CN103223018B - Flos Osmanthi Fragrantis polyphenol extract and its production and use - Google Patents

Abstract

The invention relates to a plant polyphenol extract extracted from osmanthus fragrans and its preparation method and application. Specifically, the present invention discloses a preparation method of sweet-scented osmanthus polyphenols extract, comprising the following steps: 1) Mixing sweet-scented osmanthus and extractant aqueous solution according to the solid-liquid ratio of 1g/15-60ml, and then mixing them under a pressure of 200-500KPa Under 80~200W power, microwave extraction is carried out, and the extraction time is 3~20min; repeat the above extraction; 2), the obtained filtrates are combined and then dried to obtain the osmanthus polyphenols extract. The sweet-scented osmanthus polyphenol extract can be used to prepare anti-tumor medicine or health product, and can also be used to inhibit the activity of 5α-reductase.

Description

Osmanthus fragrans polyphenol extract and its preparation method and use

technical field

The invention relates to the field of deep processing and comprehensive utilization of agricultural and sideline products. More specifically, it relates to a plant polyphenol extract extracted from osmanthus fragrans and its preparation method and application.

Background technique

Osmanthus fragrans Lour. Also known as sweet-scented osmanthus, jiulixiang, and golden millet, it is one of the top ten traditional famous flowers in my country. It is not only a famous spice plant, but also an excellent landscaping tree species, with good ecological, social and economic benefits. . Osmanthus fragrans has a long history of cultivation in my country, and it is wild in Sichuan, Yunnan, Zhejiang, Guangxi, Hubei and other provinces and regions; it is widely planted from the Huaihe River Basin to the south of the lower reaches of the Yellow River, and potted plants are mostly used in the north. There are about 40 species of sweet-scented osmanthus in the world, and 27 species in my country. It is the main distribution center of sweet-scented osmanthus, and its germplasm resources are extremely rich.

Osmanthus fragrans is a traditional fragrant flower plant. Although the flowers are small, the fragrance is long-lasting and stable. my country has a long tradition of edible osmanthus. As early as 2000 years ago, osmanthus was used to make wine and scented tea. In recent years, with the continuous expansion of osmanthus planting area and the rapid development of food industry, osmanthus cake, osmanthus sauce and other products have also been widely used mass production.

Osmanthus is rich in nutrients. The total amino acid content in osmanthus is as high as 13.78%, of which the essential amino acid for human body is 6.26%, accounting for 45.55% of the total amino acid, and the ratio of amino acid is reasonable, it is a high-quality protein. In addition, osmanthus is rich in vitamins, carotene and various trace elements, potassium, iron and zinc. The research on secondary metabolites of osmanthus fragrans mainly focuses on the extraction of essential oil and analysis of aromatic components. Li-mei Wang et al. (2009) investigated the differences in essential oil components of osmanthus fragrans at different flowering stages, and found that osmanthus osmanthus essential oil contains a variety of linalool and its oxides, α-ionone, β-ionone, nerol, etc. 20 remaining active ingredients. Chun-Di Hu et al. (2010) further found that osmanthus essential oil contains up to 73 components. Less research has been done on other biologically active substances. Chien-Ya Hung et al. (2012) isolated and identified phenolic components in osmanthus, and found that it contained 5 kinds of phenolic compounds such as rutin and verbascoside. Osmanthus fragrans has biological activities such as anti-oxidation and anti-inflammation, and has shown certain effects in neuroprotection and inhibition of melanin formation, but there is little research on the relationship between its efficacy and components.

Microwave extraction is actually mainly the heating effect of microwave on the extraction solvent and sample. Microwave heating has two mechanisms, namely ion conduction and dipole rotation. This principle enables microwaves to generate instantaneous high temperature, so the extraction time is short; microwaves will reflect, absorb and penetrate when encountering different materials during transmission. Phenomenon. When the microwave can penetrate the medium, that is, the microwave directly heats the inside of the sample, which avoids the problem of heating from a single part in the traditional heating method, so that the microwave heating has the advantage of uniform heating. At the same time, because the dielectric properties of each part of the material are often different, microwaves have the characteristic of selective heating of this material. High-pressure microwave-assisted extraction is carried out in a closed extraction tank, and its internal pressure can reach more than 1MPa, so the boiling point of the solvent is much higher than that of the solvent under normal pressure, so that the microwave extraction can reach the same solvent under normal pressure. The extraction temperature can improve the extraction efficiency without decomposing the extract to be tested.

