CN104958406B - The preparation of the active component of sugar orange tool tyrosinase inhibitory activity and purposes - Google Patents

Abstract

The invention discloses the preparation and application of the effective part of shatangju with tyrosinase inhibitory activity, and belongs to the technical field of medicine and food. Fresh Shatang Tangerine is extracted with ethanol, concentrated and rehydrated, extracted with dichloromethane and ethyl acetate in turn, concentrated and freeze-dried to obtain an effective fraction mainly containing six compounds. The invention provides that the effective part of Shatang Tangerine Tangerine has strong tyrosinase inhibitory activity, can be effectively used for the inhibition of enzymatic browning of fruits and vegetables, whitening in cosmetics, and prevention and treatment of hyperpigmentation caused by excessive melanin. Various formulations of drugs for human pigmentation disorders, melanoma, and other conditions requiring inhibition of tyrosinase activity.

Description

Preparation and application of the effective part of shatangju with tyrosinase inhibitory activity

technical field

The invention relates to the preparation and application of an effective part of shatangju with tyrosinase inhibitory activity, and belongs to the technical fields of medicine and food.

Background technique

Sand sugar orange, also known as October orange, belongs to the fruit of the genus Tangerine in the family Rutaceae. The main production areas are: Lipu (Xiuren Town), Huaiji, Guangning, and Sihui. The fruit of Shatangju is flat and round, with tumor-like protrusions on the top, sunken umbilical end of the pedicle, orange-yellow color, thin fruit wall, and easy to peel off. It is rich in vitamin C, calcium, fiber, a small amount of protein, fat and rich in glucose, fructose, sucrose, malic acid, citric acid, citric acid and carotene, thiamine, riboflavin, niacin, ascorbic acid , flavonoids and other biologically active ingredients.

At present, there are very few studies on Shatangju, and there are no literature reports on the components of its various parts (peel, tangerine core, leaves, pulp). We screened the tyrosinase inhibitory activity of the extracts of five kinds of Citrus fruits (tangerine, tangerine, lemon, Gannan orange, pomelo peel, pulp, tendon) and found that citrus tangerine It has strong tyrosinase inhibitory activity, so we further determined its main components by high performance liquid chromatography-mass spectrometry.

Tyrosinase is the key enzyme of melanin synthesis, and its expression and activity are closely related to the browning of fruits and vegetables and human pigmentation diseases. Therefore, tyrosinase inhibitors can be used as melanin inhibitors in food, cosmetics, medicine and other fields.

Contents of the invention

The object of the present invention is to provide an effective part with tyrosinase inhibitory activity, which is an effective part extracted from Shatangju Tangerine and composed of six polyphenolic compounds, including naringelin-4' -Glucoside, naringin, hesperidin, neohesperidin, citrusin and quercetin-7,3',4'-glucoside.

The total phenol content in the effective parts is 460-480 mg/g, and the total flavonoid content is 14-16 mg/g.

The total phenol content in the effective parts is 472.68 mg/g, and the total flavonoid content is 14.63 mg/g.

The contents of the six compounds in the effective fraction are 3.2%, 28.6%, 2.6%, 26.4%, 20.3%, and 2.5%, respectively.

Another object of the present invention is to provide the preparation method of the tyrosinase inhibitor active compound and its effective part, which is characterized in that it is realized by the following steps:

(1) Add 25g of Shatang Tangerine Tangerine into 500mL of 95% (v/v) ethanol, extract at 30°C with ultrasonic power of 250W for 30min, concentrate the extract to dry matter under reduced pressure, and weigh it;

(2) Dissolve 80.0 mg of the dried product in 320 mL of deionized water to obtain a raw material solution, and then use dichloromethane and ethyl acetate to extract the raw material solution twice, extraction agent: raw material solution = 2:1 (v/v);

(3) Separating the extract phase, concentrating under reduced pressure to dry matter, and obtaining the effective part extract.

Another object of the present invention is to provide the effective part of the tangerine tangerine complex that has tyrosinase inhibitory activity in the preparation of anti-enzymatic browning inhibitors of fruits and vegetables, the preparation of cosmetic whitening agents, and the prevention and treatment of melanin abnormalities. Application in the medicine of human pigmentation diseases, melanoma and other diseases that need to inhibit the activity of tyrosinase.

