阅读说明：本技术 兴安虫实提取物在制备美白类化妆品中的应用 (Application of Xingan worm extract in preparation of whitening cosmetics ) 是由 苏红 刘志宝 文程 张晓丹 何颖 余琦 于 2019-12-13 设计创作，主要内容包括：本发明涉及兴安虫实提取物的应用方法,即利用兴安虫实提取物来制备美白类化妆品。所述兴安虫实提取物中金盏菊苷E的质量百分比含量≥50％。兴安虫实提取物对细胞酪氨酸酶活性以及黑色素合成均有明显的抑制作用,可以用作化妆品中的美白添加剂。(The invention relates to an application method of a Xingan worm seed extract, namely, the Xingan worm seed extract is used for preparing whitening cosmetics. The content of calendula glycoside E in the Xingan insect seed extract is more than or equal to 50 percent by mass. The Xingan worm extract has obvious inhibition effect on cell tyrosinase activity and melanin synthesis, and can be used as a whitening additive in cosmetics.)

1. Application of Xingan Chongshi extract in preparing whitening cosmetics.

2. The application of the Xingan real extract in preparing whitening cosmetics according to claim 1, wherein the Xingan real extract contains calendoside E with the mass percent of more than or equal to 50 percent and is prepared by the following method:

taking dry whole plant of Xingan Chongshi, adding 85% ethanol solution in a ratio of 10mL ethanol solution per gram of crude drug, extracting under reflux for 2 times, each time for 1.5 hr, filtering, collecting medicinal liquid, and recovering ethanol under reduced pressure to obtain ethanol extract.

Taking an ethanol extracting solution, adding water saturated n-butanol according to the volume ratio of 1:6, extracting for 8 times, combining n-butanol layers, recovering a solvent until the solvent is dried, adding a solid onto a silica gel chromatographic column, eluting by using a methanol-chloroform mixed solvent, wherein the volume ratio of methanol to chloroform in the mixed solvent is 2:1, collecting eluates according to column volume parts, combining eluates of 5-8 column volumes, and recovering the solvent until the eluates are dried under reduced pressure to obtain the compound.

3. The application of the dahurian angelica root extract in preparing whitening cosmetics according to claim 2, wherein the content of calendoside E in the dahurian angelica root extract is more than or equal to 90% by mass, and the preparation method further comprises the following steps of preparing liquid phase separation:

chromatographic conditions are as follows: a mu Bondaparik C18 column with the specification of 10 mu m and the size of 19mm multiplied by 300mm is taken as a chromatographic column; methanol-1% glacial acetic acid with the volume ratio of 85:15 is taken as a mobile phase, the flow rate is 15mL/min, and the column temperature is 30 ℃;

dissolving the solid obtained by silica gel column chromatography with mobile phase, filtering, injecting into preparative liquid chromatograph, collecting the fraction with peak time of 11-14min, mixing, recovering solvent under reduced pressure, and drying.

Technical Field

The invention relates to application of a Xingan worm seed extract in cosmetics, in particular to application in preparing whitening cosmetics.

Background

Xingan insect (Coripermum Chinganicum Iljin) is a plant of genus Chenopodiaceae insect. The plant height is 10-50 cm, the stem is upright, the plant is cylindrical, the diameter is about 2.5 mm, and the plant is green or purple red; the base branches are longer, the lower branches are higher, the upper branches are shorter, and the upper branches are inclined. Leaf strips, 2-5 cm long and 2 mm wide, with tip gradually tapering to a small tip and base gradually narrowing, 1 pulse. Spike-like inflorescences terminal and lateral, thin cylindrical, slightly compact, from (1.5) to 4-5 cm long, 3-8 mm in diameter, usually about 5 mm; the bracts vary from needles (basal to few inflorescences) to ovoid and ovoid, with tapering or sharp tips, (1) -3 veins, with wider membranous margins. The quilt sheet 3 and the quilt sheet 1 are wide and oval, the top ends of the quilt sheet and the quilt sheet are provided with irregular thin teeth, and the far axis of the quilt sheet are small, nearly triangular and rare; stamen 5, slightly over the comforter piece. The fruit is rectangular round, inverted oval or wide ellipse, 2-4 mm long, 1.5-2 mm wide, round at the top, heart-shaped at the base, slightly flattened at the center of the back surface bulge, flat ventral surface and hairless; the stones are oval, yellowish green or beige, bright and sometimes have a few dark brown spots; the beak tip is 1/3-1/4 with long beak and is thick and short; fruit wings were clear, light yellow, opaque, full-fleshed. The flower and fruit period is 6-8 months. Produce Heilongjiang, Jilin, Liaoning, Hebei, inner Mongolia, Ningxia and Gansu. Growing in lake sand dunes, semi-fixed sand dunes or grasslands.