According to the head of the WHO International Cancer Research Council (IARC), the global cancer incidence rate will increase by 50% to 15 million per year by 2020. The clinical treatment methods for malignant tumors have been developed so far, such as surgical resection, chemotherapy, radiotherapy, etc., but these treatment methods will cause great damage to the normal body while killing tumor cells, with great side effects. It is of great research significance to reduce the dosage of clinical medication or the intensity of surgery through the auxiliary effect of natural active substances. In view of the limitations of the research on the active ingredients of osmanthus fragrans and their biological activities, it is of great significance to expand the research on the active ingredients of osmanthus fragrans and apply them to the prevention and treatment of tumors.

5α-reductase (5α-reductase) is a membrane protease dependent on reducing coenzyme II (NADPH), which can catalyze the conversion of testosterone (T) into dihydrotestosterone (DHT). T and DHT act on the same androgen receptor, However, the catalytic activity of DHT is stronger and has a stronger affinity for the androgen receptor, so the catalytic transformation of 5α-reductase can amplify the androgen response, which may cause pseudohermaphroditism, acne, androgenetic alopecia, female Symptoms such as hirsutism are also inseparable from the occurrence of benign prostatic hyperplasia and even prostate cancer, so it is of great significance to find high-efficiency 5α-reductase inhibitors. Researchers have found that certain phytochemicals such as myricetin, dalbergol, and impatiens extract (Matsuda H, 2001; Shirota O, 2003; Ishiguro K, 2000.) have a certain degree of 5α-reductase inhibitory activity, One of the main active ingredients of Qianliekang, a Chinese patent medicine commonly used in the treatment of benign prostatic hyperplasia, has also been proved to be a polyether in rapeseed pollen. Studies have shown that natural phytochemicals are high-quality sources of 5α-reductase inhibitors.

Contents of the invention

The technical problem to be solved by the present invention is to provide an osmanthus fragrans polyphenol extract and its preparation method and application.

In order to solve the problems of the technologies described above, the invention provides a preparation method of sweet-scented osmanthus polyphenols extract, comprising steps:

1), using high-pressure microwave extraction method:

After mixing osmanthus fragrans and extractant aqueous solution according to the solid-liquid ratio of 1g/15~60ml, under the pressure of 200~500KPa, the power of 80~200W is used for microwave extraction, and the extraction time is 3~20min; filter to obtain filter cake and first filtrate;

Replace the sweet-scented osmanthus with the filter cake and repeat the above microwave extraction and filtration at least once to obtain the filtrate again;

The volume concentration of the extractant in the aqueous extractant solution is 70% to 90%;

2) Combine the first filtrate and the second filtrate obtained in step 1) and dry them to obtain the osmanthus fragrans polyphenols extract.

As the improvement of the preparation method of the sweet-scented osmanthus polyphenol extract of the present invention: the extractant is acetone, methanol or ethanol.

As a further improvement of the preparation method of the osmanthus fragrans polyphenol extract of the present invention: the drying in step 2) is at least one of vacuum drying, freeze drying, and film drying.

As a further improvement of the preparation method of the sweet-scented osmanthus polyphenol extract of the present invention: in step 1), the microwave extraction pressure is 300-450KPa, and the power is 80-120W.

As a further improvement of the preparation method of the sweet-scented osmanthus polyphenols extract of the present invention: the aqueous extractant solution is an aqueous acetone solution with a volume concentration of 80%, a solid-liquid ratio of 1 g/45ml, a pressure of 300KPa, and 100W microwave extraction for 3min.

The present invention also provides an osmanthus fragrans polyphenol extract prepared by the above method at the same time: the total phenol weight content in the osmanthus fragrans polyphenols extract is 30%-70%.

As an improvement of the osmanthus fragrans polyphenol extract of the present invention: in the total phenols, the weight content of phenolic acid is 0.5%-2%, and the weight content of phenylethanol glycoside is 50%-80%.

