Preparation method and application of fructus chebulae extract

Abstract

The invention belongs to the technical field of daily cosmetics, and particularly relates to a preparation method and application of a fructus chebulae extract. The medicine terminalia fruit extract contains gallic acid and medicine terminalia fruit tannic acid, and the content of the gallic acid and the content of the medicine terminalia fruit tannic acid are larger than or equal to 20 wt% on the basis of the dry weight of the medicine terminalia fruit extract. In the invention, the myrobalan extract can simultaneously enrich two active matters, namely gallic acid and myrobalan tannic acid to the greatest extent through the type of the composite macroporous resin, the transfer rate of the myrobalan extract is improved by 10.64% compared with that of a single resin, and the yield of the obtained myrobalan extract freeze-dried powder is improved by 13.99% compared with that of the single resin. Besides, the prepared fructus chebulae extract has the effects of inhibiting 5alpha-reductase, melanin secretion, streptococcus mutans and porphyromonas gingivalis, so that the fructus chebulae extract is proved to have the effects of oil control, sunscreen, whitening, brightening and bacteriostasis, and possibility and basis are provided for applying the fructus chebulae extract to the field of daily cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of daily cosmetics, and particularly relates to a preparation method and application of a terminalia chebula extract. Background Art

[0002] Terminalia chebula is the dried mature fruit of Terminalia chebula Retz. or Terminalia chebula Retz, var. tomentella Kurt., a plant of the Combretaceae family, and Xiqingguo is the dried young fruit of Terminalia chebula, a plant of the Combretaceae family. The chemical components of the two medicinal materials are mainly tannins, triterpenes, phenolic acids and fatty acids, among which the tannin content is as high as 23% to 37%. "The King of Tibetan Medicine" - Terminalia chebula. In the Yongkang Town of Yongde County, there grows a tree called Terminalia chebula, which is known as the "King of Tibetan Medicine". In the classic work of Tibetan medicine "Jingzhu Materia Medica", Terminalia chebula is called the "King of Tibetan Medicine" and is the most commonly used Tibetan medicine.

[0003] Patent CN110772550A prepares ellagic acid by pulp powder, ethanol reflux, and single macroporous resin NKA-9 or HPD600. Since the polyphenolic components of gallic acid and chebula tannic acid are more polar than ellagic acid, the preparation method of this patent has a low adsorption rate of active ingredients of gallic acid and chebula tannic acid, and the obtained extract is mainly ellagic acid. Patent CN111658683A provides an application of a chebula water extract in the preparation of a drug for treating and / or preventing Malassezia infection-related skin diseases, which is mainly used in Malassezia infection-related skin diseases including pityriasis versicolor, Malassezia folliculitis and seborrheic dermatitis. The chemical components contained in chebula are rich and diverse, mainly including tannins, phenolic acids, triterpenes, flavonoids, volatile oils and other components. Modern pharmacological studies have shown that its extract has multiple pharmacological activities such as antioxidant, anti-diabetic, antibacterial, antiviral, anti-inflammatory, and analgesic. However, in the field of daily chemical products, there are few studies on the application of Terminalia chebula extract in oil control, sun protection, whitening and oral care.

[0004] Therefore, the art is in urgent need of developing applications of terminalia chebula extract in oil control, sun protection, whitening and oral care. Summary of the invention

[0005] The present invention aims to provide application of a terminalia chebula extract in oil control, sun protection, whitening and oral care, and specifically relates to a preparation method and application of the terminalia chebula extract.

[0006] In a first aspect of the present invention, a terminalia chebula extract is provided, wherein the terminalia chebula extract comprises gallic acid and terminalia tannic acid, wherein the total content of the gallic acid and terminalia tannic acid is greater than 20 wt %, based on the dry weight of the terminalia chebula extract.

[0007] In another preferred embodiment, the total content of gallic acid and terminalia chebula tannic acid is ≥25wt%, based on the dry weight of terminalia chebula extract.

[0008] In another preferred embodiment, the total content of gallic acid and terminalia chebula tannic acid is 20wt%-60wt%, preferably 20wt%-50wt%, more preferably 20wt%-40wt%, based on the dry weight of terminalia chebula extract.

[0009] In another preferred embodiment, the content of gallic acid is 0.5wt%-3wt%, preferably 1wt%-2.5wt%, more preferably 1.5wt%-2wt%, based on the dry weight of the Terminalia chebula extract.

[0010] In another preferred embodiment, the content of the terminalia chebula tannic acid is 15wt%-40wt%, preferably 20wt%-35wt%, more preferably 25wt%-30wt%, based on the dry weight of the terminalia chebula extract.

[0011] In another preferred embodiment, the terminalia chebula extract further comprises terminalia chebula tannic acid and / or ellagic acid, wherein the content of terminalia chebula tannic acid is 10-20wt%, more preferably 15-20wt%.

[0012] In another preferred embodiment, the terminalia chebula extract further comprises terminalia chebula tannic acid and / or ellagic acid, wherein the content of the ellagic acid is 1-10wt%, more preferably 1-5wt%.

[0013] In a second aspect of the present invention, a method for preparing the Terminalia chebula extract according to the first aspect of the present invention is provided, the method comprising the steps of: (1) Extraction: Extract the terminalia chebula fruit with ethanol, collect the filtrate and concentrate it to obtain a crude terminalia chebula extract; (2) Purification: taking the crude extract obtained in step (1), eluting it with a combination of S-8 and NKA-9 macroporous resins, wherein the elution comprises: first eluting with water, collecting the water washing liquid, then eluting with ethanol, and collecting the ethanol analytical liquid; (3) taking the water washing solution obtained in step (2), using ethanol as the eluent, and continuing to elute with the combined macroporous resin of S-8 and NKA-9 to obtain an ethanol analytical solution; (4) The ethanol analytical solutions obtained in steps (2) and (3) are combined and freeze-dried to obtain the Terminalia chebula extract.

[0014] In another preferred embodiment, the ethanol is an ethanol aqueous solution with a concentration of 60 v / v %-70 v / v %.