According to records of medicinal plants in desert areas, the whole grass of Xingan Cordyceps can be used as a Chinese medicament, and has light, slightly bitter and cool taste; has effects of clearing away damp-heat and promoting urination; it can be used for treating dysuria, heat, pain, and jaundice. However, the insects are not generally used clinically as a local medicine, and no literature report about the phytochemical component or the modern pharmacological activity exists in the prior art.

Xingan insect is widely distributed in Heilongjiang and inner Mongolia areas, and has abundant resources. In animal husbandry, however, the worms are often discarded as weeds, which results in waste of resources. Therefore, the development and utilization of the Xingan insect is an urgent technical problem to be solved.

Calendula glycoside E (Calendiloside E), also known as Desgluco-chikusetsusaponin, etc., CAS number 26020-14-4, molecular formula C₃₆H₅₆O₉The structural formula is as follows:

modern researches show that the calendula glycoside E has various pharmacological activities and has wide clinical application prospects.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application method of a Xingan worm seed extract, namely, the Xingan worm seed extract is used for preparing whitening cosmetics.

In order to solve the technical problems, the invention adopts the following technical scheme:

application of Xingan Chongshi extract in preparing whitening cosmetics.

Preferably, the content of the calendula glycoside E in the Xingan insect seed extract is more than or equal to 50 percent by mass, and the method is as follows:

taking dry whole plant of Xingan Chongshi, adding 85% ethanol solution in a ratio of 10mL ethanol solution per gram of crude drug, extracting under reflux for 2 times, each time for 1.5 hr, filtering, collecting medicinal liquid, and recovering ethanol under reduced pressure to obtain ethanol extract.

Taking an ethanol extracting solution, adding water saturated n-butanol according to the volume ratio of 1:6, extracting for 8 times, combining n-butanol layers, recovering a solvent until the solvent is dried, adding a solid onto a silica gel chromatographic column, eluting by using a methanol-chloroform mixed solvent, wherein the volume ratio of methanol to chloroform in the mixed solvent is 2:1, collecting eluates according to column volume parts, combining eluates of 5-8 column volumes, and recovering the solvent until the eluates are dried under reduced pressure to obtain the compound.

Further preferably, the content of the calendula glycoside E in the Xingan insect seed extract is more than or equal to 90 percent by mass, and the preparation method further comprises the following steps of preparing liquid phase separation:

chromatographic conditions are as follows: a mu Bondaparik C18 column with the specification of 10 mu m and the size of 19mm multiplied by 300mm is taken as a chromatographic column; methanol-1% glacial acetic acid with the volume ratio of 85:15 is taken as a mobile phase, the flow rate is 15mL/min, and the column temperature is 30 ℃;

dissolving the solid obtained by silica gel column chromatography with mobile phase, filtering, injecting into preparative liquid chromatograph, collecting the fraction with peak time of 11-14min, mixing, recovering solvent under reduced pressure, and drying.

The whitening effect of the Xingan Chongshi extract can be proved by the following tests.

Test examples the effect of the extracts of the fruits of the Sinomenia xingan on the tyrosinase activity and melanin synthesis in the melanocytes of mouse B16

1. Material

Mouse B16 melanoma cell line; DMEM medium; (ii) trypsin; L-DOPA.

Arbutin (98%), purchased externally.

Xingan Chongshi extract (XT50) was prepared as described in example 1.

Xingan Chongshi extract (XT90) was prepared as described in example 2.

Microplate reader, BIO-RAD USA.

