthermore, in step 4), the macroporous resin is a D101 macroporous adsorption resin; the volume fraction of ethanol in the ethanol aqueous solution is 5%-100%, specifically, the volume fraction of ethanol in the ethanol aqueous solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

Furthermore, in step 5), the small pore resin is MCI GEL CHP20/P120 small pore adsorption resin; the volume fraction of methanol in the methanol aqueous solution is 5%-100%, specifically, the volume fraction of methanol in the methanol aqueous solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

Furthermore, in step 6), the hydrophobic filler is Toyopearl HW-40 resin; the volume fraction of methanol in the methanol aqueous solution is 5%-100%, specifically, the volume fraction of methanol in the methanol aqueous solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

Further, in step 7), the conditions of the semi-preparative high performance liquid chromatography are: (when separation formula I) the chromatographic column is selected as Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 80:20; the detection wavelength is 220nm; or (when separation formula II) the chromatographic column is selected as Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 85:15; the detection wavelength is 220nm.

The present invention also provides the use of the flavonoid glycoside compounds in the Camellia chrysantha in the preparation of anti-tumor drugs, wherein the tumor is cervical cancer, liver cancer or non-small cell lung cancer.

Compared with the prior art, the present invention has the following beneficial effects:

1. In the present invention, golden camellia is used as raw material, and two flavonoid glycosides are obtained from golden camellia for the first time through the steps of extraction, extraction, separation and purification. The two flavonoid glycoside compounds are identified as follows: 3,5,7,4′-tetrahydroxyflavonol-3-O-β- _D -glucopyranosyl-(1→3)-[β- _D -xylopyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-co umaryl-β- _D -glucopyranoside (Formula I); 3,5,7,4′-tetrahydroxyflavonol-3-O-α- _L -arabinopyranosyl-(1→3)-[β- _D -xylopyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-coumaryl-β- _D -glucopyranoside These compounds provide chemical basis and material reference for the comprehensive development of Camellia chrysantha medicinal materials and the research on the pharmacological activity of natural plants.

2. In vitro pharmacological activity experiments show that the flavonoid glycosides of the present invention have obvious inhibitory effects on Hela (human cervical cancer cells), HepG2 (human liver cancer cells), and A549 (human non-small cell lung cancer cells), and have good prospects in the field of tumor treatment.

The method for separating flavonoid glycoside compounds from golden camellia flowers of the present invention is practical and feasible for industrial production, and provides a new approach for the comprehensive development and utilization of golden camellia resources. At the same time, the separated flavonoid glycoside compounds have good biological activity and are expected to be applied to the fields of medicine, health products and cosmetics. Therefore, the present invention has important practical application value and market prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a HR-ESI-MS mass spectrum of Formula I of the present invention;

FIG2 is a ¹ H-NMR spectrum of Formula I of the present invention;

FIG3 is a ¹³ C-NMR spectrum of Formula I of the present invention;

FIG4 is a DEPT135 spectrum diagram of Formula I of the present invention;

FIG5 is a ¹ H- ¹ H COSY spectrum of Formula I of the present invention;

FIG6 is a HSQC spectrogram of Formula I of the present invention;

FIG7 is a HMBC spectrum diagram of Formula 1 of the present invention;

FIG8 is a NOESY spectrum diagram of Formula I of the present invention;

FIG9 is a TOCSY spectrum diagram of Formula I of the present invention;

FIG10 is a HR-ESI-MS mass spectrum of formula II of the present invention;

FIG11 is a ¹ H-NMR spectrum of Formula II of the present invention;

FIG12 is a ¹³ C-NMR spectrum of Formula II of the present invention;

FIG13 is a DEPT135 spectrum diagram of Formula II of the present invention;

FIG14 is a ¹ H- ¹ H COSY spectrum of Formula II of the present invention;

FIG15 is a HSQC spectrum diagram of formula II of the present invention;

FIG16 is a HMBC spectrum diagram of formula II of the present invention;

Figure 17 is a NOESY spectrum diagram of formula II of the present invention;

FIG. 18 is a TOCSY spectrum diagram of Formula II of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention are described in detail and clearly below. It is obvious that the embodiments disclosed here are only part of the present invention, not all of it. All other embodiments based on the embodiments of the present invention and obtained by those skilled in the art without creative work are within the protection scope of the present invention.

