CN116725905A - Essence containing sweet wormwood volatile oil and application thereof - Google Patents

Abstract

The invention discloses an essence containing sweet wormwood volatile oil, which contains sweet wormwood volatile oil prepared by using mother liquor after extracting sweet wormwood essence, and the sweet wormwood volatile oil has the effects of inhibiting inflammatory factors and repairing skin barriers and is high in safety. The invention further provides other components, clinical efficacy and skin microecology analysis of the essence, and the essence is proved to be high in safety, has the effects of improving skin sensitivity, relieving redness, improving skin compactness, preserving moisture and repairing, is particularly suitable for sensitive muscle groups, can improve skin microecology, and has huge market and application value.

Description

Essence containing sweet wormwood volatile oil and application thereof

Technical Field

The invention relates to the field of cosmetics, in particular to the field of skin repair and improvement of skin sensitivity, and especially relates to an essence containing sweet wormwood herb volatile oil and application thereof.

Background

Sensitive skin is mainly characterized in that the skin is easy to have symptoms such as burning, stinging, itching, tightness and the like after being stimulated by the outside, and the symptoms such as erythema, scales, telangiectasia and the like are accompanied or not. Skin sensitivity is more likely to occur in people with low skin barrier function and increased transepidermal water loss values. Factors such as physics, chemistry, life style and psychology can cause sensitive skin to occur, and can cause adverse effects on psychological conditions of people, and the quality of life is reduced.

Herba Artemisiae Annuae is aerial part of Artemisia annua of Compositae, has effects of clearing summer heat, and preventing malaria, and can be used for treating summer heat, fever due to yin deficiency, night fever, fever due to fatigue, malaria, hyperpyrexia and jaundice. Modern researches prove that the sweet wormwood has the effects of resisting malaria, resisting tumors, relieving fever, easing pain, resisting bacteria, resisting viruses and the like, wherein the sweet wormwood is extracted and separated from the sweet wormwood to serve as an antimalarial specific component, and has breakthrough significance in malaria treatment. It is worth noting that, in the sweet wormwood, other chemical components such as volatile oil are also included besides sweet wormwood, and in the conventional industrial purification of sweet wormwood, the rest part is treated as waste after the sweet wormwood is extracted and obtained, so that other effective components are wasted, and the waste treatment cost is increased.

The herba Artemisiae Annuae volatile oil (INCI name: herba Artemisiae Annuae (ARTEMISIA ANNUA) extract) is light yellow clear liquid, contains various active ingredients such as arteketone, isoaarteketone, eucalyptol, L-camphora, caryophyllene, pinene, etc., has antibacterial, antipyretic, antitussive, antiasthmatic, etc., and can be used for treating upper respiratory tract infection, chronic bronchitis, neurodermatitis, dermatomycosis, etc. The sweet wormwood essential oil can be combined with other substances and also can be used for treating skin diseases and caring skin, for example, chinese patent 202111159910.0 (publication number: CN 1136662978A) discloses that a composition of sweet wormwood essential oil and white tea essential oil can be used for treating eczema.

Disclosure of Invention

In order to fully utilize the residue of the artemisia annua after extracting the artemisinin, the mother liquor after extracting the artemisinin is further processed to obtain volatile oil components, namely the artemisia annua volatile oil. In one aspect, the application provides a process for extracting sweet wormwood volatile oil from sweet wormwood residues; on the other hand, the obtained sweet wormwood volatile oil is applied to the field of cosmetics, and the essence containing the sweet wormwood volatile oil is provided, and the clinical efficacy of the essence is researched, so that the essence has the effects of reducing the percutaneous moisture loss value, increasing the skin moisture content, improving the skin firmness and resisting and repairing the allergy; meanwhile, the application researches the skin microecology change after the essence is used, and discovers that the essence has the effect of improving the skin microecology.

The application aims at solving the problems through the following technical scheme:

in one aspect, the application provides an essence containing sweet wormwood volatile oil, which comprises the following components: the sweet wormwood volatile oil is prepared by using mother liquor after extracting sweet wormwood element.

The mother liquor after extracting the artemisinin is further used for extracting the artemisia apiacea volatile oil, thereby changing waste into valuable, fully utilizing natural resources and reducing waste treatment cost. Furthermore, the inventors have studied to find that: the obtained sweet wormwood volatile oil has the effects of safety, no stimulation, relieving, repairing skin barrier and the like, and is especially suitable for preparing essence.