The effective fraction with tyrosinase inhibitory activity extracted from Shatangju Tangerine in the present invention contains six kinds of flavonoids, which can be used as active ingredients for inhibiting tyrosinase activity, without or adding food additives and auxiliary materials, cosmetics The excipients or excipients accepted on the pharmacy are made into preparations according to the preparation method of the corresponding dosage form.

The dosage form includes solid powder, aqueous solution, alcohol solution, emulsion, facial mask, cataplasm, injection, infusion, powder injection, granule, tablet, electuary, powder, oral liquid, sugar-coated agent, film-coated tablet tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, buccal preparations, granules, pills, ointments, pills, sprays, dropping pills, disintegrants, oral disintegration, pellets, etc. .

The present invention extracts the effective part with tyrosinase inhibitory activity from Shatangju Tangerine, which is a composition containing six polyphenolic compounds, and the effective part has stronger activity than other Citrus fruit extracts It can be used in enzymatic browning inhibitors of fruits and vegetables, cosmetic whitening agents, prevention and treatment of human pigmentation diseases caused by abnormal melanin, melanoma and other diseases that need to inhibit tyrosinase activity. application in medicine.

Description of drawings

Figure 1 is the UPLC/Q-TOF-MS ultraviolet and total ion chromatograms of the effective part extract of Shatangju with tyrosinase inhibitory activity; (a): UV chromatogram; (b): low collision energy total Ion current map; (c): High collision energy total ion current map.

Figure 2 is a schematic diagram of the extracted mass spectrum and molecular fragmentation analysis of the effective part of Shatangju with tyrosinase inhibitory activity; Figures (a) to (f) are the mass spectra and molecular structures of the compounds corresponding to chromatographic peaks 1 to 6, respectively.

Figure 3 Determination of rutin standard curve for total phenols.

Figure 4 Determination of total flavonoids rutin standard curve.

Detailed ways

The essence and beneficial effects of the present invention will be further described in detail below in conjunction with examples, and these examples are only used to illustrate the present invention rather than limit the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The preparation of embodiment 1 Shatang Tangerine Tangerine Extract

Fresh tangerine tangerines were extracted with 95% ethanol at a solid-liquid ratio (m/v) of 1:20 at a power of 250w, ultrasonically extracted at 30°C for 30 minutes, and the extract was concentrated under reduced pressure to a dry product, and weighed; Suspend with deionized water, the ratio of material to liquid is 1:4 (m/v) to obtain the raw material solution, and then use dichloromethane and ethyl acetate to extract twice each, extraction agent: raw material solution = 2:1 (v/v) ; Separating the extract phase, concentrating under reduced pressure to dry matter, and weighing to obtain the extract of the effective part.

Ultra-high performance liquid chromatography-mass spectrometry detection conditions: Instrument: Waters Maldi Synapt Q-TOF MS; Chromatograph UHPLC: Waters Acquity UPLC; Detector UV detector: Waters Acquity PDA; Column: Acquity BEH C18 (1.7μm; 50× 2.1mm I.D.).

Mobile phase: mobile phase A is acetonitrile, B phase is 0.1mol/L formic acid; elution gradient: 0min, 5%A; 8min, 30%A; 10min, 80%A; 13min, 100%A; 14min, 100% %A; 14.1 min, 5%A. Flow rate, 0.3 mL/min. Mass spectrometry conditions: electrospray ion source, negative ion scan mode; cone voltage, 30V; capillary voltage, 3.0kV; channel 1 collision energy, 6eV; channel 2 collision energy, 20eV; ion source temperature, 100°C; desolvation temperature, 400°C; scanning range, 50-1500Da.