[0015] In another preferred embodiment, the macroporous resin is selected from the group consisting of S-8, NKA-9, or a combination thereof. In another preferred embodiment, the method comprises the following steps:

[0016] (1) providing a raw material of Terminalia chebula, adding ethanol with a concentration of 50-80 v / v% for extraction, collecting the extract, concentrating it, and obtaining a crude extract of Terminalia chebula extract; diluting the crude extract of Terminalia chebula extract with water to obtain a sample solution; (2) loading the sample solution onto a macroporous resin for resin adsorption, and eluting, collecting the elution effluent containing gallic acid and chebulic acid, thereby obtaining a chebulic acid extract rich in gallic acid and chebulic acid, wherein the macroporous resin is a composite macroporous resin of S-8 and NKA-9, and the elution comprises: first eluting with water, collecting the water washing solution, then eluting with ethanol, collecting the ethanol analytical solution, thereby obtaining an eluate rich in gallic acid and chebulic acid; and (3) Optionally, the eluate is concentrated to obtain a Terminalia chebula extract rich in gallic acid and terminalia tannic acid.

[0017] In another preferred embodiment, the content of the crude extract of Terminalia chebula extract in the sample solution is 40-60 vv%.

[0018] In another preferred embodiment, the content of the crude extract of Terminalia chebula extract in the sample solution is 45-55 vv%.

[0019] In another preferred embodiment, the volume ratio of the crude extract of Terminalia chebula extract to the composite macroporous resin is 1:(2-6).

[0020] In another preferred embodiment, the volume ratio of the crude extract of Terminalia chebula extract to the composite macroporous resin is 1:(3-5).

[0021] In another preferred embodiment, the method for preparing the Terminalia chebula extract according to the first aspect of the present invention comprises the steps of: (1) Extraction: providing a raw material of Terminalia chebula fruit, adding ethanol at a concentration of 60 v / v% to 70 v / v% for reflux extraction, collecting the filtrate, and concentrating it to obtain a crude extract of Terminalia chebula extract; (2) Purification: The crude extract obtained in step (1) is loaded onto a combined macroporous resin of S-8 and NKA-9, and the elution procedure is performed: first eluting with water, collecting the water washing liquid, then eluting with ethanol at a concentration of 60 v / v%-70 v / v%, and collecting the ethanol analytical liquid; (3) taking the water washing solution obtained in step (2), repeating the elution procedure in step (2) 2-4 times, and combining the ethanol analytical solution obtained each time; (4) The ethanol solution obtained in step (3) is freeze-dried to obtain the Terminalia chebula extract.

[0022] In another preferred embodiment, the ratio of S-8 to NKA-9 in the composite macroporous resin of S-8 and NKA-9 is 6:1 to 1:6 (w:w).

[0023] In another preferred embodiment, the ratio of S-8 to NKA-9 in the composite macroporous resin of S-8 and NKA-9 is 1 to 3-5 (w:w).

[0024] In another preferred embodiment, the ratio of S-8 to NKA-9 in the composite macroporous resin of S-8 and NKA-9 is (2-1)~(2-1) (w:w).

[0025] In another preferred embodiment, the ratio of S-8 to NKA-9 in the composite macroporous resin of S-8 and NKA-9 is 3-5 to 1 (w:w).

[0026] In another preferred embodiment, in the terminalia chebula extract rich in gallic acid and terminalia tannic acid, the transfer rates of gallic acid and terminalia tannic acid and F1 are ≥80wt%, preferably, F1 is ≥90wt%.

[0027] In another preferred embodiment, the transfer rate F1 is calculated according to the following formula Q1: F1=Fm+Fh Q1 In the formula, Fm is the transfer rate of gallic acid in the chebula extract rich in gallic acid and chebula tannic acid; Fh is the transfer rate of chebulic acid in the chebulic acid extract rich in gallic acid and chebulic acid.

[0028] In another preferred embodiment, the transfer rate Fm is calculated according to the following formula Q2: Fm=[Wm / W 浸膏 ]×100% Q2 Where: Wm is the mass of gallic acid in the chebula extract rich in gallic acid and chebula tannic acid; W 浸膏 It is the mass of crude extract of Terminalia chebula extract.

[0029] In another preferred embodiment, the transfer rate Fh is calculated according to the following formula Q3: Fh=[Wh / W 浸膏 ]×100% Q3 Where: Wh is the mass of chebulic acid in the chebulic acid- and chebulic acid-rich extract; W 浸膏 It is the mass of crude extract of Terminalia chebula extract.

[0030] In another preferred embodiment, the method further comprises vacuum drying or freeze drying the eluate rich in gallic acid and terminalia chebula tannic acid to obtain a powdered terminalia chebula extract.

[0031] In another preferred embodiment, in the terminalia chebula extract rich in gallic acid and terminalia tannic acid, the yield of gallic acid and terminalia tannic acid H1 is ≥20wt%, preferably, H1 is ≥25wt% (based on dry weight).

[0032] In another preferred embodiment, the yield and H1 are calculated according to the following formula Q4: H1=[Wh / W 原料 ]×100% Q4 Where: Wh is the mass of the Chebula chebula extract enriched with gallic acid and Chebula tannic acid.

[0033] W 原料 The quality of the Terminalia chebula raw materials.

[0034] In another preferred embodiment, step (2) in the method comprises: (S1) loading the sample solution onto the composite macroporous resin for resin adsorption; (S2) first eluting with water, collecting the water washing solution A, and then eluting with 60-70 wt% ethanol, collecting the ethanol analytical solution B; (S3) taking the washing solution A obtained in step (S1) as the loading solution A1, loading it onto the composite macroporous resin for resin adsorption; (S4) repeating steps (S2-S3) 2-4 times, collecting the ethanol solution obtained each time, or mixing the ethanol solution obtained each time in any proportion, and concentrating to obtain a terminalia chebula extract rich in gallic acid and terminalia chebula tannic acid; Wherein, the macroporous resin is a composite macroporous resin of S-8 and NKA-9.

[0035] In another preferred embodiment, in step (S2) of the method, a washing liquid A corresponding to 2BV of washing is collected.