As a further improvement of the osmanthus polyphenols extract of the present invention: in the phenolic acids, the weight content of chlorogenic acid is 40% ~ 70%, and the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 25 ~ 50:5 ~15: 6~10; among the phenylethanol glycosides, the weight content of verbascoside is 80%~95%.

Remarks: The phenolic acids described in the present invention include chlorogenic acid, caffeic acid and p-coumaric acid, and the phenylethanol glycosides include verbascoside.

The present invention also provides the use of the sweet-scented osmanthus polyphenol extract prepared by the above method, and its application in the preparation of antitumor drugs or health care products.

The present invention also provides another use of the sweet-scented osmanthus polyphenols extract prepared by the above method, for inhibiting 5α-reductase activity (that is, the application in the preparation of 5α-reductase activity inhibitors).

The sweet-scented osmanthus polyphenol extract of the present invention can be made into composition, contains 0.01wt%～99.9wt% (better, 0.1wt%-90wt%) sweet-scented osmanthus polyphenol extract of the present invention in the composition, and Contains acceptable carriers (including those acceptable in pharmaceuticals, food or cosmetics).

After in-depth research, the inventor extracted and separated the active ingredients of osmanthus fragrans, and obtained a polyphenol extract that can effectively fight cancer --- osmanthus fragrans polyphenols extract. Research on the extract shows that the extract mainly Contains chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside.

As used herein, sweet-scented osmanthus refers to the flower of sweet-scented osmanthus, a plant of the genus Osmanthus in the family Oleaceae. The osmanthus fragrans that can be used as the raw material of the present invention can be osmanthus fragrans fresh flowers, osmanthus fragrans dried flowers, osmanthus fragrans tea and other products using osmanthus fragrans as raw materials or residues of osmanthus fragrans after distillation extraction. The preferred raw materials are osmanthus fresh flowers and dried osmanthus flowers.

The raw materials used in the present invention may or may not be pretreated. A preferred raw material is sweet osmanthus in powder form. For example, collect fresh sweet-scented osmanthus or other products added with sweet-scented osmanthus, dry to a moisture content (weight) below 10%, and pulverize to about 20-40 mesh powder.

The osmanthus species that can be used in the present invention and the place of production are not particularly limited, and can be osmanthus of different varieties, such as golden osmanthus, silver osmanthus, etc., and can also come from different places of production such as Guangxi, Zhejiang, etc. The fragrant golden osmanthus is preferred.

The sweet-scented osmanthus polyphenols extract of the present invention should be extracted by high-pressure microwave technology, although it can also be extracted by other methods.

A preferred method is to extract polyphenolic compounds from sweet-scented osmanthus and its related products or residues of sweet-scented osmanthus by using high-pressure microwave extraction technology. In a preferred example, the extractant is 80% (volume concentration) acetone, the extraction pressure is 200-500KPa, the solid-liquid ratio is 1:15-1:60 (g/ml), and the microwave extraction is 3-20min.

In a more preferred example, about 20-40 mesh osmanthus powder (moisture weight content ≤ 10%) is added to the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument, and the extractant is selected as 80% (volume concentration) Acetone, solid-liquid ratio 1:15-1:45 (g/ml), control pressure 200-500KPa, 80-200W microwave treatment for 3-20min, centrifuge, repeat extraction more than 2 times, filter and combine supernatant (i.e., filtrate ), the supernatant was concentrated by vacuum rotary evaporation, and dried at low temperature to obtain osmanthus polyphenols extract.

The osmanthus polyphenol extract of the present invention is a polyphenol mixture mainly composed of verbascoside. Detect with Folin's phenol method, take chlorogenic acid as standard substance, measure total phenolic content in sweet-scented osmanthus polyphenols extract to be 30%～70%, better 40%～60%; The mass fraction of phenylethanol glycosides is 50% to 80%, more preferably 60% to 75%; the composition of phenolic acids and phenylethanol glycosides is analyzed by HPLC, wherein the content of chlorogenic acid in phenolic acids is 40% to 70%, more preferably The best 45%~65%, the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 25~50:5~15:6~10, more preferably 25~45:5~12:7~9, benzene The content of verbascoside in ethanol glycosides is 80%-95%, more preferably 82%-92%.

