CN110787094B - Application of Xingan worm extract in preparation of whitening cosmetics - Google Patents

Abstract

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.

Description

Application of Xingan worm extract in preparation of whitening cosmetics

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 (Corispermum changanicum Iljin) is a plant of genus Eugenia of family Chenopodiaceae. 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, pale yellow, opaque, full-fledged. 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 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 fruit extract is more than or equal to 50 percent by mass, and the method comprises the following steps:

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 the 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. Sonicating in ice bath, adding 10. mu.L 10mM L-DOPA to each well, incubating at 37 deg.C for 60min, and measuring absorbance at 490nm。

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 ℃ 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 cell)/(control well absorbance value/number of control wells cell) ] × 100%

2.5 data processing

Data are presented 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 ratio (%)	Melanin synthesis inhibition ratio (%)
			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 Chongshi extract can be used as additive for preparing whitening cosmetics in the form of powder, aqua, cream, etc.

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

Example 1 preparation of Xingan Worm seed extract (XT50)

Taking 10Kg of dry whole plant of Xingan Chongshi, adding 85% ethanol solution in a proportion of 10mL of ethanol solution per gram of crude drug, reflux extracting for 2 times, 1.5 hours each time, filtering, collecting liquid medicine, 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 (the ethanol extracting solution: the water saturated n-butanol), extracting for 8 times, taking an n-butanol layer, recovering the solvent to dry, adding the solid to 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 eluent by column volume parts, combining the eluent with the column volume of 5-8 parts, and recovering the solvent to dry under reduced pressure to obtain 6.26g of a Xingan insect extract (XT50), wherein the mass percent content of calendula E is 52.8%.

Example 2 preparation of Xingan Chongshi extract (XT90)

4g of XT50 prepared in example 1 were taken and further separated and purified by preparative liquid phase as follows:

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 XT50 with mobile phase, filtering, injecting into preparative liquid chromatograph, collecting the fractions with peak time of 11-14min, mixing, recovering solvent under reduced pressure, and drying to obtain white powder 1.53g, wherein the calendula glycoside E content is 91.7% by mass.

Example 3 preparation of aqueous solutions of Xingan Worm fruit extract

The preparation method comprises the following steps: and (3) taking 0.2gXT50 (or XT90), adding 500mL of deionized water for dissolving, and filling to obtain the finished product.

The application method comprises the following steps: is directly applied on skin.

Example 4 preparation of a Xingan Worm seed extract cream

Prescription: 4g of glyceryl stearate, 0.3g of sodium dodecyl sulfate, 3g of stearic acid, 7g of white vaseline, 3g of liquid paraffin, 1g of propylene glycol, 5g of glycerol, 25g of distilled water and 0.2g of XT50 (or XT 90).

The preparation method comprises the following steps: taking glyceryl stearate, stearic acid, white vaseline and liquid paraffin as oil phases, placing in a beaker, and heating in a water bath to melt; putting glycerol, propylene glycol and distilled water into another beaker as a water phase, heating to 75 ℃, and adding sodium dodecyl sulfate for dissolution; slowly pouring the water phase into the oil phase while stirring until the water phase is condensed to obtain an emulsion matrix; weighing the Xingan worm seed extract and the spice, adding the Xingan worm seed extract and the spice into the matrix, and uniformly stirring to obtain the Xingan worm seed essence.

It should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (2)

1. The application of the Xingan worm extract in 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, and the method is as follows:

taking dry whole herbs of Xingan Chongshi, adding 85% ethanol solution in a volume percentage according to the proportion that each gram of crude drug is added with 10mL of ethanol solution, performing reflux extraction for 2 times, 1.5 hours each time, filtering, taking liquid medicine, and recovering ethanol under reduced pressure to obtain ethanol extract;

taking the ethanol extract, adding water saturated n-butanol according to the volume ratio of 1:6, extracting for 8 times, combining n-butanol layers, recovering the solvent until the solvent is dried, adding the solid onto a silica gel chromatographic column, eluting with a methanol-chloroform mixed solvent, wherein the volume ratio of methanol to chloroform in the mixed solvent is 2:1, collecting the eluent according to column volume parts, combining the eluent of the 5 th to 8 th column volume parts, and recovering the solvent until the solvent is dried under reduced pressure to obtain the methanol-chloroform-based chromatographic solid.

2. The application of the Xingan real extract in preparing whitening cosmetics according to claim 1, is characterized in that the content of calendula glycoside E in the Xingan real 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.