[0036] In another preferred embodiment, in step (S2) of the method, ethanol analysis liquid B corresponding to 4BV of alcohol wash is collected.

[0037] In another preferred embodiment, in step (S3) of the method, the volume ratio of the water washing solution A to the composite macroporous resin is 1:(3-5).

[0038] In another preferred embodiment, step (2) in the method comprises: (S1) loading the sample solution onto the composite macroporous resin for resin adsorption; (S2) Elution with water, first washing with 2BV of water, collecting the washing solution A, then analyzing with 4BV of 65% ethanol, collecting the ethanol analysis solution A1; (S3) The washing liquid A was purified with 4 times the amount of composite macroporous resin; (S2) was repeated to obtain the washing liquid B and ethanol analytical solution B1; (S4) The washing liquid B was purified with 4 times the amount of composite macroporous resin; (S2) was repeated to obtain washing liquid C and ethanol analytical solution C1; (S5) Collecting the ethanol analysis solutions A1, B1 and C1, or mixing the ethanol analysis solutions obtained each time in any proportion, and concentrating them to obtain a terminalia chebula extract rich in gallic acid and terminalia chebula tannic acid.

[0039] In another preferred embodiment, the elution procedure in step (2) is repeated 3 times, and the elution procedure includes the steps of: (2-1) First, elute with ethanol to collect the water washing solution 1, then elute with ethanol at a concentration of 60 v / v %-70 v / v %, and collect the ethanol analytical solution 1; (2-2) Take the water washing solution 1 obtained in step (2-1), first elute with ethanol, collect the water washing solution 2, then elute with ethanol at a concentration of 60 v / v%-70 v / v%, and collect the ethanol analytical solution 2; (2-3) Take the water washing solution 2 obtained in step (2-1), first elute with 5% ethanol, then elute with 60 v / v %-70 v / v % ethanol, collect the ethanol analysis solution 3, and combine the ethanol analysis solutions obtained each time.

[0040] In another preferred embodiment, the Terminalia chebula fruit in step (1) is a Terminalia chebula young fruit.

[0041] In another preferred embodiment, the Terminalia chebula fruit in step (1) is a Terminalia chebula young fruit or a Terminalia chebula full fruit.

[0042] In another preferred embodiment, the terminalia chebula fruit in step (1) is a terminalia chebula young fruit, or a terminalia chebula ripe fruit, or a terminalia chebula pulp (pitted).

[0043] In another preferred embodiment, the extraction in step (1) is heating reflux extraction.

[0044] In another preferred embodiment, the reflux extraction time in step (1) is 1.5-2.5 h.

[0045] In another preferred embodiment, in step (2), the amount of the combined macroporous resin is S-8:NKA-9=10:1~10:1 (w:w).

[0046] In another preferred embodiment, the amount of the combined macroporous resin in step (2) is S-8:NKA-9=6:1~1:6 (w:w).

[0047] In another preferred embodiment, the mass ratio of the crude extract to the combined macroporous resin in step (2) is 20%-150%.

[0048] In the third aspect of the present invention, there is provided a use of the Terminalia chebula extract as described in the first aspect of the present invention, which is used for preparing daily products or daily chemical products with oil control, sun protection, whitening or antibacterial effects.

[0049] In another preferred embodiment, the composition is used to inhibit 5a-reductase.

[0050] In another preferred embodiment, the composition also has an oil control effect.

[0051] In another preferred embodiment, the inhibition rate of the composition on 5a-reductase is ≥35%.

[0052] In another preferred embodiment, the composition is used for sun protection.

[0053] In another preferred embodiment, the sun protection index SPF value of the composition is ≥5.5.

[0054] In another preferred embodiment, the composition is used to reduce melanin content.

[0055] In another preferred embodiment, the product further has one or more characteristics selected from the following group: (1) Used to inhibit 5α-reductase; (2) SPF value >5.5; (3) Reduce melanin content: (4) Inhibiting microorganisms selected from the group consisting of Streptococcus mutans and Porphyromonas gingivalis.

[0056] In a fourth aspect of the present invention, a composition is provided, comprising: (1) the Terminalia chebula extract as described in the first aspect of the present invention; and (2) additional ingredients that can be used in daily chemical products.

[0057] In another preferred embodiment, the composition is used for a purpose selected from the group consisting of oil control, sun protection, whitening, and antibacterial.

[0058] In another preferred embodiment, the composition is a sunscreen composition.

[0059] In another preferred embodiment, the composition is an anti-dandruff composition.

[0060] In another preferred embodiment, the composition is used to inhibit microorganisms selected from the group consisting of Streptococcus mutans, Porphyromonas gingivalis, or a combination thereof.

[0061] In another preferred embodiment, the composition is a cream or an emulsion.

[0062] In another preferred embodiment, the composition is selected from the following group: facial cream, skin care water, isolation cream, lotion, facial mask, toothpaste, shampoo, mouthwash, and sunscreen.

[0063] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The HPLC spectrum of the Terminalia chebula extract described in the examples of the present invention is shown.

[0065] Figure 2 The inhibition rate of chebula extract on 5α-reductase is shown.

[0066] Figure 3 The effect of Terminalia chebula extract on the viability of mouse B16-F10 melanoma cells was shown.

[0067] Figure 4 The effect of Terminalia chebula extract on melanin secretion in mouse B16-F10 cells is shown. DETAILED DESCRIPTION

[0068] The inventors have conducted extensive and in-depth research, and through a large number of experimental screenings, unexpectedly discovered for the first time that ethanol is used as an extraction solvent to perform reflux extraction on myrobalan, the solvent is safe and non-toxic, and the type of composite macroporous resin can simultaneously enrich the two active substances of gallic acid and myrobalan tannic acid to the greatest extent, and the content of gallic acid and myrobalan tannic acid in the obtained myrobalan extract is ≥20wt%. In the present invention, mixed macroporous resin purification is used in the purification process, and the transfer rate of the obtained active substances gallic acid and myrobalan tannic acid is increased by 10.64% compared with that of a single resin, and the yield of the obtained myrobalan extract freeze-dried powder is increased by 13.99% compared with that of a single resin. In addition, the prepared myrobalan extract has the effect of inhibiting 5α-reductase, melanin secretion, Streptococcus mutans and Porphyromonas gingivalis, indicating that the myrobalan extract has the effects of oil control, sun protection, whitening and brightening, and antibacterial, and provides a possibility and basis for its application in the field of daily cosmetics. On this basis, the inventors have completed the present invention.