The sweet-scented osmanthus polyphenol extract of the invention has the biological activity of anti-tumor and inhibition of 5α-reductase activity, so it can be used to prepare anti-tumor or 5α-reductase inhibitory medicines, health care products and food. Therefore, the present invention also provides a composition (comprising pharmaceutical composition, health product composition, food composition and cosmetic composition etc.) Or adjuvant therapy for tumor cell proliferation.

After obtaining the sweet osmanthus polyphenols extract, it can be mixed with pharmaceutically, food science or health care products or cosmetically acceptable carriers, excipients or diluents by conventional methods to form the pharmaceutical composition of the present invention, food Composition or health product composition. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.

Taking pharmaceutical compositions or health product compositions as examples, they can be in solid state (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes. The content of the osmanthus fragrans polyphenols extract of the present invention is usually 1%-99% by weight of the composition, preferably 2%-96%, more preferably 5%-90%, most preferably 15%-80%.

Nutraceutical compositions can be in single or multi-dose form. According to the administration dose, it usually contains 10-2000 mg/dose, preferably about 20-1000 mg/dose, more preferably 50-500 mg/dose.

The health product composition of the present invention can be administered through conventional routes, preferably orally. The pharmaceutical formulation should match the mode of administration. The application amount of the health care product of the present invention, calculated by active substance, is usually about 0.1-500 mg/kg body weight of osmanthus fragrans polyphenols extract per day, preferably about 0.5-50 mg/kg body weight. In addition, the preparations according to the invention can also be used together with other auxiliary preparations, for example, including various existing anticancer active substances or antioxidative active substances.

The present invention has the following advantages:

(a) Provide an osmanthus fragrans polyphenol extract with broad sources, clear composition, high quality, controllable quality, wide application and various physiological and pharmacological activities.

(b) The efficient extraction of polyphenols in Osmanthus fragrans and its related products was realized by high-pressure microwave extraction technology, and the operation process is simple.

To sum up, polyphenols are a class of compounds containing polyhydric phenolic hydroxyl groups, which are widely distributed in nature. There are many kinds of plant polyphenols, mainly including phenolic acids, flavonoids, etc. Studies have shown that they have various physiological activities such as scavenging free radicals, anti-oxidation, alleviating atherosclerosis, antibacterial agents, and anti-tumor. Edible flowers contain a large amount of polyphenols, and as one of the common edible flowers, osmanthus is rich in varieties and has great potential for development. Therefore, it is of great significance to obtain polyphenol extracts from osmanthus and expand its health care functions.

The present invention realizes the high-efficiency extraction of polyphenols in sweet-scented osmanthus and its related products through ultrasonic-assisted extraction technology, the operation process is simple, and it is found that the extract has good anti-cancer effects, and has wide application prospects in food, health care products and medicines .

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is the spectrum of HPLC separation results of chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside reference substance and sweet-scented osmanthus sample. The spectrum shows that 1 and 10.1min are chlorogenic acid, 2 and 12.8min are caffeic acid, and 3 , 19.3min for p-coumaric acid, 4, 30.7min for verbascoside.

Fig. 2 is the HPLC separation result spectrum of osmanthus osmanthus polyphenols extract, wherein B is the 8-22min enlarged effect diagram in A. Corresponding to the spectrum of the reference substance, about 1 and 10.1 minutes are chlorogenic acid, about 2 and 12.8 minutes are caffeic acid, about 3 and 19.3 minutes are p-coumaric acid, and about 4 and 30.7 minutes are verbascoside;

Figure 3 is the structural diagrams of chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside, respectively.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed.

The general method of detection is:

1) Determination of total phenol content in osmanthus polyphenols extract - Folin phenol method

With chlorogenic acid as the control, total phenols were determined by Folin's phenol method. Take 500ul sample (diluted to a certain multiple, that is, each ml sample contains 2mg of osmanthus polyphenol extract) or standard solution (chlorogenic acid methanol solution with a concentration of 10-150μg/ml), add 50ul 2M folinol, mix Evenly react for 5-8 minutes, add 1ml of 1M NaOH, dilute to 6ml with water, react at room temperature for 90 minutes, measure the absorbance at 760nm wavelength, and calculate the chlorogenic acid equivalent concentration of total phenols.