Table 1 Detection results of ultra-high performance liquid chromatography-mass spectrometry

Example 2 The UPLC-MS mass spectrum and molecular fragmentation analysis of the main components of Shatangju Tangerine Extract

As shown in Figure 2(a), the m/z of the negative ion of peak No. 1 is 741, which is the [MH] ^-peak , indicating that the molecular weight of peak No. 1 is 742, and its main fragments are 597, 433, 271, 151, due to the parent The nuclear ion is subjected to a collision voltage, and the CO bond between the glycoside and the parent nuclear ion is easier to break during the bombardment process, so we judge that the glucoside at the 4' position is broken first, and the m of the [MH-glu] ^-peak (glu, glucose) /z is 597, after which the rhamnoside and glucoside CO bonds in the rutinose on the A ring are broken, the m/z of the [MH-glu-rha] ^-peak (rha, rhamnose) is 433, and the further 7 The CO bond of the [MH-glu-rut] ^-peak (rut, rutinose) is broken at m/z 271, and the [MH-glu-rut] ^-peak occurs Retro Diels-Alder Fragmentation gave a chromatographic peak with m/z 151. The compound was identified as naringerin rutin-4'-glucoside according to molecular weight and molecular fragment fragmentation mode.

As shown in Figure 2(b), the m/z of the negative ion of the No. 2 peak is 579, which is the [MH] ^-peak , indicating that the molecular weight of the No. 2 peak is 580, and its main fragments are 271 and 151. First, the collision causes the glycosidic CO bond at the 7th position of the A ring to be broken, and the m/z of the [MH-rut ^] -peak is 271, and the [MH-rut] ^-peak undergoes Retro Diels-Alder cleavage to obtain Chromatographic peak with m/z 151. The compound was identified as naringin according to molecular weight and molecular fragment fragmentation mode.

As shown in Figure 2 (c～d), the m/z of No. 3 peak and No. 4 peak is negative ion is 609, is [MH] ^- peak, shows that the molecular weight of No. 2 peak is 610, and its main fragment is 301, 151. First, the collision causes the glycoside CO bond ^at the 7th position of the A ring to be broken, and a molecule of rutinoside is lost ^. -Alder) cracked to give m/z as the chromatographic peak of 151. Because the relative molecular masses are the same, we speculate that the two substances are isomers. However, the pair of isomers, hesperidin and neohesperidin, are widely found in citrus fruits, and the molecular weight and fragmentation mode are consistent with this, and the peak time of hesperidin will be earlier than that of neohesperidin. Therefore, it is judged that peak No. 3 is hesperidin, and peak No. 4 is neohesperidin.

As shown in Figure 2(e), the m/z of the negative ion of peak No. 5 is 593, which is the [MH] ^-peak , indicating that the molecular weight of peak No. 5 is 594, and its main fragments are 309 and 285. First, the collision causes the glycoside CO bond at the 7th position of the A ring to be broken, and the neohesperidin sugar is lost. The m/z of the [MH-308] ^-peak is 285, which is the parent nucleus ion of isoserarin, and the neohesperidin is lost The sugar anion m/z is 309. The compound was determined to be citrusin according to the molecular weight and molecular fragment fragmentation mode.

As shown in Figure 2(f), the m/z of the negative ion of peak No. 6 is 771, which is the [MH] ^-peak , indicating that the molecular weight of peak No. 5 is 772, and its main fragments are 609, 469, and 301. At low collision voltage, the first collision results in the breakage of the sophorotrioside CO bond at the 7 position of the A ring, and the [MH-3glu] ^-peak has an m/z of 301, while at high collision voltage, we get more fragments. First, one glucoside of sophorotriose is broken (162Da), the m/z of the [MH-glu] ^-peak is 609, the CO bond of sophorotrioside at the 7th position of the A ring is broken, and the m/z of the [MH-3glu ^] -peak is 301, a sophorotriose fragment with m/z 469. According to the molecular weight and molecular fragment fragmentation mode, it was determined that the compound might be hesperetin-7-sophorotriose.

Example 3 Evaluation of the tyrosinase inhibitory activity of the effective part extract of Shatangju Tangerine

The extracts of the effective parts of Shatang Tangerine Tangerine were made into sample solutions with a concentration of 1 mg/mL with DMSO reagent, 300 μL of the sample solution was drawn into a test tube, and 700 μL of phosphate buffer solution with a pH value of 6.8 was added. Then add 1mL of 0.1mg/mL L-tyrosine solution and 1mL of 200U/mL tyrosinase solution prepared by phosphate buffer solution with a pH value of 6.8, and measure the absorbance at 492nm after rapid shaking and mixing. Then put it into a water bath and react at 37°C for 20 minutes and measure its absorbance again. The blank control is DMSO solution without sample, and the positive control is kojic acid. Calculate the inhibition rate corresponding to each concentration sample by the following calculation formula:

Inhibition rate (%)=[(A2-A1)-(B2-B1)]/(A2-A1)×100%

A1 is the absorbance of the blank control group at 0 minutes, A2 is the absorbance of the blank group after 20 minutes, B1 is the absorbance of the sample at 0 minutes, and B2 is the absorbance of the sample group after 20 minutes.

