Inula aquilaria extract as well as preparation method and application thereof

Abstract

The invention discloses an aqueous inula flower extract as well as a preparation method and application thereof, the extract is obtained by carrying out ethanol reflux extraction and resin adsorption elution on aqueous inula flower, and the extract mainly comprises six sesquiterpenoids, namely 8-epihelenalin, aromaticin, ergolide, carabrone, 2, 3-dihydroxy-aromaticin and tomentosin, and is prepared from the following raw materials in parts by weight: 1, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, 2, 3-dihydroxy-aromaticin, the inula aquilaria extract disclosed by the invention has a remarkable inhibition effect on hyaluronidase activity and mast cell degranulation activity, the highest inhibition rate of hyaluronidase can reach 93.7%, the lowest release rate of beta-Hex during mast cell degranulation can reach 11.22%, and the inula aquilaria extract has an excellent soothing effect and has a wide application prospect in preparation of cosmetics with a soothing function.

Description

Technical Field

[0001] The invention relates to an Inula fusca flower extract and also relates to a preparation method and application of the Inula fusca flower extract. Background Art

[0002] Because sensitive skin has characteristics of damaged barrier function or abnormal nerve function, it is more difficult to resist the invasion of external irritants and allergens than normal skin, and is prone to a series of skin inflammatory reactions caused by irritation or allergies, further damaging the skin barrier structure and nerve endings, leading to a vicious cycle. Green and functional cosmetics are becoming more and more popular. With the increasing application of plant extracts in cosmetics, more and more varieties are being used.

[0003] At present, the common plant extract raw materials on the market mainly include aloe extract, green tea extract, ginseng extract, etc. These raw materials play an important role in personal care products and skin care products due to their unique efficacy and uses. They can be used as whitening agents, moisturizers and soothing agents. Many Chinese herbal extracts, such as Centella asiatica extract and Licorice extract, are also widely used in the cosmetics field due to their excellent moisturizing, repairing and anti-aging effects. However, some plant extracts have potential quality and safety issues caused by allergenicity, instability, pesticide residues or pollution. For example, curcumin compounds with good antioxidant effects will degrade when exposed to light, heat and oxygen conditions; in addition, the complex extraction process, high cost and unclear active ingredients also limit the further promotion and application of some products.

[0004] Inulaehelianthus-aquatica C.Y.WuexLing., a plant of the genus Inulaehelianthus in the Asteraceae family, is included in the Sichuan Provincial Standard for Traditional Chinese Medicines and is mainly produced in Sichuan, Gansu, Yunnan and Guizhou. Its inflorescence has the effects of descending qi, eliminating phlegm, promoting qi circulation and stopping vomiting, and is used for wind-cold cough, phlegm accumulation, chest and diaphragm fullness, wheezing and coughing with excessive phlegm, vomiting and belching, and hardness under the heart. In the prior art, the sesquiterpene components of Inulaehelianthus-aquatica C.Y.WuexLing. are often used in the research of anti-tumor activity and anti-inflammatory aspects, and new applications thereof can be further developed. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a water angelica flower extract with soothing effect, and also to provide a preparation method and application of the water angelica flower extract.

[0006] Technical solution: The present invention discloses an extract of Inula japonica L., which is obtained by subjecting Inula japonica L. to ethanol reflux extraction and resin adsorption elution, and mainly comprises 6 sesquiterpenoid compounds: 8-epihelenalin, aromaticin, ergolide, carabrone, 2,3-dihydro-aromaticin and tomentosin, and its structural formula is as follows:

[0007]

[0008] The total content of the sesquiterpenes is 22% to 65% of the extract of Inula japonica var. aquatica; the contents of the sesquiterpenes are respectively: 8-epihelenalin 2% to 10%, aromaticin 1% to 6%, ergolide 5% to 12%, carabrone 8% to 15%, 2,3-dihydro-aromaticin 3% to 12%, and tomentosin 3% to 10%.

[0009] Wherein, the weight ratio of carabrone to tomentosin is 2:1 to 1:2.

[0010] Wherein, the weight ratio of aromaticin to carabrone is 1:5 to 1:2.

[0011] The preparation method of the Inula japonica extract comprises the following steps:

[0012] (1) taking water and adding 5 to 20 times the volume of ethanol to the flower of Inula japonica, performing reflux extraction, and recovering the ethanol to obtain an extract;

[0013] (2) The extract is suspended in 2 to 3 times the volume of ethanol aqueous solution, adsorbed by a macroporous resin, and the solvent is recovered to obtain the Inula japonica flos extract.

[0014] Wherein, in step (1), the ethanol concentration is 80% to 100%, and the reflux extraction is performed 2 to 3 times, each time for 1 to 3 hours.