[0069] Terminology Terminalia chebula extract In the present invention, the active ingredients in the terminalia chebula extract include gallic acid and terminalia chebula tannic acid, and the structural formulas thereof are shown below.

[0070]

[0071] In the present invention, the terminalia chebula extract can simultaneously enrich two active substances, gallic acid and terminalia tannic acid, to the greatest extent through the type of composite macroporous resin, and the content of gallic acid and terminalia tannic acid in the terminalia chebula extract is ≥20wt%, based on the dry weight of the terminalia chebula extract.

[0072] Preparation method of terminalia chebula extract Weigh the raw material Terminalia chebula or Cibotium barometz, add 6 times (feeding amount mass KG / solvent volume L) of 65% ethanol, reflux at 70°C-85°C for 2h; extract twice; filter; concentrate under reduced pressure to recover alcohol (55°C-80°C, vacuum pressure 0.060-0.080Mpa) to obtain a crude extract; purify the crude extract with 4 times the feed amount of S-8:NKA-9=1:4 mixed macroporous resin. After the crude extract is mixed with 1 volume of water, the sample is loaded, washed with 2BV of water to obtain washing liquid A, and then analyzed with 4BV of 65% ethanol to obtain analytical solution A1; washing liquid A is purified with 4 times the feed volume of S-8: NKA-9 = 1:4 mixed macroporous resin, washed with 2BV of water to obtain washing liquid B, and then analyzed with 4BV of 65% ethanol to obtain analytical solution B1; washing liquid B is purified with 4 times the feed volume of S-8: NKA-9 = 1:4 mixed macroporous resin, washed with 2BV of water to obtain washing liquid B, and then analyzed with 4BV of 65% ethanol to obtain analytical solution C1. The analytical solutions AI, B1 and C1 can be concentrated separately, or AI, B1 and C1 can be mixed and concentrated in any proportion to obtain the extract; vacuum drying or freeze drying is performed to obtain the terminalia chebula extract powder.

[0073] Uses of Terminalia chebula extract The terminalia chebula extract of the present invention is enriched with two active compounds, gallic acid and terminalia chebula tannic acid, and has rich active effects, including but not limited to: a) Oil control: The Terminalia chebula extract can inhibit the activity of 5α-reductase with an inhibition rate of ≥35%, thereby having an oil control effect; b) Sunscreen: The SPF value of the Terminalia chebula extract is 5.67, the measured value of the UVA PFA value is 2.01, and the PA value is +, which has a certain sunscreen effect; c) Whitening: The terminalia chebula extract can inhibit the secretion of melanin by mouse melanoma cells, which is equivalent to kojic acid and has whitening effect; d) Broad-spectrum antibacterial effect: The Terminalia chebula extract has the effect of inhibiting Streptococcus mutans and Porphyromonas gingivalis, and the MIC concentration is lower than 0.5wt%, which has antibacterial effect.

[0074] Therefore, the terminalia chebula extract obtained by the method described in the present application can be used to prepare daily use or daily chemical products for oil control, sun protection, whitening and brightening, and antibacterial. Preferably, in the daily use or daily chemical products, the terminalia chebula extract is used in an amount of 0.05 to 99 wt %.

[0075] Compared with the prior art, the present invention has the following beneficial effects: 1. In the present invention, ethanol is used as the extraction solvent in the preparation process of the terminalia chebula extract, and the solvent is safe and non-toxic. The type of composite macroporous resin can simultaneously enrich the two active substances of gallic acid and terminalia tannic acid to the greatest extent, and the content of the active ingredients gallic acid and terminalia tannic acid in the terminalia chebula extract is ≥20wt%.

[0076] 2. In the present invention, the extraction method of the terminalia chebula extract is a heating reflux extraction method, which is simple, has low equipment requirements, low cost, and is suitable for industrial production.

[0077] 3. In the present invention, a mixed macroporous resin is used for purification, and the transfer rate of the active substances gallic acid and terminalia chebula tannic acid obtained is increased by 10.64% compared with that of a single resin; and the yield of the terminalia chebula extract freeze-dried powder obtained is increased by 13.99% compared with that of a single resin.

[0078] 4. In the present invention, the Terminalia chebula extract has the effect of inhibiting 5α-reductase, and the inhibition rate of 5α-reductase is ≥35%, thereby having the effect of controlling oil.

[0079] 5. In the present invention, the Terminalia chebula extract has the effect of inhibiting Streptococcus mutans and Porphyromonas gingivalis.

[0080] 6. In the present invention, the Terminalia chebula extract has the effect of inhibiting the secretion of melanin, which is equivalent to the effect of kojic acid, a commonly used whitening ingredient.

[0081] 7. In the present invention, the prepared Terminalia chebula extract has the effects of oil control, sun protection, whitening and brightening, and antibacterial, which provides the possibility and basis for its application in the field of daily cosmetics.

[0082] The present invention will be further described below in conjunction with specific examples. 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. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0083] Example 1: Testing of active ingredients in raw materials 0.5 g of chebula young fruit powder and fruit powder were accurately weighed through a 24-mesh sieve, added with 50 mL of 50% methanol solution by mass percentage and ultrasonicated for 30 min, the extract was diluted 10 times and filtered. The contents of gallic acid and chebula tannic acid (by dry weight) were tested by high performance liquid chromatography.

[0084] Table 1 Active ingredient content in raw materials

[0085] From the test data in Table 1, it can be seen that the contents of gallic acid and terminalia tannic acid in young Terminalia chebula fruits are higher than those in fruit fruits. Therefore, young Terminalia chebula fruits can be preferably used as raw materials for the extraction of active ingredients.