The inhibitory rate of the effective part extract of Shatangju Tangerine at a concentration of 100 μg/mL is 26.17%, and the positive control kojic acid (monomer substance) is 69.19%, indicating that Shatangju extract has a strong tyrosinase inhibitory effect .

Example 4 Analysis of Total Phenols and Total Flavonoids Content Determination of the Effective Parts Extract of Shatang Tangerine Tangerine Tangerine

(1) Determination of total phenols: Take rutin standard solution 0.05mL, 0.1mL, 0.2mL, 0.4mL, 0.8mL, 1.6mL, 3.2mL respectively in test tubes, add 2mL of aluminum nitrate nonahydrate solution and 3mL of potassium acetate solution , and add 70% aqueous ethanol to a total volume of 10 mL, while making a blank. Shake well, let stand at room temperature for 30 minutes, measure absorbance at a wavelength of 420nm, and draw a standard curve. The standard curve drawn by rutin is shown in Figure 3: regression equation y=29.02x-0.0013, correlation coefficient R ² =0.9999. The results showed that the absorbance had a good linear relationship with the concentration of rutin in the range of 0-0.02 mg/L.

Dissolve the sample in 70% ethanol aqueous solution to make 1mg/mL test solution, absorb 1.0mL sample test solution, add 2mL of aluminum nitrate nonahydrate solution, 3mL of potassium acetate solution, 4mL of 70% ethanol aqueous solution, shake well, and let it stand at room temperature. Set aside for 30 minutes, centrifuge at 4000r/min for 10 minutes to take the supernatant, and measure the absorbance at a wavelength of 420nm.

The measured total phenolic content in the effective part extract of Shatangju Jujupi is 472.68mg/g.

(2) Determination of total flavonoids: Dissolve the sample in 70% ethanol aqueous solution to form a 1 mg/mL test solution, draw 1.0 mL of the sample test solution, add 1 mL of Folin’s phenol reagent, 3 mL of 7.5% sodium carbonate, mix well, and distilled water Dilute to 10mL, heat in a water bath at 45°C for 60min, and measure absorbance at a wavelength of 765nm.

Using gallic acid monohydrate solutions with concentrations of 0, 10, 20, 30, 40, 50 mg/L as standard solutions (dissolved in 70% ethanol), a standard curve was drawn. The standard curve drawn for gallic acid is shown in Figure 4: regression equation y=0.0046x-0.0221, correlation coefficient R ² =0.9992. The results showed that there was a good linear relationship between the absorbance and the concentration of gallic acid in the range of 0-80 mg/L.

The measured total flavonoid content in the effective part extract of Shatangju Tangerine is 14.63mg/g.

The content of total phenols and total flavonoids in the effective part extract of Shatangju Tangerine are higher, indicating that its tyrosinase inhibitory activity is mainly due to these polyphenols and flavonoids.

The preparation of the whitening cream of embodiment 5 tangerine extract

Tangerine Tangerine Extract 0.5%, Grape Seed Extract 2.0%, Stearic Acid 5.0%, Stearyl Alcohol 4.0%, Isopropyl Myristate 18.0%, Glyceryl Monostearate 10.0%, Hydroxide Sodium 0.2%, sodium sulfite 0.01%, V _C 0.5%, appropriate amount of preservatives, appropriate amount of spices, deionized water added to 100%.

Preparation method: 5.0% stearic acid, 4.0% stearyl alcohol, 18.0% isopropyl myristate, 10.0% glyceryl monostearate, 0.2% sodium hydroxide, 0.01% sodium sulfite, heat to 75°C, stir well . When the temperature drops to 40°C, add 0.5% tangerine tangerine extract, 2.0% grape seed extract, 0.5% V _C , appropriate amount of preservatives, appropriate amount of spices, stir evenly, and the product is ready.