2) Determination of the content of phenylethanol glycosides in osmanthus polyphenols extracts——UV spectrophotometry

Using verbascoside as a control, the total ethanol glycosides were determined by UV spectrophotometry. A certain amount of sample (containing 8 mg of osmanthus polyphenols extract per ml sample) was adsorbed by D-101 macroporous resin and eluted successively with water 25 times the sample volume and 20% (v/v) ethanol, and discarded. solution, and then eluted with 50 times the volume of 70% ethanol (v/v), collected the eluate, evaporated to dryness, and constant volume with methanol. The absorbance was measured at a wavelength of 330nm, and the equivalent concentration of verbascoside was calculated.

3) Analysis of active components of osmanthus polyphenols extract - high performance liquid chromatography (HPLC)

The components of osmanthus fragrans polyphenols extract were separated and analyzed by HPLC separation method.

HPLC separation method: use Phenomenex Synergi 4μ hydro-RP 80A (250*4.6mm) chromatographic column; mobile phase: A (acetic acid aqueous solution, pH2.74), B (acetonitrile), column temperature 38 ℃, flow rate 0.8ml/min; Ultraviolet scanning was performed between 200-600nm, and the gradient elution procedure was as follows:

Table 1 Gradient elution program

time/min A% B% 0 95 5 2 92 8 15 84 16 35 75 25 50 5 95 51 0 100 55 0 100 56 95 5 60 95 5

The sweet-scented osmanthus powder described in the following examples refers to the powder obtained by drying fresh sweet-scented osmanthus to a water content (weight) below 10% and pulverizing to about 20-40 meshes, with a weight of 100 g.

Embodiment 1,

Add about 20-40 mesh sweet-scented osmanthus powder into the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument. The extractant is 80% acetone (that is, the acetone aqueous solution with a volume concentration of 80%), and the ratio of solid to liquid is 1:15 (g/ml), control the pressure 200KPa, 80W microwave treatment for 3min, filter, and repeat the extraction twice (that is, replace the sweet-scented osmanthus powder with the filter residue, repeat the above microwave treatment and filtration twice in turn), combine the filtrate obtained from the three extractions, The filtrate was concentrated by vacuum rotary evaporation at 45°C, and the concentrated solution was pre-frozen at -80°C and vacuum freeze-dried to constant weight to obtain the polyphenol extract of osmanthus fragrans with a yield of 15.1%.

The components of the extract were determined and analyzed, and the results are shown in Figures 1 and 2. Figures 1 and 2 show that the extract contains chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. Colorimetric method and HPLC method were used to analyze the composition content of osmanthus fragrans polyphenols extract, and the total phenol content was 30.5±1.3%. Among the total phenols, the content of chlorogenic acid is 0.25±0.01%, the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 30:5:6; the content of phenylethanol glycoside is 65.2±3.2%, and verbascoside in phenylethanol glycoside The content is 87.6±1.1%.

Embodiment 2,

Add about 20-40 mesh osmanthus powder into the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument, use 80% acetone as the extractant, the ratio of solid to liquid is 1:25 (g/ml), and the control pressure is 200KPa, 80W Microwave treatment for 5 minutes, filter, and then repeat the extraction twice (that is, replace the osmanthus powder with the filter residue, repeat the above microwave treatment and filtration twice in turn), combine the filtrates obtained from the three extractions, and concentrate the filtrate by vacuum rotary evaporation at 45 °C. After pre-freezing at -80°C, vacuum freeze-dry to constant weight to obtain osmanthus polyphenols extract with a yield of 23.5%.

Composition determination and analysis were carried out on the extract, which contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. Colorimetric method and HPLC method were used to analyze the composition content of osmanthus fragrans polyphenols extract, and the total phenol content was 59.7±1.1%. Among the total phenols, the content of chlorogenic acid is 0.4±0.015%, the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 35:15:6; the content of phenylethanol glycoside is 71.5±1.9%, and verbascoside The content is 85.7±2.3%.