Example 1 Preparation of flavonoid glycosides

The preparation method of flavonoid glycoside compounds in golden camellia comprises the following steps:

1) Take Camellia chrysantha flowers, dry them, extract them three times with a 70% ethanol aqueous solution at 60° C. under hot reflux, combine the extracts, and concentrate under reduced pressure to remove ethanol to obtain Camellia chrysantha ethanol extract;

2) adding water to the Camellia chrysantha alcohol extract of step 1) and stirring until a suspension is formed;

3) extracting the suspension obtained in step 2) with an equal volume of ethyl acetate three times, filtering the extracted suspension, and concentrating under reduced pressure to remove part of the water to obtain a Camellia chrysantha aqueous extract;

4) using D101 macroporous adsorption resin to adsorb the water extract of Camellia chrysantha obtained in step 3), and desorbing the water extract with ethanol aqueous solutions having volume fractions of 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence to obtain fractions Fr.1 to Fr.12 containing flavonoid glycosides;

5) The fraction Fr.5 of step 4) is adsorbed by MCI GEL CHP20/P120 small pore adsorption resin, and desorbed by methanol aqueous solution with volume fractions of 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence to obtain secondary fractions Fr.5.1 to Fr.5.7 containing flavonoid glycoside compounds;

6) The secondary fraction Fr.5.2 of step 5) is adsorbed by Toyopearl HW-40 resin, and desorbed with methanol aqueous solution with volume fractions of 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% in sequence to obtain sub-fractions Fr.5.2.1 to Fr.5.2.9 containing flavonoid glycoside compounds;

7) The subfraction Fr.5.2.1 of step 6) is separated and purified by semi-preparative high performance liquid chromatography to obtain the compound represented by formula I, wherein the chromatographic conditions are: the chromatographic column is a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of acetonitrile and water, the volume ratio of acetonitrile to water is 80:20; the detection wavelength is 220nm;

8) The subfraction Fr.5.2.2 of step 6) is separated and purified by semi-preparative high performance liquid chromatography to obtain the compound represented by formula II, wherein the chromatographic conditions are: the chromatographic column is a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 85:15; the detection wavelength is 220nm.

Example 2 Structural analysis and identification of Formula I

Formula I is a yellow powder (methanol), positive ion HR-ESI-MS gives m/z 1035.3014 [M+H] ⁺ (C ₄₇ H ₅₅ O ₂₆ ⁺ , calculated value is 1035.2981), and the molecular formula of Formula I is determined to be C ₄₇ H ₅₄ O ₂₆ by high resolution mass spectrometry. After thin layer development and color development, it is determined that the compound may be flavonoids.

¹ H-NMR (CD ₃ OD, 600 MHz) observed two aromatic proton signals δ _H 6.17 (1H, br s, H-6), 6.35 (1H, br s, H-8), and a pair of A ₂ B ₂ coupled proton signals δ _H 6.89 (2H, d, J = 8.3 Hz, H-3′, H-5′), 7.98 (2H, d, J = 8.3 Hz, H-2′, H-6′), indicating that the aglycone of formula I is 3,5,7,4′-tetrahydroxyflavone.

Four sugar terminal carbon-hydrogen signals δ _H/C 5.53/100.7, 4.42/104.8, 4.54/102.2, 4.35/106.3 can be seen in the HSQC spectrum. Acid hydrolysis and derivatization identified three sugars: _D -glucose, _D -xylose and _L -rhamnose. Combined with HMBC and TOCSY spectrum analysis, it was determined that formula I contained 2 glucoses, 1 xylose and 1 rhamnose. The configuration of the sugar was determined by the coupling constant of the terminal protons, _D -xylose and 2 _D -glucoses were β-type, and _L -rhamnose was α-type. A group of 1,4-disubstituted aromatic proton signals δ _H 6.81 (2H, d, J = 8.2 Hz, H-3", 5"), 7.47 (2H, d, J = 8.2 Hz, H-2", 6") and trans-vinyl proton signals δ _H 6.39 (1H, d, J = 15.9 Hz, H-8"), 7.70 (1H, d, J = 15.9 Hz, H-7") can also be seen in NMR. In HMBC, H-8" is respectively related to C-1" and C-9";H-7" is respectively related to C-2"/6" and C-8", indicating that Formula I contains a pE-coumaryl fragment.

HMBC defines the connection positions and methods between monosaccharides, between coumaroyl and monosaccharides, and between glycosides and aglycones: the C-1 position of xylose is connected to the C-3 position of rhamnose through an oxygen bridge, the C-1 position of rhamnose, the C-1 position of glucose (2), and the C-9″ position of coumaroyl are connected to the C-6, C-3, and C-2 positions of glucose (1) through oxygen bridges, respectively, and the C-1 position of glucose (1) is connected to the C-3 position of the aglycone through an oxygen bridge.