[0015] Wherein, in step (2), the reflux extraction is performed by eluting with 30% ethanol until it becomes colorless, and then collecting the 60% to 80% ethanol water elution fraction.

[0016] Wherein, in step (2), the model of the macroporous resin is D101 or XAD-16, etc.

[0017] The Inula japonica flos extract can also be used in the preparation of cosmetics with soothing effects.

[0018] Among them, the application is specifically that the extract of Inula japonica var. aquatica achieves a soothing effect by inhibiting hyaluronidase activity and mast cell degranulation.

[0019] The cosmetic is an essence containing an extract of Inula japonica var. argyi, and the content is as follows: 1% to 3% of an extract of Inula japonica var. argyi, 1% to 5% of 1,2-hexanol, 5% to 10% of glycerol, 5% to 10% of butylene glycol, 5% to 15% of 1,3-propylene glycol, 0.1% to 0.5% of p-acetophenone, 0.1% to 0.5% of carbomer, and 0.3% to 0.8% of arginine.

[0020] Principle of the invention: The Inula japonica extract of the present invention can inhibit the activity of hyaluronidase and reduce the production of small molecule hyaluronic acid, thereby achieving water retention and soothing effects; and has a significant inhibitory effect on mast cell degranulation, significantly reducing the release of β-Hexosaminidase (β-Hex); the Inula japonica extract is further used in cosmetics, and can effectively achieve soothing effects by inhibiting the activity of hyaluronidase and mast cell degranulation.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the water Chaoyang Inula flower extract of the present invention has a significant inhibitory effect on hyaluronidase activity and mast cell degranulation activity, the hyaluronidase inhibition rate can reach as high as 93.7%, and the β-Hex release rate during mast cell degranulation can reach as low as 11.22%; it has excellent soothing effect and has great application prospects in cosmetic additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the high performance liquid chromatogram of the Inula japonica extract;

[0023] Figure 2 This is the HPLC chromatogram comparing the extract of Inula japonica L. and six sesquiterpenoid reference substances;

[0024] Figure 3 This is the result diagram of the anti-hyaluronidase activity of the Inula japonica flower extract;

[0025] Figure 4 The extract of Inula japonica var. chebula affects the results of toluidine blue staining of mast cell degranulation;

[0026] Figure 5 Effect of Inula japonica L. extract on β-Hex release from mast cells. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0028] Example 1

[0029] The Inula japonica flower extract of the present invention is prepared by:

[0030] (1) Take 0.5 kg of dried flowers of Inula japonica chinensis, add 5 L of 95% ethanol, reflux extract twice, each time for 2 hours, filter the extract with gauze, combine the extracts, and concentrate under reduced pressure to obtain 48 g of extract;

[0031] (2) After being suspended in 96 mL of 30% ethanol, the mixture was adsorbed on a macroporous resin D101 and eluted with 30% ethanol until colorless. The elution fractions with 60% to 80% ethanol were collected and the solvent was recovered to obtain 15.8 g of Inula japonica chinensis extract, which was recorded as Inula japonica chinensis extract A.

[0032] Example 2

[0033] The Inula japonica flower extract of the present invention is prepared by:

[0034] (1) Take 0.5 kg of dried flowers of Inula japonica chinensis, add 3 L of 85% ethanol, reflux extract twice, each time for 1.5 hours, filter the extract with gauze, combine the extracts, and concentrate under reduced pressure to obtain 42 g of extract;

[0035] (2) After being suspended in 84 mL of 30% ethanol, the mixture was adsorbed on a macroporous resin D101 and eluted with 30% ethanol until colorless. The elution fractions with 60% to 75% ethanol were collected and the solvent was recovered to obtain 11.3 g of Inula japonica chinensis extract, which was recorded as Inula japonica chinensis extract B.

[0036] Example 3

[0037] The Inula japonica flower extract of the present invention is prepared by:

[0038] (1) Take 0.5 kg of dried flowers of Inula japonica chinensis, add 4 L of 90% ethanol, and reflux extract 3 times, each time for 1 hour. Filter the extract with gauze, combine the extracts, and concentrate under reduced pressure to obtain 55 g of extract;

[0039] (2) After being suspended in 110 mL of 30% ethanol, the mixture was adsorbed on a macroporous resin XAD-16 and eluted with 30% ethanol until colorless. The elution fractions with 60% to 80% ethanol were collected and the solvent was recovered to obtain 14.0 g of Inula japonica chinensis extract, which was recorded as Inula japonica chinensis extract C.