Embodiment 3,

Add about 20-40 mesh sweet-scented osmanthus powder into the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument. The extractant is 80% acetone, the ratio of solid to liquid is 1:35 (g/ml), and the control pressure is 300KPa. 80W microwave treatment for 8min, filter, and then repeat the extraction 2 times (that is, replace the osmanthus powder with filter residue, repeat the above microwave treatment and filtration 2 times in turn), combine the filtrate obtained from the 3 extractions, and concentrate the filtrate by vacuum rotary evaporation at 45°C, the concentrated solution After pre-freezing at -80°C, vacuum freeze-dry to constant weight to obtain osmanthus polyphenols extract with a yield of 25.1%.

Composition determination and analysis were carried out on the extract, which contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. Colorimetric method and HPLC method were used to analyze the composition content of osmanthus fragrans polyphenols extract, and the total phenol content was 60.8±2.1%. Among the total phenols, the content of chlorogenic acid is 0.4±0.01%, the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 40:15:7; the content of phenylethanol glycoside is 61.6±0.9%, and verbascoside in phenylethanol glycoside The content is 87.1±2.5%.

Embodiment 4,

Add about 20-40 mesh sweet-scented osmanthus powder into the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument. The extractant is 80% acetone, the ratio of solid to liquid is 1:40 (g/ml), and the control pressure is 400KPa. Microwave treatment at 100W for 8 minutes, filter, and repeat the extraction twice (that is, replace the osmanthus powder with filter residue, repeat the above microwave treatment and filtration twice in turn), combine the filtrate obtained from the three extractions, and concentrate the filtrate by vacuum rotary evaporation at 45°C. After pre-freezing at -80 ℃, vacuum freeze-drying to constant weight, the polyphenol extract of osmanthus fragrans was obtained, with a yield of 28.0%.

Composition determination and analysis were carried out on the extract, which contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. Colorimetric method and HPLC method were used to analyze the composition content of osmanthus fragrans polyphenols extract, and the total phenol content was 51.5±1.3%. Among the total phenols, the content of chlorogenic acid is 0.8±0.01%, and the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 45:12:6; the content of phenylethanol glycoside is 68.8±1.7%, and verbascoside in phenylethanol glycoside The content is 88.5±2.1%.

Example 5

Add about 20-40 mesh sweet-scented osmanthus powder into the inner cup made of polytetrafluoroethylene material of the intelligent microwave digestion instrument. The extractant is 80% acetone, the ratio of solid to liquid is 1:45 (g/ml), and the control pressure is 300KPa. Microwave treatment at 100W for 3 minutes, filter, and repeat the extraction twice (that is, replace the osmanthus powder with the filter residue, repeat the above microwave treatment and filtration twice in turn), combine the filtrate obtained from the three extractions, and concentrate the filtrate by vacuum rotary evaporation at 45°C. After pre-freezing at -80°C, vacuum freeze-dry to constant weight to obtain the polyphenol extract of sweet-scented osmanthus with a yield of 30.4%.

Composition determination and analysis were carried out on the extract, which contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. The colorimetric method and HPLC method were used to analyze the composition content of the polyphenol extract of osmanthus fragrans, and the total phenol content was 63.6±3.2%. Among the total phenols, the content of chlorogenic acid is 0.25±0.01%, the mass ratio of chlorogenic acid, caffeic acid and p-coumaric acid is 50:15:10; the content of phenylethanol glycoside is 60.1±1.5%, and verbascoside in phenylethanol glycoside The content is 89.3±1.3%.

The obtained total phenolic content of this embodiment 5 is the highest, reaching 19.3% of the dried sweet-scented osmanthus.

In order to fully prove the technical advantages of the present invention, the inventor has also carried out the following comparative examples:

Ratio 1-1~1-Comparative example 1-4, only change the extractant from 80% acetone to 60% acetone, 100% acetone, 80% methanol, and 80% ethanol; the rest are the same as in Example 5.

Composition determination and analysis were carried out on the extract, which contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. The specific content is as described in Table 2.

Table 2

Proportion 2-1. Change the control pressure 300KPa, 100W microwave treatment for 3 minutes to "control pressure 200KPa, 100W microwave treatment for 3 minutes", and the rest are the same as in Example 5.

Proportion 2-2, change the control pressure 300KPa, 100W microwave treatment for 3 minutes to "control pressure 500KPa, 100W microwave treatment for 3 minutes", and the rest are the same as in Example 5.