According to the 1D and 2D NMR spectra of formula I, see Figures 1 to 9, the compound was assigned to all carbon and hydrogen signals, and the data are shown in Table 1. In summary, formula I was identified as 3,5,7,4′-tetrahydroxyflavonol-3-O-β- _D -glucopyranosyl-(1→3)-[β- _D -xyl opyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-coumaryl-β- _D -glucopyranoside

Table 1 ¹ H (600 MHz) and ¹³ C (151 MHz) NMR data of Formula I (deuterated reagent: CD ₃ OD)

Example 3 Structural analysis and identification of Formula II

Formula II is a yellow powder (methanol), and positive ion HR-ESI-MS gives m/z 1005.2885 [M+H] ⁺ (C ₄₆ H ₅₃ O ₂₅ ⁺ , calculated value is 1005.2876). The molecular formula of Formula II is determined to be C ₄₆ H ₅₂ O ₂₅ by high resolution mass spectrometry.

Comparison of NMR data showed that the structures of Formula II and I were very similar. ¹ H-NMR (CD ₃ OD, 600 MHz) showed δ _H 6.90 (2H, d, J = 8.6 Hz), 7.98 (2H, d, J = 8.6 Hz), indicating that the aglycone of Formula II was the same as that of Formula I.

Four sugar terminal carbon-hydrogen signals δ _H/C 5.57/100.7, 4.34/105.3, 4.54/102.2, and 4.36/106.3 were observed in HSQC. Acid hydrolysis and derivatization confirmed the types of sugars as _D -glucose, _D -xylose, _L -rhamnose, and _L -arabinose. The configuration of the sugars was determined by the coupling constants of the terminal protons, with _D -glucose and _D -xylose being β-type, and _L -rhamnose and _L -arabinose being α-type. A group of 1,4-disubstituted aromatic proton signals δ _H 6.80 (2H, d, J = 8.0 Hz, H-3", 5"), 7.46 (2H, d, J = 8.0 Hz, H-2", 6") and trans-vinyl proton signals δ _H 6.39 (1H, d, J = 15.9 Hz, H-8"), 7.69 (1H, d, J = 15.9 Hz, H-7") can also be seen in NMR. In HMBC, H-8" is respectively related to C-1" and C-9";H-7" is respectively related to C-2"/6" and C-8", indicating that Formula II contains a pE-coumaryl fragment.

The HMBC correlation determined the position and mode of connection between monosaccharides and between glycosides and aglycones: the C-1 position of xylose was connected to the C-3 position of rhamnose through an oxygen bridge, the C-1 position of rhamnose, the C-1 position of arabinose, and the C-9″ position of coumaroyl were connected to the C-6, C-3, and C-2 positions of glucose through oxygen bridges, and the C-1 position of glucose was connected to the C-3 position of aglycone through an oxygen bridge.

According to the 1D and 2D NMR spectra of formula II, see Figures 10 to 18, the compound was assigned to all carbon and hydrogen signals, and the data are shown in Table 2. In summary, formula II was identified as 3,5,7,4′-tetrahydroxyflavonol-3-O-α- _L -arabinopyranosyl-(1→3)-[β- _D -xylopyranosyl-(1→3)-α- _L -rhamnopyranosyl-(1→6)]-2-OpE-coumaryl-β- _D -glucopyranoside.

Table 2 ¹ H (600 MHz) and ¹³ C (151 MHz) NMR data of Formula II (deuterated reagent: CD ₃ OD)

Example 4 Test on the inhibitory activity of two flavonoid glycoside compounds on tumor cell proliferation

Weigh appropriate amounts of Formula I and II, add 1 mL of sterile water containing 1% dimethyl sulfoxide to dissolve, and dilute to 0.5, 1, 2, 4, 8, 16, 32 μM test solution using DMEM medium or RPMI-1640 medium. Cisplatin (DDP) and 5-fluorouracil (5-Fu) are positive controls.

HepG2 (human liver cancer cells) in the logarithmic growth phase were seeded at about 8×10 ³ cells/well, A549 (human non-small cell lung cancer cells) at about 5×10 ³ cells/well, and Hela (human cervical cancer cells) at about 3×10 ³ cells/well in a 96-well plate and incubated in a 37°C, 5% CO ₂ saturated humidity incubator. After 24 hours, the culture medium in all wells was removed, and the culture medium containing different concentrations of compounds was added and incubated for 48 hours. 10 μL of 5 mg/mL MTT solution was added to each well and incubated for 4 hours. The culture medium containing MTT in each well was removed, 150 μL DMSO was added, and the OD value of each well was measured at 570 nm. The inhibition rate of the compound on tumor cells was calculated according to formula 1, and the IC ₅₀ value was calculated using SPSS software.