The preparation of embodiment 6 granulated sugar tangerine extract whitening facial mask

Tangerine Tangerine Extract 1.5%, Green Tea Extract 2.0%, Polyvinyl Alcohol 12.0%, Butylene Glycol 10.0%, Glycerin 8.0%, Polyoxyethylene Lauryl Ether (AEO-9) 2.0%, V _C 0.2% , an appropriate amount of preservatives, an appropriate amount of spices, and deionized water to 100%.

Preparation method: Mix and stir 12.0% polyvinyl alcohol and water, heat to 80°C to dissolve, then add 10.0% butanediol, 8.0% glycerin, 2.0% polyoxyethylene lauryl ether (AEO-9) under stirring, When the temperature drops to 40°C, add 1.5% tangerine orange extract, 2.0% green tea extract, appropriate amount of preservatives, appropriate amount of spices, and then spread it on a non-woven fabric to obtain the product.

Claims (9)

1. The effective part of Shatangju with tyrosinase inhibitory activity is characterized in that it is an effective part containing six compounds extracted from Shatangju Tangerine, including naringerin rutin-4'-glucoside, Naringin, hesperidin, neohesperidin, citrusin and quercetin-7, 3', 4'-glucoside, the extraction of the effective parts is the volume percentage of Shatang tangerine tangerine assisted by ultrasound It is extracted with 95% ethanol, and the extract is concentrated under reduced pressure, then extracted with dichloromethane and ethyl acetate respectively, and the extract phase is concentrated to dryness under reduced pressure. 2. The effective part according to claim 1, characterized in that the total phenol content is 460-480 mg/g, and the total flavonoid content is 14-16 mg/g. 3. The effective part according to claim 1, characterized in that the contents of the six compounds are respectively 3.2%, 28.6%, 2.6%, 26.4%, 20.3%, and 2.5%. 4. effective part according to claim 1, is characterized in that, the extraction step of described effective part is: (1) Add 25g of shatangtangerine to 500mL of ethanol with a volume percentage of 95%, extract at 30°C with an ultrasonic power of 250W for 30min, and concentrate the extract to dryness under reduced pressure; (2) Dissolve 80.0mg of the dried product in 320mL of deionized water to obtain a raw material solution, and then use dichloromethane and ethyl acetate to extract the raw material solution twice, and the volume ratio of the extractant to the raw material solution is 2:1; (3) Separating the extract phase, concentrating under reduced pressure to dry matter, and obtaining the effective part extract. 5. A method for preparing the effective part of the sugar orange with tyrosinase inhibitory activity according to claim 1, characterized in that, comprising the following steps: (1) Add 25g of shatangtangerine to 500mL of ethanol with a volume percentage of 95%, extract at 30°C with an ultrasonic power of 250W for 30min, and concentrate the extract to dryness under reduced pressure; (2) Dissolve 80.0mg of the dried product in 320mL of deionized water to obtain a raw material solution, and then use dichloromethane and ethyl acetate to extract the raw material solution twice, and the volume ratio of the extractant to the raw material solution is 2:1; (3) Separating the extract phase, concentrating under reduced pressure to dry matter, and obtaining the effective part extract. 6. The application of the effective part of the sugar orange with tyrosinase inhibitory activity described in claim 1 in the preparation of anti-enzymatic browning inhibitors of fruits and vegetables. 7. The application of the effective part of the sugar orange with tyrosinase inhibitory activity described in claim 1 in the preparation of whitening cosmetics. 8. The effective part of the sugar orange with tyrosinase inhibitory activity described in claim 1 is used in the preparation of prevention and treatment of human pigmentation diseases caused by abnormal melanin, melanoma and other diseases that need to inhibit tyrosinase activity application in medicine. 9. The application according to claim 8, wherein the dosage forms of the medicine include solid powder, aqueous solution, alcohol solution, emulsion, facial mask, cataplasm, injection, infusion, powder injection, granule, Tablets, sugar-coated agents, capsules, buccal preparations, pills, ointments, elixirs, sprays, drop pills, and oral disintegrations.