[0040] Example 4

[0041] A water inula flower extract essence:

[0042] The essence is prepared as follows: 1% water Inula japonica extract (obtained in Example 1), 1% 1,2-hexanol, 5% glycerol, 5% butylene glycol, 6% 1,3-propylene glycol, 0.3% p-acetophenone, 0.3% carbomer, 0.5% arginine, and the balance is water.

[0043] The components of the Inula japonica extract of the present invention are analyzed:

[0044] The extract A of Inula japonica obtained in Example 1 was taken and analyzed by high performance liquid chromatography; the chromatographic method was: Agilent Zorbax SB-C18 chromatographic column (250×4.6 mm, 5 μm); water was used as mobile phase A, acetonitrile was used as mobile phase B, and the column temperature was 30°C; the flow rate was 0.8 mL / min; the detection wavelength was 210 nm; the injection volume was 5 μL; the gradient conditions were shown in Table 1.

[0045] Table 1. HPLC gradient conditions

[0046]

[0047] like Figure 1 Shown is the high performance liquid chromatogram of the Inula japonica extract.

[0048] Determination of the content of sesquiterpenoid components in the Inula japonica extract of the present invention:

[0049] Through the previous study on the chemical composition of the extract of Inula japonica Thunb., combined with high performance liquid chromatography (the same method as above), six main sesquiterpenoid components in the extract of Inula japonica Thunb. were identified, such as Figure 2 As shown, they are 8-epihelenalin (peak 1), aromaticin (peak 2), ergolide (peak 3), carabrone (peak 4), 2,3-dihydro-aromaticin (peak 5) and tomentosin (peak 6).

[0050] Content determination showed that the contents of the six sesquiterpenes in the Inula japonica extract obtained in Example 1 were: 3.2% 8-epihelenalin, 2.5% aromaticin, 8.7% ergolide, 10.2% carabrone, 6.6% 2,3-dihydro-aromaticin and 4.5% tomentosin.

[0051] Research on the inhibitory effect of the Inula japonica extract of the present invention on hyaluronidase activity:

[0052] Hyaluronic acid is the most abundant component in the extracellular matrix. It regulates the secretion of cytokines and affects cell adhesion, growth, proliferation and differentiation. Therefore, it plays an important role in maintaining skin moisture and elasticity, wound healing and angiogenesis. Hyaluronidase is a specific cleavage enzyme of hyaluronic acid. It can reduce the activity of hyaluronic acid in the body. It has a strong correlation with the release of histamine by mast cells and is a participant in allergic reactions. Inhibiting the activity of hyaluronidase can ensure the normal content and function of hyaluronic acid. Therefore, examining the inhibitory activity of hyaluronidase can be used to evaluate the soothing effect of active substances.

[0053] The Elson-Morgan method was used to determine the inhibitory effect of the extract A of Inula japonica on hyaluronidase activity. 20 μL of the sample of each concentration (acetic acid buffer solution was used as the blank) and 100 μL of 900 U / mL hyaluronidase solution were added to the EP tube in sequence, mixed evenly and incubated at 37°C for 0.5 h, 180 μL of 2.5 mg / mL hyaluronic acid solution was added and mixed evenly and incubated at 37°C for 0.5 h, 50 μL of 0.8 M disodium tetraborate aqueous solution was added, and the mixture was incubated at 95°C for 5 min. After cooling to room temperature, 400 μL of 100 mg / mL DMBA solution was added, mixed evenly and incubated at 37°C for 5 min, and the absorbance was measured at 590 nm. The inhibition rate was calculated according to formula (1).

[0054] Hyaluronidase inhibition rate = [(AB)-(CD)] / (AB)×100%

[0055] In the formula: A is the OD value of the control solution (acetic acid buffer solution is used instead of the sample); B is the OD value of the control blank solution (acetic acid buffer solution is used instead of the sample and enzyme solution); C is the OD value of the water Chaoyang extract solution with different concentrations; D is the OD value of the sample blank (acetic acid buffer solution is used instead of the enzyme solution).

[0056] Table 2. Inhibitory effects of different concentrations of Inula japonica extract on hyaluronidase (n=3)

[0057]

[0058] The results showed that hyaluronidase can reduce the activity of hyaluronic acid and hydrolyze hyaluronic acid, thereby reducing its water retention effect, but the extract of Inula japonica var. truncatula can inhibit its activity, thereby achieving the effect of water retention. As shown in Table 2, with the increase of the mass concentration of the extract of Inula japonica var. truncatula, its inhibition rate has been significantly enhanced, such as Figure 4 As shown, IC 50 It is 1.19 mg / mL, so the Inula japonica flos extract of the present invention has a very strong hyaluronidase inhibitory effect.