For ratio 2-3, change the control pressure 300KPa, 100W microwave treatment for 3 minutes to "control pressure 300KPa, 200W microwave treatment for 3 minutes", and the rest are the same as in Example 5.

For ratio 2-4, change the control pressure 300KPa, 100W microwave treatment for 3 minutes to "control pressure 300KPa, 80W microwave treatment for 20 minutes", and the rest are the same as in Example 5.

Comparative example 2-5, change the ratio of solid to liquid from 1:45 (g/ml) to 1:20 (g/ml), and the rest are the same as in Example 5.

Comparative example 2-6, change the ratio of solid to liquid from 1:45 (g/ml) to 1:60 (g/ml), and the rest are the same as Example 5.

The extracts obtained in Comparative Examples 2-1 to 2-6 were subjected to component determination and analysis, and the extracts all contained chlorogenic acid, caffeic acid, p-coumaric acid and verbascoside. The specific content is as described in Table 3.

table 3

Experiment 1. Inhibitory effect of osmanthus polyphenols extract on tumor cell proliferation

① Cell lines and culture conditions

Human breast cancer cells (MCF-7, MDA), human prostate cancer cells (PC-3, LN-CaP), culture medium is RPMI-1640, containing 10% inactivated fetal bovine serum, 5% carbon dioxide incubator at 37℃ nourish.

② MTT colorimetric method to detect cell proliferation rate

Collect the logarithmic phase cells, adjust the concentration of the cell suspension to 50,000 cells/ml, add 100ul of the cell suspension to each well, put the cells into an incubator for culture, and administer the drug the next day after attaching to the wall, and put the cells into the incubator for 24 hours. After the drug effect is over, add 20ul MTT (5mg/ml) to each well, incubate for 3-4h, terminate the culture, and carefully suck off the culture medium in the well. Add 150ulDMSO to each well, incubate at 37°C for 10 minutes or shake at low speed on a shaker for 10 minutes. Then use a microplate reader to detect the absorbance (A) value of each hole at 490nm and record the results. The cell growth curve is drawn with the time as the abscissa and the absorbance value as the ordinate. The inhibitory rate (IR) of the drug on cell proliferation was calculated according to the formula. IR=[1-(the mean A value that medication group measures/the mean A value that control hole measures)] * 100%, and carry out regression analysis by SPSS17.0 software, calculate _IC50 value (half-lethal inhibitory concentration), to sweet-scented osmanthus Polyphenol extracts were evaluated.

The sweet-scented osmanthus polyphenol extract in table 4, each embodiment and comparative example is to each tumor cell IC ₅₀ value

_IC50 (ug/ml) MCF-7 MDA PC-3 LN-CaP Example 1 690.9 503.7 250.4 318.1 Example 2 630.5 490.2 189.8 220.2 Example 3 604.5 450.3 140.2 193.9 Example 4 653.2 499.8 177.6 211.1 Example 5 534.4 399.3 136.6 140.2 Comparative example 1-1 598.7 453.8 250.4 318.1 Comparative example 1-2 624.5 509.2 169.8 220.2 Comparative example 1-3 604.4 465.3 160.2 193.9 Comparative example 1-4 673.2 498.9 177.6 211.1 Comparative example 2-1 544.4 429.3 156.3 180.2 Comparative example 2-2 590.9 501.1 250.4 288.1 Comparative example 2-3 610.5 491.8 189.8 251.8 Comparative example 2-4 684.5 480.3 190.2 310.9 Comparative example 2-5 693.2 489.8 224.6 218.1 Comparative example 2-6 552.4 423.5 146.6 194.2

Experiment 2. Inhibitory effect of osmanthus polyphenols extract on 5α-reductase activity

① Preparation of rat 5α-reductase

Three clean-grade female SD rats were taken, fasted without food and water overnight, and then killed by dislocation of the cervical vertebrae. The liver was taken out and cut into pieces on ice. Use the pre-cooled buffer solution to make a 1:5 homogenate in a glass homogenizer, centrifuge at 10,000 g for 15 min at 4 °C, take the cytoplasmic part and centrifuge for 1 h at 100,000 g, discard the supernatant, and precipitate It is the coarse particle extract. Add a certain volume of buffer, and use a homogenizer to homogenate the suspension. Aliquot and store in -80°C refrigerator.