In some embodiments, the preparation of the sweet wormwood volatile oil comprises:

s1, removing a solvent in a mother solution after artemisinin is extracted by adopting a microwave heating and reduced pressure distillation mode to obtain a concentrate;

s2, taking the concentrate in the step S1, adding an organic solvent, mixing and dissolving, and removing surface wax to obtain crude sweet wormwood oil solution;

s3, taking the crude sweet wormwood oil solution in the step S2, and removing the solvent by adopting a microwave heating and reduced pressure distillation mode to obtain a crude sweet wormwood oil product;

s4, adding distilled water into the crude sweet wormwood product in the step S3, distilling in a microwave heating and reduced pressure distillation mode, and collecting a first fraction at 70-100 ℃; adjusting the intensity of microwaves, raising the temperature to 110-120 ℃, and collecting a second fraction;

and S5, combining the first fraction and the second fraction collected in the step S4, standing for layering, and collecting an oil layer.

The oil layer is herba Artemisiae Annuae volatile oil, and the obtained herba Artemisiae Annuae volatile oil can be further purified to improve purity of herba Artemisiae Annuae volatile oil and reduce impurity content.

Preferably, the preparation step of the sweet wormwood volatile oil further comprises the following steps: s6: adding silica gel into the oil layer collected in the step S5, stirring, standing, filtering, and collecting filtrate.

Preferably, the essence contains 0.01-0.25% of sweet wormwood volatile oil;

preferably, the essence also contains other functional components, and the components and the sweet wormwood volatile oil can mutually promote to better play the skin care effect, and specifically comprise the following steps: purslane extract, lactobacillus/soybean milk fermentation product filtrate.

Preferably, the content of the sweet wormwood volatile oil is 0.01% -0.25%, the content of the purslane extract is 0.001% -5.0%, and the content of the lactobacillus/soybean milk fermentation product filtrate is 0.005% -2.0%.

Preferably, the content of the sweet wormwood volatile oil is 0.05-0.25%, the content of the purslane extract is 1.0-5.0%, and the content of the lactobacillus/soybean milk fermentation product filtrate is 1.0-1.5%.

Optimally, the content of the sweet wormwood volatile oil is 0.1%, the content of the purslane extract is 3.0%, and the content of the lactobacillus/soybean milk fermentation product filtrate is 1.0%.

In some embodiments, the concentrate consists of butylene glycol, glycerin, panthenol, purslane extract, polydimethylsiloxane, 1, 2-pentanediol, 1, 2-hexanediol, fructooligosaccharides, p-hydroxyacetophenone, caprylic/capric triglyceride, C14-22 alcohols, shea (BUTYROSPERMUM PARKII) fruit fat, hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer, arginine, C12-20 alkyl glucosides, ceramide NP, dioctyl dodecanol lauroyl glutamate, dipotassium glycyrrhizinate, sweet wormwood volatile oil, troxerutin, trehalose, lactobacillus/soybean milk fermentation product filtrate, white pool (LIMNANTHES ALBA) seed oil, hydrogenated lecithin, behenyl polyether-20, carbomer, acrylic acid (ester) class/C10-30 alkanol acrylate cross-linked polymer, xanthan gum, acid bean (TAMARINDUS INDICA) seed polysaccharide, sunflower (HELIANTHUS ANNUUS) seed oil, phytosterol/octyldodecanol lauroyl glutamate, phytosphingosine and water.

In some embodiments, the concentrate consists of 3.0% butylene glycol, 3.0% glycerol, 2.5% panthenol, 3.0% purslane extract, 2.0% dimethicone, 1.0%1, 2-pentanediol, 1.0%1, 2-hexanediol, 1.0% fructo-oligosaccharides, 0.5% p-hydroxyacetophenone, 0.5% caprylic/capric triglyceride, 0.48% C14-22 alcohol, 0.47% shea (BUTYROSPERMUM PARKII) fruit fat, 0.2% hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, 0.14% arginine, 0.12% C12-20 alkyl glucoside, 0.1% ceramide NP, 0.1% dioctyl dodecanol lauroyl glutamate, 0.1% dipotassium glycyrrhizinate, 0.1% sweet wormwood volatile oil, 0.1% troke, 0.1% trehalose, 1.0% lactobacillus/fermentation product filtrate, 0.1% white pool (42) phospholipids, 0.05% hydrogenated vegetable seed oil (62.05%) 0.05% caprylic acid, 0.05% soy sauce, 0.06% 8% phytol, 0.05% soy bean curd seed oil, 0.05% 8% phytol, 0.05% phytol, 0.06% water, 8% phytol, and (62% phytol, 0.05% phytol, 10% glycon, and (52) seed oil).