[0086] Example 2: Extraction solvent selection In this embodiment, the young fruits of Terminalia chebula are used as raw materials, and Terminalia chebula is extracted by different solvents, and then the contents of gallic acid and terminalia tannic acid in each extract are tested by high performance liquid chromatography. The extraction steps include:

[0087] 2g of chebula young fruit powder was accurately weighed through a 24-mesh sieve, 200mL of water, 65% ethanol solution by mass percentage concentration and 75% ethanol solution by mass percentage concentration were added and heated to reflux for 1 hour, the extract was filtered, concentrated, and the concentrate was freeze-dried to obtain a chebula extract. The contents of gallic acid and chebula tannic acid were tested by high performance liquid chromatography. The results are shown in Table 2.

[0088] Table 2 Content of active ingredients in each solvent extract

[0089] From the data in Table 2, it can be seen that the total content of gallic acid and terminalia tannic acid in the terminalia chebula extract obtained by ethanol extraction is more than 30% higher than that of the water-extracted extract, among which the terminalia chebula extract obtained by 65% ​​ethanol extraction has a higher total content, and the total extraction rate is as high as 91%.

[0090] Example 3: Purification of Terminalia chebula Extract In this embodiment, the Terminalia chebula young fruit was used as raw material, and 65% ethanol was used for extraction to obtain a Terminalia chebula extract crude extract, and then different types of macroporous resins were used for purification to obtain the Terminalia chebula extract.

[0091] 3.1 Preparation of crude extract of Terminalia chebula Solvent extraction: Take 302.56g of the raw material Terminalia chebula young fruit (uncrushed), add 6 times (feeding amount mass KG / solvent volume L) mass percentage of 65% ethanol solution, heat and reflux for 2 hours, extract twice, combine the two extracts, filter, and concentrate to obtain 458.95g of Terminalia chebula extract crude extract. The crude extract contains 8mg / ml of gallic acid and 86.38mg / ml of Terminalia chebula tannic acid.

[0092] 3.2 Purification of crude extract of Terminalia chebula by single-model macroporous resin Purification: Take the crude extract of the terminalia chebula extract prepared in 3.1, dilute it with water at a ratio of 1:1, and apply it to macroporous resins D101, HPD300, HPD100, AB-8, HP20, S-8 and NKA-9 (the amount of resin is 4 times the total amount of the crude extract), first elute with water, the water elution volume is 2.5BV, then elute with 65% ethanol solution by mass percentage, the ethanol elution volume is 4BV, collect the ethanol analytical solution, and freeze-dry to obtain the terminalia chebula extract. The content of gallic acid and terminalia chebula tannic acid was tested by high performance liquid chromatography.

[0093] Table 3 Data of purification of a single type of macroporous resin

[0094] Note: Adsorption rate = (the amount of active matter in the extract before column loading - the amount of active matter in the sample loading and water elution) / the amount of active matter in the extract before column loading * 100% Resolution rate = active matter resolved by ethanol / (active matter in the extract before column loading - active matter in loading and water elution) From the data in Table 3, it can be seen that the transfer rate and yield of gallic acid and terminalia chebula tannic acid in the terminalia chebula extract purified by using macroporous resins S-8 and NKA-9 alone are the highest, so these two macroporous resins are considered as better purification resins in the future.

[0095] 3.3 Purification of crude extract of Terminalia chebula extract by combined macroporous resin Take the crude extract of Terminalia chebula extract prepared in 3.1, dilute the crude extract with water at a ratio of 1:1, and load it on a combination of macroporous resins of types S-8:NKA-9=1:4, S-8:NKA-9=1:1 and S-8:NKA-9=4:1 (the total amount of mixed resin is 4 times the total amount of crude extract) for elution.

[0096] Elution procedure: firstly elute with water, the water elution volume is 2.5BV, collect the water washing solution 1, then elute with 65% by mass ethanol solution, the ethanol elution volume is 4BV, collect the ethanol analytical solution 1; Take water wash 1, first elute with water, the water elution volume is 2.5BV, collect water wash solution 2, then elute with 65% ethanol solution by mass percentage concentration, the ethanol elution volume is 4BV, collect ethanol analytical solution 2; Take water washing liquid 2, first elute with water, the water elution volume is 2.5BV, then elute with ethanol solution with a mass percentage concentration of 65%, the ethanol elution volume is 4BV, and collect ethanol analysis liquid 3.

[0097] The 3 analytical solutions were collected and freeze-dried to obtain the terminalia chebula extract. The contents of gallic acid and terminalia chebula tannic acid were tested by high performance liquid chromatography.

[0098] Table 4 Data of combined macroporous resin purification

[0099] It can be seen from the data in Table 4 that after the crude extract of Terminalia chebula extract was purified using a combination of two macroporous resins, S-8 and NKA-9, the transfer rates and yields of gallic acid and terminalia chebula tannic acid in the Terminalia chebula extract were significantly improved, with the transfer rates and yields reaching over 91%, and the yields increasing to over 26%. Specifically, the maximum transfer rates and yields of S-8 alone were 78.29%, and the maximum transfer rates and yields of NKA-9 alone were 83.49%. However, the combination of the two resins in equal proportions (1:1) unexpectedly increased the transfer rates and yields, reaching 91.09%>78.29% / 2+83.49% / 2=80.89%. The purification result after the combination was significantly higher than the simple superposition of the two. In terms of yield, the same conclusion can be drawn, and the yield of the combined macroporous resins is significantly higher than the superposition of the simple effects of the two. The HPLC spectrum of the Terminalia chebula extract obtained from Example 3-3-1 is shown in the figure below. Figure 1 shown.

[0100] The components, peak time and content of the Terminalia chebula extract are shown in Table 5 below: Table 5 Components, peak time and content in Terminalia chebula extract

[0101] Example 4: Evaluation experiment of oil control efficacy In this example, the Terminalia chebula extract prepared in Example 3-3-1 was used to evaluate the oil control effect by using the inhibition rate of 5α-reductase as an indicator. 4.1 Positive Control Finasteride solution was used as the positive control, and the test concentration was 0.93 mg / ml.