The inhibition rate was calculated as follows:

Inhibition rate (%) = [1-( _A2 - _A0 )/( _A1 - _A0 )] × 100%, where: _A2 is the test group added with the culture medium containing the test solution; _A1 is the control group added with the culture medium without the test solution; _A0 is the blank control group without any liquid and cells.

Table 3 Results of inhibition of cell proliferation by two flavonoid glycosides (IC ₅₀ , μM±SD)

It can be seen that the two flavonoid glycoside compounds have obvious inhibitory effects on Hela (human cervical cancer cells), HepG2 (human liver cancer cells), and A549 (human non-small cell lung cancer cells), and can be used as treatment or for research on drugs for treating tumors.

The above description is a detailed description of the preferred feasible embodiments of the invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims (10)

1. The flavone glycoside compound in golden camellia is characterized in that the structure is shown as a formula I and/or a formula II:

2. a method for preparing flavone glycoside compounds in golden camellia according to claim 1, which is characterized by comprising the following steps:

1) Collecting camellia chrysantha flower, drying, performing hot reflux extraction by using an ethanol aqueous solution with the volume fraction of 70%, mixing the extracting solutions, and concentrating under reduced pressure to remove ethanol to obtain an ethanol extract of camellia chrysantha flower;

2) Adding water into the golden camellia alcoholic extract obtained in the step 1) and stirring until a suspension is formed;

3) Extracting the suspension in the step 2) by using ethyl acetate with the same volume, filtering the extracted suspension, and concentrating under reduced pressure to remove part of water to obtain a golden camellia water extract;

4) Adsorbing the camellia chrysantha water extract of the step 3) by using macroporous resin, and desorbing by using ethanol water solutions with different volume fractions to obtain fractions containing flavone glycoside;

5) Adsorbing the fraction obtained in the step 4) by using a small-pore resin, and desorbing by using methanol water solutions with different volume fractions to obtain a secondary fraction containing flavone glycoside compounds;

6) Adsorbing the secondary fraction in the step 5) by a hydrophobic filler, and desorbing by methanol water solutions with different volume fractions to obtain a sub-fraction containing flavone glycoside compounds;

7) Separating and purifying the sub-fraction in the step 6) by semi-preparative high performance liquid chromatography to obtain the flavone glycoside compound.

3. The method for preparing flavonoid glycoside compounds in golden camellia according to claim 2, wherein in the step 4), the macroporous resin is D101 macroporous adsorption resin; the volume fraction of the ethanol in the ethanol water solution is 5% -100%.

4. The method for preparing flavonoid glycoside compounds in golden camellia according to claim 3, wherein in the step 4), the volume fraction of ethanol in the ethanol aqueous solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.

5. The method for preparing flavonoid glycoside compounds in golden camellia according to claim 2, wherein in the step 5), the small pore resin is MCI GEL CHP/P120 small pore adsorption resin; the volume fraction of methanol in the aqueous methanol solution is 5% -100%.

6. The method for preparing flavonoid glycoside compounds in camellia nitidissima according to claim 5, wherein in the step 5), the volume fraction of methanol in the aqueous methanol solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%.

7. The method for preparing flavonoid glycoside compounds in golden camellia according to claim 2, wherein in the step 6), the hydrophobic filler is Toyopearl HW-40 resin; the volume fraction of methanol in the aqueous methanol solution is 5% -100%.

8. The method for preparing flavonoid glycoside compounds in camellia nitidissima according to claim 7, wherein in the step 6), the volume fraction of methanol in the aqueous methanol solution is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%.

9. The method for preparing flavonoid glycoside compounds in golden camellia according to claim 2, wherein in the step 7), the conditions of the semi-preparative high performance liquid chromatography are as follows: the chromatographic column is a Welch Ultimate XB-C18 semi-preparation column, the mobile phase is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 80:20; the detection wavelength is 220nm; or selecting Welch Ultimate XB-C18 semi-preparative column from chromatographic column, wherein the mobile phase is a mixed solution of acetonitrile and water, and the volume ratio of acetonitrile to water is 85:15; the detection wavelength was 220nm.

10. The use of flavonoid glycoside compounds in golden camellia according to claim 1 in preparing antitumor drugs, wherein the tumors are cervical cancer, liver cancer or non-small cell lung cancer.