In one embodiment of the application, the essence is applied to a subject with sensitive skin, and as a result, it is found that: after 28 days of use, the lactic acid stinging score of the subject is reduced, the visible-CR erythema value is reduced, the skin redness condition is improved, the heme value is obviously reduced, the percutaneous moisture loss value is obviously reduced, the water content of the stratum corneum is obviously increased, the skin compactness parameter F4 is more approximate to 1, and the subject self-evaluates and use experience score is good, so that the essence has the effects of skin soothing, moisturizing, repairing and compacting, and is accepted and favored by the subject.

Further, after the essence is used, the change condition of skin micro-ecology of a subject is researched, and the essence has the effect of improving the skin micro-ecology, so that the alpha diversity of skin microbial communities can be increased, the relative abundance of bacteria of the phylum bacteroides is obviously increased, and the relative abundance of bacteria of the pseudomonadaceae and the pseudomonadaceae is obviously reduced.

The application has the advantages that: the application prepares the sweet wormwood volatile oil by using the mother liquor after extracting sweet wormwood element, and provides an optimized preparation method. The herba Artemisiae Annuae volatile oil has effect of inhibiting inflammatory factor. The application further provides the composition and clinical efficacy of the essence and the influence on skin microecology, and proves that the essence has high safety, has the effects of improving skin sensitivity, relieving, improving skin firmness and moisturizing and repairing, can improve the microecology environment of the skin, is especially suitable for sensitive muscle groups, and has great market value.

Drawings

FIG. 1 shows the results of the test of the effect of sweet wormwood volatile oil on the activity of human primary skin keratinocytes;

FIG. 2 results of experiments of the effect of sweet wormwood essential oil on human primary skin keratinocyte FLG gene expression;

FIG. 3 results of experiments of the effect of sweet wormwood essential oil on human primary skin keratinocyte IL-8 expression;

FIG. 4 results of a test of the effect of sweet wormwood essential oil on expression of human primary skin keratinocyte TLSP gene;

FIG. 5 shows the results of an experiment of the effect of sweet wormwood herb volatile oil on the activity of macrophage Raw264.7;

FIG. 6 shows the results of experiments on the effect of volatile oil of Artemisia annua on the expression of inflammatory factor NO secreted by macrophages;

FIG. 7 shows the results of experiments on the effect of volatile oil of Artemisia annua on the expression of inflammatory factor IL-6 secreted by macrophages;

the results of the assay of the composition of FIG. 8 on the effect of human primary skin keratinocyte viability;

the results of the test of the effect of the composition of FIG. 9 on the expression of FLG gene in human primary skin keratinocytes;

the results of the test of the effect of the composition of FIG. 10 on the expression of inflammatory factor NO secreted by macrophages;

FIG. 11 is a test of the effect of the composition on macrophage secretion of inflammatory factor IL-6 expression;

FIG. 12 comparison of differences in lactic acid stinging test scores;

FIG. 13 comparison of differences in VISIA erythema values;

fig. 14 is a picture of subject VISIA before and after use of the product;

FIG. 15 skin heme value differential analysis;

FIG. 16 comparison of skin moisture loss and moisture content;

FIG. 17 comparison of skin elasticity and firmness;

fig. 18 subject clinical symptom scores before and after product use;

fig. 19 subjective symptom differential analysis of subjects after product use;

FIG. 20 comparison of facial skin flora Alpha diversity of subjects before and after serum use;

in the figure: * Indicating that there is a significant difference, P < 0.05; * Indicating that there was a very significant difference, P < 0.01.

Detailed Description

The application is further described below with reference to the drawings and examples. It should be apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and should not be used to limit the scope of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application. Materials, reagents and the like used, unless otherwise specified, are commercially available.

In order to fully utilize the mother liquor left after the production of artemisinin, the application further extracts and separates the mother liquor to obtain the artemisia apiacea volatile oil. The mother liquor after extracting the artemisinin is derived from the mother liquor obtained by an artemisinin extracting method well known in the art; for example: extracting residual mother liquor after artemisinin is extracted according to an artemisinin extraction method in Chinese patent 201410636189.3 (publication number: CN 104327093B); the mother liquor which is remained after the extraction of the artemisinin is the mother liquor which contains ether solvent, preferably petroleum ether, such as petroleum ether mother liquor after the extraction of the artemisinin in Chinese patent 201310550808.2 (publication number: CN 104628739A). The inventor optimizes the extraction process to ensure that the preparation yield of the sweet wormwood volatile oil is high and the active ingredients are high.