[0102] 4.2 Experimental procedures 4.2.1 Sample treatment: Weigh 0.2028, 0.1995, and 0.2018 g of Terminalia chebula extract samples respectively and dilute to 10 mL with PBS solution; 4.2.2 Sample concentration: 20 mg / mL.

[0103] 4.2.3 Sample process: Sample tube A: Take 1 mL of sample solution into a test tube, add 1 mL of 5α-reductase solution, 1 mL of NADPH solution, and 1 mL of testosterone solution, and mix well; B Sample background tube: Take 1 mL of sample solution into a test tube, add 1 mL of 5α-reductase solution, 1 mL of NADPH solution, and 1 mL of PBS solution, and mix well; C solvent tube: add 1 mL 5α-reductase solution, add 1 mL NADPH solution, add 1 mL testosterone solution, add 1 mL testosterone solution, and mix well; D solvent background tube: add 1 mL 5α-reductase solution, 1 mL NADPH solution, 2 mL PBS solution, and mix well; Allow each reaction solution to react at 37°C for 20 minutes, remove an appropriate amount of sample solution, and measure the absorbance at 340 nm.

[0104] 4.3 Calculation The ability of the test sample to inhibit 5α-reductase is calculated and the statistical difference P value is calculated to determine whether the sample has oil control effect.

[0105] Calculation formula:

[0106] ASample: indicates the absorbance of the sample tube; B Sample substrate: indicates the absorbance of the sample background tube; C: represents the average absorbance of the solvent tube; D: represents the absorbance of the solvent background tube.

[0107] 4.4 Results Table 6 Inhibition rate test results

[0108] The test results are characterized by the inhibition rate of the test sample on 5α-reductase, which is compared with the control with oil-control effect. If the inhibition rate of the positive control is higher than that of the blank control group and the difference is statistically significant (P<0.05), the test results are valid. If the inhibition rate of the test sample is higher than that of the blank control group and the difference is statistically significant (P<0.05), it means that the test sample has oil-control effect.

[0109] From Table 6 and Figure 2 It can be seen that the inhibition rate of 5α-reductase in the three parallel positive control groups was higher than that in the blank control group, and the difference was statistically significant (P<0.05), indicating that the results of this experiment were valid. The inhibition rate of chebula extract in this application as the experimental sample group was higher than that in the blank control group, and the difference was statistically significant (P<0.05). The average inhibition rate of the three experimental sample groups reached 39.82%, proving that the chebula extract has the effect of controlling oil.

[0110] Example 5: Sunscreen effect test of Terminalia chebula extract In this example, the Terminalia chebula extract prepared in Example 3-3-1 was used to verify the sunscreen effect.

[0111] 5.1 Test Instrument Sun Protection Factor Tester SPF-290AS 5.2 Test Environment Environmental requirements: Temperature: 25.0℃±5℃; Humidity: 50%±10%.

[0112] 5.3 Testing Process (1) Fix the PMMA plate on the sample plate.

[0113] (2) First measure the background curve of the blank plate, then use a spotter to evenly apply the sample on the PMMA plate and apply it with a finger with a latex cot at a concentration of 2 mg / cm 2 Apply the sample gently and evenly on a 25 cm 2 Area size PMMA board.

[0114] (3) Place the coated sample plate in a dark environment at a temperature of (25±5)℃ and a relative humidity of (50±10)% to dry naturally for 20 min.

[0115] (4) Turn on the power and preheat the instrument for 30 minutes. After confirming that it is normal, use the SPF-290AS analyzer to select 6 points on the PMMA plate for testing. The software that draws the MPF of 6 light wavelengths is used for calculation and analysis to obtain the SPF value and standard deviation of a test, as well as the SPF value of each scanning point.

[0116] (5) Each sample should be tested at least 3 times.

[0117] 5.4 Judgment criteria The Sun Protection Factor (SPF) is a specific value that indicates the ability of a product to protect against UVB rays. It is a protective indicator for evaluating the ability of sunscreen cosmetics to protect the skin from sunburn. The larger the SPF value, the better the effect of preventing sunburn, which means that the sunscreen product can prolong the time the skin is sunburned by UVB. Generally speaking, sunscreens with SPF values ​​between 2-6 are considered low-grade sunscreens, while products with SPF values ​​between 6-8 are considered medium-grade sunscreens.

[0118] Other sunscreen effect test standards are shown in Table 7 and Table 8.

[0119] Table 7 Sun protection standards 1

[0120] Table 8 Sun protection standards 2

[0121] PA is a measure of the ability of sunscreen products to protect against UVA (ultraviolet rays), which is the main cause of skin aging and tanning.

[0122] 5.5 Test results Table 9 Sunscreen test data

[0123] From the data in Table 9, it can be seen that the SPF value of the terminalia chebula extract of the present application is 5.67, which is a low-grade sunscreen product, the measured value of the UVA PFA value is 2.01, and the PA value is +. This indicates that the terminalia chebula extract of the present application has a certain sunscreen effect. This is an outstanding effect among the pure plant extract series of sunscreen products.

[0124] Example 6: Experiment on the effect of Terminalia chebula extract on melanin secretion by melanoma cells In this example, the Terminalia chebula extract prepared in Example 3-3-1 was used to conduct an experiment on the effect of melanin secretion in mouse B16-F10 melanoma cells. 6.1 Determination of the maximum safe concentration of Terminalia chebula extract by MTT method 1) Inoculation: When the cell density is ≥70%, discard the old culture medium, collect the cells, mix and transfer to a 2 mL centrifuge tube, centrifuge, and discard the supernatant; add 1 mL of complete culture medium again, pipette and mix, dilute appropriately and count, adjust the cell density to a certain concentration based on the counting results, inoculate the cells into a 96-well plate at a volume of 200 μL per well, and return to the incubator for incubation (37°C, 5% CO2).

[0125] 2) Experimental grouping: The experiment set up a blank control group and an experimental group, and set up 3 cell-free wells as zero wells. Six concentration gradients were set for each sample in the experimental group, and 3 replicate wells were set under each concentration gradient; 3) Solution preparation: According to the experimental design in Table 10, use basal culture medium to prepare different concentrations of the test substances.