② Determination of 5α-reductase activity

a. 5α-reductase activity assay system: 0.5mL 20μM phosphate buffer (pH=6.5), 0.1mL 50% ethanol (reaction tube) or sample solution (sample tube), 150μL 0.5mg/mL testosterone, 250μL 0.8mg/mL mL NADPH, add 0.5mL enzyme extract with concentrations of 0, 0.96, 1.92, 2.88, 3.84, 4.80mg/mL. After reacting at 37°C for 30 minutes, 2.5 mL of dichloromethane was added to stop the reaction, and then 0.25 mL of 100 μg/mL propylparaben was added as an internal standard, shaken for 60 s, and centrifuged at 400 g for 10 min.

b. Determination of enzyme activity by HPLC: remove about 1ml of organic phase and evaporate to dryness, dissolve the residue in 1.5ml of methanol, take 10μL of high performance liquid phase injection to measure the ratio of residual testosterone to internal standard peak area and record it as r reaction, r reaction value Low indicates high enzyme activity. Chromatographic conditions: luna C18 (2) column (4.6×250mm, 5μm); column temperature: 40°C; injection volume: 10μL; flow rate: 1mL/min; mobile phase: 65% methanol, detection wavelength: 242nm.

Inhibition rate of sample to 5α-reductase = (r _sample - r _reaction ) / (r _blank - r _reaction ) × 100%;

Note: r _blank --- add dichloromethane before the reaction starts to inactivate the enzyme, and measure the value of the enzyme blank tube;

c. The same method was used to test finasteride as a control, and the finasteride equivalent of osmanthus polyphenols extract was calculated.

Osmanthus fragrans polyphenols extract in table 5, each embodiment and comparative example is to 5α-reductase inhibition

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (7)

1. the purposes of sweet-scented osmanthus polyphenols extract, it is characterized in that: the application in the medicine or the health product that is used for the preparation of antitumor, described tumor is breast cancer or prostate cancer; The preparation method of sweet-scented osmanthus polyphenols extract is to comprise following step: 1), using high-pressure microwave extraction method: Mix osmanthus fragrans and extractant aqueous solution according to the solid-liquid ratio of 1g/15-60ml, then carry out microwave extraction under the pressure of 200-500KPa and the power of 80-200W, and the extraction time is 3-20min; filter to obtain the filter cake and the first filtrate; Replace the sweet-scented osmanthus with the filter cake and repeat the above microwave extraction and filtration at least once to obtain the filtrate again; The volume concentration of the extractant in the extractant aqueous solution is 70% to 90%; The extractant is acetone, methanol or ethanol; 2), combining the first filtrate and the second filtrate obtained in step 1) and then drying to obtain the osmanthus fragrans polyphenols extract. 2. the purposes of sweet-scented osmanthus polyphenols extract according to claim 1 is characterized in that: the drying in described step 2) is at least one in decompression drying, freeze drying, film drying. 3. The use of osmanthus fragrans polyphenols extract according to claim 2, characterized in that: the microwave extraction pressure in the step 1) is 300-450KPa, and the power is 80-120W. 4. the purposes of sweet-scented osmanthus polyphenols extract according to claim 3 is characterized in that: extractant aqueous solution is the acetone aqueous solution of volume concentration 80%, solid-liquid ratio 1g/45ml, and pressure is 300KPa, and 100W microwave extracts 3min. 5. The use of the osmanthus polyphenol extract according to any one of claims 1 to 4, characterized in that: the total phenol weight content in the osmanthus polyphenol extract is 30% to 70%. 6. the purposes of sweet-scented osmanthus polyphenols extract according to claim 5 is characterized in that: in described total phenol, the weight content of phenolic acid is 0.5%～2%, the weight content of phenylethanol glycoside is 50%～80% %. 7. the purposes of sweet-scented osmanthus polyphenols extract according to claim 6 is characterized in that: in described phenolic acid, the weight content of chlorogenic acid is 40%～70%, and chlorogenic acid, caffeic acid and p-couma bean The mass ratio of the acids is 25-50:5-15:6-10; the weight content of verbascoside in the phenylethanol glycosides is 80%-95%.