2. Method of producing a composite material

2.1 preparation of test drugs

XT50 was taken and diluted to 0.02, 0.2, 2mg/mL with deionized water, respectively, and labeled as XT50 low, medium, and high dose groups.

XT90 was taken and diluted to 0.01, 0.1, 1mg/mL with deionized water, respectively, and labeled as XT90 low, medium, and high dose groups.

Taking arbutin, diluting to 2mg/mL with deionized water, and marking as arbutin group.

2.2 mouse B16 melanocyte culture

Under aseptic conditions, 1 × 10⁵Mouse B16 melanocytes per mL were inoculated in a DMEM medium flask containing 10% (volume parts) calf serum at 37 deg.C with 5% CO₂The cells were cultured in an incubator and were digested with 0.25% trypsin every 3 days for subculture.

2.3 determination of intracellular tyrosinase Activity

After the cells had grown to log phase, they were digested with 0.25% pancreatin and adjusted to 2X 10 cells⁴one/mL, seeded in 96-well plates at 100. mu.L per well. Culturing in an incubator for 24h, removing supernatant, and adding the drug to be tested. 8 holes are set for each concentration, and the control group is added with no drug and is replaced with the same amount of maintenance liquid. Incubate for 3 days. After 3 days of drug action, the supernatant was discarded and washed twice with PBS pH6.8, and 90. mu.L of PBS containing 1% Triton X-100 was added to each well. Sonicate in ice bath, add 10. mu.L of 10mM L-DOPA to each well, incubate at 37 ℃ for 60min, measure absorbance values at 490nm for each well.

Inhibition ═ 100% (1-mean absorbance value of drug/mean absorbance value of control).

2.4 determination of intracellular Melanin content

B16 melanocytes at 1 × 10⁵one/mL of the density was seeded in 6-well plates. 3mL of the cells in each well are incubated for 24h, the supernatant is discarded, the drug to be tested is added, after the drug acts for 3d, the supernatant is discarded, the cells are washed by PBS, 0.5mL of pancreatin digestive cells are added into each well for 3min, 2mL of maintenance solution is added to stop the digestion, and each group of cells is counted respectively. Centrifuging the cell suspension, removing the supernatant, adding 1mol/L NaOH solution into the precipitate, and heating at 80 deg.C for 30 min. The absorbance at 475nm was measured by a spectrophotometer.

Melanin synthesis inhibition rate ═ 1- (drug well absorbance value/number of drug wells)/(control well absorbance value/number of control wells) ] × 100%

2.5 data processing

Data are expressed as mean values.

3. Results

Since high concentrations of drugs have a significant effect on cell growth, to avoid interference of drug cytotoxicity on the enzymatic activity, this experiment employed drug concentrations that did not affect normal cell physiology.

As can be seen from Table 1, the extracts of various fruits of Xingan Chongshi have obvious inhibition effects on the activity of cell tyrosinase and the synthesis of melanin, and show a certain dose-effect relationship. Among them, the inhibition rate of the XT50 high dose group is better than that of the positive control group arbutin.

The results also show that the higher the purity of the calendoside E at the corresponding dosage, the better the whitening effect is, and therefore the material basis of the whitening effect of the Xingan real extract can be related to the calendoside E not only.

TABLE 1 Effect on tyrosinase Activity and melanin Synthesis in mouse B16 melanocytes

	Tyrosinase inhibition (%)	Melanin synthesis inhibition (%)
			XT50 low dose group	53.8	49.7
XT50 Medium dose group	63.4	60.6
			XT50 high dose group	87.6	84.3
XT90 low dose group	59.7	54.8
			XT90 Medium dose group	61.5	57.7
XT90 high dose group	81.4	76.9
			Arbutin group	83.5	78.8

According to different preparation methods, the mass percent of the Xingan worm seed extract in the cosmetic can be 0.01-50%.

The Xingan insect extract can be used as an additive for preparing whitening cosmetics in the forms of powder, water, cream and the like.

The invention is further illustrated below with reference to specific embodiments in order to better illustrate the solution, but the scope of protection claimed by the invention is not limited to the following examples.

Detailed Description