[0126] Table 10 Concentration setting of Terminalia chebula extract

[0127] 4) Administration: After 24 hours, take out the 96-well plate, discard the old culture medium, add 200 μL of basal culture medium to each well of the zero well and blank control group, and add 200 μL of sample prepared with basal culture medium to each well of the experimental group, and plate three replicate wells per group and concentration. Then return to the incubator for culture (37°C, 5% CO2).

[0128] 5) Detection: 24 hours after drug treatment, take out the 96-well plate, add 20 μL of MTT working solution (5 mg / mL) to each well, return to the incubator and continue to culture for 4 hours, then discard the liquid in the wells, add 150 μL DMSO to each well again, shake for 10 minutes, and measure the absorbance (OD value) at a wavelength of 490 nm.

[0129] 6) Calculation of relative cell viability: Cell viability % = (OD value of the experimental group - OD value of the zero-adjustment well) / (OD value of the blank control group - OD value of the zero-adjustment group) * 100%.

[0130] 6.2 Effect of Terminalia chebula extract on melanin secretion in mouse B16-F10 melanoma cells 6.2.1 Experimental design Experimental design was performed according to Table 11 Table 11 Experimental design of the effect of melanoma cell secretion of melanin

[0131] 6.2.2 Experimental process 1) Inoculation: When the cell density is ≥70%, discard the old medium, collect the cells, mix and transfer to a 2 mL centrifuge tube, centrifuge, and discard the supernatant; add 1 mL of complete medium again, pipette and mix, dilute appropriately and count, adjust the cell density to a certain concentration based on the counting results, inoculate the cells into a 6-well plate at a volume of 2 mL per well, and return to the incubator for incubation (37°C, 5% CO2).

[0132] 2) Experimental grouping: grouping is performed as shown in Table 11, and the plates are arranged with 3 replicate wells per concentration in each group; 3) Solution preparation: According to the cell viability test results obtained in 6.1, select an appropriate concentration as the test concentration of the experimental group samples; use kojic acid (100 μg / mL) as the positive control for the experiment.

[0133] 4) Administration: After 24 hours, the 6-well plate was removed, the old culture medium was discarded, the cells were washed with PBS, and then 3 replicate wells were added to each concentration in each group. The negative control group (NC) was divided into 3 replicate wells, and 2 mL of basal culture medium was added to each well. The positive control group (PC) was divided into 2 mL of kojic acid (100 μg / mL) was added to each well. The experimental group was divided into 3 replicate wells, and 2 mL of the corresponding concentration sample was added to each well. Then the cells were returned to the incubator for co-culture (37°C, 5% CO2).

[0134] 5) Melanocyte collection: After 24 hours of drug treatment, remove the 6-well plate, discard the supernatant, collect the cells into a 2 ml EP tube, centrifuge, discard the supernatant, add 200 μL of 1 mol / L sodium hydroxide solution to each tube, place in an 80°C water bath for 1 hour, vortex to mix, take 150 μL from each well into a 96-well plate, use 1 mol / L sodium hydroxide solution as a blank, and measure the absorbance (OD) at a wavelength of 405 nm.

[0135] 6) Result settlement: Melanin content = (OD value of experimental group - OD value of blank well) / (OD value of negative control group - OD value of blank well) * 100%.

[0136] 6.3. Analysis of experimental results 6.3.1 Determination of the maximum safe concentration of Terminalia chebula extract by MTT method Table 12 Test data of maximum safe concentration of Terminalia chebula extract

[0137] As shown in Table 12 and Figure 3 As shown, when the concentration of Terminalia chebula extract is ≤125.0 μg / ml, the cell viability is ≥90%, so the appropriate concentration can be selected within the concentration range of ≤125.0 μg / ml for preparing the composition.

[0138] 6.3.2 Effect of Terminalia chebula extract on melanin secretion in mouse B16-F10 melanoma cells Test results such as Figure 4 As shown: Compared with the negative control group (NC), the melanin secretion content of cells treated with the positive control group (kojic acid) was significantly decreased (*P < 0.05), indicating that the experimental conditions were effective; compared with the negative control group (NC), the melanin secretion content of cells treated with 100 μg / mL of Terminalia chebula extract was significantly decreased (*P < 0.05), and the difference was statistically significant.

[0139] The reduction in the melanin content secreted by cells treated with 100 μg / mL of Terminalia chebula extract was comparable to that of the positive control (100 μg / mL of kojic acid), demonstrating that the Terminalia chebula extract described in the present application has the effect of inhibiting melanin secretion.

[0140] Example 7: Experimental study on the effect of Terminalia chebula extract on Streptococcus mutans and Porphyromonas gingivalis In this example, the Terminalia chebula extract prepared in Example 3-3-1 was used to conduct an experiment on the effect of Streptococcus mutans and Porphyromonas gingivalis. 7.1 Test equipment (1) Strain Streptococcus mutans BNCC 336931 and Porphyromonas gingivalis ATCC 33277 (2) Prepare hydrolyzed casein agar medium (MH): weigh 38 g MH agar medium and dissolve it in 1000 ml water. Heat until boiling and dissolve. Then sterilize it by high pressure at 121°C for 15 min and place it in a water bath at 45°C to 50°C for later use. This medium will be used for the control test.

[0141] (3) 0.03 mol / L phosphate buffer, pH 7.2.

[0142] (4) Pipette (1μl~10μl).

[0143] (5) Water bath thermostat at 45℃~50℃.

[0144] (6) Pipette, test tube, and petri dish.

[0145] (7) 37°C incubator.

[0146] 7.2 Operation steps (1) Preparation of mutans Streptococcus and Porphyromonas gingivalis solution: Aseptically take 5 ml or 5 g (after solid grinding) of sample, respectively, and place it in 45 ml of sterile phosphate buffer. Shake thoroughly to dissolve and prepare a 10% uniformly dispersed solution or suspension.