[0059] Research on the inhibitory effect of the Inula japonica extract of the present invention on mast cell degranulation:

[0060] Mast cells degranulate after being stimulated by immune and non-immune stimuli. Immune stimulants mainly include IgE, which degranulates mast cells by binding to the high-affinity receptor FcεRI. Anti-DNP-IgE / DNP-HSA is often used to activate the IgE signaling pathway to induce mast cell degranulation. The toluidine blue staining method was used to observe the effect of Inula japonica extract A on the morphological changes of mast cells during degranulation.

[0061] like Figure 5 As shown, the mast cells in the control group were normal spindle-shaped, bluish-purple and darker in color, while the mast cells in the model group were irregular in shape and lighter in color after degranulation. The irregular mast cells in the Inula japonica extract group decreased and became darker in color. The mast cells in the high-dose Inula japonica extract group (5 mg / mL) were similar in morphology to those in the control group.

[0062] β-Hexosaminidase (β-Hex) is a presynthesized substance released after mast cell degranulation. Only when the body is allergic will the content of β-Hex increase significantly, so β-Hex can be used as a marker for mast cell degranulation. Figure 5 As shown, compared with the control group, the β-Hex release rate of mast cells in the model group increased to 22.66% (P<0.0001), and after the low dose (1 mg / mL) and high dose (5 mg / mL) of the water Chaoyang Inula flower extract, the β-Hex release rate of mast cells was 13.03% and 11.22%, which was significantly lower than that of the model group (P<0.0001). The results show that the water Chaoyang Inula flower extract can inhibit the anti-DNP-IgE / DNP-HSA-induced mast cell degranulation and significantly reduce the release of β-Hex; therefore, the water Chaoyang Inula flower extract of the present invention achieves a soothing effect on the skin by inhibiting the hyaluronidase activity and mast cell degranulation activity, and has a broad application prospect in the preparation of soothing functional cosmetics.

Claims

1. A water inula flower extract, characterized in that The extract is obtained by ethanol reflux extraction and resin adsorption elution of Inula japonica L., and mainly contains 6 sesquiterpenoid compounds: 8-epihelenalin, aromaticin, ergolide, carabrone, 2,3-dihydro-aromaticin and tomentosin, and its structural formula is as follows:

2. The Inula japonica extract according to claim 1, characterized in that The total content of the sesquiterpenes is 22% to 65% of the extract of Inula japonica L.; the contents of the sesquiterpenes are respectively: 8-epihelenalin 2% to 10%, aromaticin 1% to 6%, ergolide 5% to 12%, carabrone 8% to 15%, 2,3-dihydro-aromaticin 3% to 12%, and tomentosin 3% to 10%.

3. The Inula japonica extract according to claim 1, characterized in that The weight ratio of carabrone to tomentosin is 2:1 to 1:

2.

4. The Inula japonica extract according to claim 1, characterized in that The weight ratio of the aromaticin to carabrone is 1:5 to 1:

2.

5. A method for preparing the Inula japonica extract according to claim 1, characterized in that: The following steps are involved: (1) taking water and adding 5 to 20 times the volume of ethanol to the flower of Inula japonica, performing reflux extraction, and recovering the ethanol to obtain an extract; (2) The extract is suspended in 2 to 3 times the volume of ethanol aqueous solution, adsorbed by a macroporous resin, eluted with ethanol aqueous solution, and the solvent is recovered to obtain the Inula japonica flos extract.

6. The preparation method according to claim 5, characterized in that: In step (1), the ethanol concentration is 80% to 100%, and the reflux extraction is performed 2 to 3 times, each time for 1 to 3 hours.

7. The preparation method according to claim 5, characterized in that: In step (2), after the macroporous resin is adsorbed, 30% ethanol is used to elute until it becomes colorless, and then 60% to 80% ethanol water elution fractions are collected; the model of the macroporous resin is D101 or XAD-16, etc.

8. Use of the Inula chinensis flower extract according to claim 1 in preparing cosmetics with soothing effects.

9. The use according to claim 8, characterized in that: The application is specifically that the extract of Inula japonica var. aquatica achieves a soothing effect by inhibiting hyaluronidase activity and mast cell degranulation.

10. The use according to claim 8, characterized in that: The cosmetic is an essence containing water ginseng extract, and the content is as follows: 1% to 3% water ginseng extract, 1% to 5% 1,2-hexanol, 5% to 10% glycerol, 5% to 10% butylene glycol, 5% to 15% 1,3-propylene glycol, 0.1% to 0.5% p-acetophenone, 0.1% to 0.5% carbomer, 0.3% to 0.8% arginine, and the balance is water.

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