[0147] (2) Preparation of culture medium containing antimicrobial agents: dilute the 10% antibacterial solution or suspension with PBS in multiples to produce test solutions of different concentrations, and place in a water bath at a constant temperature of 45°C to 50°C for later use.

[0148] (3) Preparation of double-concentration culture medium: Weigh 76g MH agar medium and dissolve it in 1000ml water. Heat until boiling and dissolved. Then sterilize it with 121℃ pressure steam for 15min and place it in a 45℃~50℃ water bath for later use. This culture medium will be used to dilute the antibacterial solution or suspension.

[0149] (4) Preparation of culture medium containing antibacterial solution: Take 10 ml of serially diluted antibacterial solution and add it to the plate. Add 10 ml of double MH agar medium in a 45℃~50℃ water bath to the plate, shake the plate while adding to mix the antibacterial solution and culture medium thoroughly, and wait for solidification before use.

[0150] (5) Use a pipette to take 1 μl to 2 μl of the bacterial suspension (containing approximately 107 cfu / ml) and inoculate it on a plate containing antibacterial (inhibitory) culture medium. The diameter of the bacterial liquid circle formed after inoculation is about 5 mm to 8 mm (the bacterial quantity at each point is approximately 104 cfu).

[0151] (6) Inoculate an MH agar plate without antibacterial (inhibitory) ingredients in the same way as a positive control.

[0152] (7) Place the inoculated plate in a 35°C incubator, invert and incubate for 18 to 24 hours, and observe the results.

[0153] 7.3 Judging Rules The minimum inhibitory concentration at which colony growth is completely inhibited is the MIC of the sample against the test bacteria. Single colony growth can be ignored.

[0154] 7.4 Experimental Results Table 13 Experimental data on the effects of Terminalia chebula extract on Streptococcus mutans and Porphyromonas gingivalis

[0155] It can be seen from the data in Table 13 that the Terminalia chebula extract of the present application has an inhibitory effect on Streptococcus mutans and Porphyromonas gingivalis, and the minimum inhibitory concentration (MIC) is 0.312wt% and 0.078wt%, respectively.

[0156] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of 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 claims attached to this application.

Claims

1. A Terminalia chebula extract, characterized in that The terminalia chebula extract contains gallic acid and terminalia tannic acid, wherein the total content of the gallic acid and terminalia tannic acid is greater than 20 wt %, based on the dry weight of the terminalia chebula extract.

2. The Terminalia chebula extract according to claim 1, characterized in that The extract further comprises terminalia chebula tannic acid and / or ellagic acid, wherein the terminalia chebula tannic acid is present in an amount of 10-20 wt %, and / or the ellagic acid is present in an amount of 1-10 wt %.

3. The method for preparing the Terminalia chebula extract according to claim 1, characterized in that: The method comprises the steps of: (1) Extraction: Extract the terminalia chebula fruit with ethanol, collect the filtrate and concentrate it to obtain a crude terminalia chebula extract; (2) Purification: taking the crude extract obtained in step (1), eluting it with a combination of S-8 and NKA-9 macroporous resins, wherein the elution comprises: first eluting with water, collecting the water washing liquid, then eluting with ethanol, and collecting the ethanol analytical liquid; (3) taking the water washing solution obtained in step (2), using ethanol as the eluent, and continuing to elute with the combined macroporous resin of S-8 and NKA-9 to obtain an ethanol analytical solution; (4) The ethanol analytical solutions obtained in steps (2) and (3) are combined and freeze-dried to obtain the Terminalia chebula extract.

4. The method for preparing the Terminalia chebula extract according to claim 1, characterized in that: The method comprises the steps of: (1) Extraction: providing a raw material of Terminalia chebula fruit, adding ethanol at a concentration of 60 v / v% to 70 v / v% for reflux extraction, collecting the filtrate, and concentrating it to obtain a crude extract of Terminalia chebula extract; (2) Purification: The crude extract obtained in step (1) is loaded onto a combined macroporous resin of S-8 and NKA-9, and the elution procedure is performed: first eluting with water, collecting the water washing liquid, then eluting with ethanol at a concentration of 60 v / v%-70 v / v%, and collecting the ethanol analytical liquid; (3) taking the water washing solution obtained in step (2), repeating the elution procedure in step (2) 2-4 times, and combining the ethanol analytical solution obtained each time; (4) The ethanol solution obtained in step (3) is freeze-dried to obtain the Terminalia chebula extract.

5. The method according to claim 3, characterized in that The elution procedure in step (2) is repeated 3 times, and the elution procedure includes the steps of: (2-1) First, elute with ethanol to collect the water washing solution 1, then elute with ethanol at a concentration of 60 v / v %-70 v / v %, and collect the ethanol analytical solution 1; (2-2) taking the water washing solution 1 obtained in step (2-1), firstly eluting with ethanol, collecting the water washing solution 2, and then eluting with ethanol at a concentration of 60 v / v%-70 v / v%, collecting the ethanol analytical solution 2; (2-3) Take the water washing solution 2 obtained in step (2-1), first elute with 5% ethanol, then elute with 60 v / v %-70 v / v % ethanol, collect the ethanol analysis solution 3, and combine the ethanol analysis solutions obtained each time.

6. The method according to claim 3, characterized in that The terminalia chebula fruit described in step (1) is a terminalia chebula young fruit or a terminalia chebula fruit.

7. The method according to claim 3, characterized in that In step (2), the amount of the combined macroporous resin is S-8:NKA-9=10:1~10:1 (w:w).

8. The use of the Terminalia chebula extract according to claim 1, characterized in that: Used to prepare daily products or daily chemical products with oil control, sun protection, whitening or antibacterial effects.

9. The use according to claim 8, characterized in that The product also has one or more characteristics selected from the following group: (1) Used to inhibit 5α-reductase; (2) Sun protection factor SPF value ≥ 5.5; (3) Reduce melanin content: (4) Inhibiting microorganisms selected from the group consisting of Streptococcus mutans and Porphyromonas gingivalis.

10. A composition, characterized in that The composition comprises: (1) the Terminalia chebula extract according to claim 1; and (2) additional ingredients that can be used in daily chemical products.

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