It should be noted that, the volatile oil of sweet wormwood in the application is prepared from mother liquor after extracting sweet wormwood, and the volatile oil extracted by directly using sweet wormwood, namely the dry overground part of sweet wormwood, is different, and the composition and the proportion of the volatile oil are also different. For example, it is known from example 4 of the present application that the sweet wormwood essential oil of the present application mainly comprises sweet wormwood ketone (33.24%), sweet wormwood triene (15.42%), camphor (5.25%), artenol (5.11%), etc., while it is known from chinese patent 202111159908.3 (publication No. 113773909 a) that sweet wormwood essential oil extracted directly from sweet wormwood mainly comprises eucalyptol (27.47%), caryophyllene (27.47%), α -pinene (19.56%), β -pinene (16.03%), etc. It may be that the volatile oil part of the sweet wormwood is volatilized and lost after the sweet wormwood is subjected to the process of extracting the sweet wormwood, in addition, the column separation process exists in the process of extracting the sweet wormwood, and part of components may be lost in the separation process, so that the sweet wormwood volatile oil prepared from the mother liquor after extracting the sweet wormwood is different from the volatile oil extracted by directly using the sweet wormwood. In addition, it may be caused by differences in the extraction process of the volatile oil.

The sweet wormwood volatile oil has different compositions from the volatile oil extracted by directly using sweet wormwood, and the functions of the sweet wormwood volatile oil are naturally different. Further, the inventor of the application researches the in vitro efficacy of the obtained sweet wormwood volatile oil, including related researches of cytotoxicity, skin repair, anti-inflammation, anti-sensitivity and the like, and discovers that the sweet wormwood volatile oil has anti-inflammation and anti-sensitivity effects.

Furthermore, the sweet wormwood volatile oil, the purslane extract and the lactobacillus/soybean milk fermentation product filtrate are combined for use, so that the sweet wormwood volatile oil and the lactobacillus/soybean milk fermentation product filtrate have higher safety and anti-inflammatory and repairing effects.

Further, the application provides essence containing sweet wormwood volatile oil or the composition.

In some embodiments, the concentrate consists of butylene glycol, glycerin, panthenol, purslane extract, polydimethylsiloxane, 1, 2-pentanediol, 1, 2-hexanediol, fructooligosaccharides, p-hydroxyacetophenone, caprylic/capric triglyceride, C14-22 alcohols, shea (BUTYROSPERMUM PARKII) fruit fat, hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer, arginine, C12-20 alkyl glucosides, ceramide NP, dioctyl dodecanol lauroyl glutamate, dipotassium glycyrrhizinate, sweet wormwood volatile oil, troxerutin, trehalose, lactobacillus/soybean milk fermentation product filtrate, white pool (LIMNANTHES ALBA) seed oil, hydrogenated lecithin, behenyl polyether-20, carbomer, acrylic acid (ester) class/C10-30 alkanol acrylate cross-linked polymer, xanthan gum, acid bean (TAMARINDUS INDICA) seed polysaccharide, sunflower (HELIANTHUS ANNUUS) seed oil, phytosterol/octyldodecanol lauroyl glutamate, phytosphingosine and water.

Clinical experiments of the essence are carried out, the safety of use, the improvement condition of skin and the like are examined, the safety is good after the essence is used for 28 days, the skin condition of a subject is improved, and the essence comprises the following components: improving skin sensitivity, relieving, moisturizing, repairing, and tightening.

Furthermore, the application also researches the skin microecological change of sensitive skin after the essence is used. After the essence is used for 7 days, the relative index of alpha diversity of the skin microbial community is obviously increased, the relative abundance of bacteria of the phylum bacteroideae is obviously increased, the relative abundance of bacteria of the pseudomonadaceae and the pseudomonadaceae is obviously reduced, and the essence can improve the micro-ecological environment of the skin.

Sweet wormwood oil and sweet wormwood volatile oil: the sweet wormwood oil and sweet wormwood volatile oil in the application are volatile oil components prepared from mother liquor after extracting artemisinin.

Lactobacillus/soymilk fermentation product filtrate: the LACTOBACILLUS/soybean milk fermentation product filtrate (English name: LACTOBACILLUS/soybean milk FERMENT FILTRATE) in the international cosmetic raw material standard Chinese name catalog (INCI) is a common cosmetic raw material. It is rich in vitamins and proteins, and active ingredients, and has the main effects of humectant in cosmetics and skin care products, risk factor of 1, and high safety.

Purslane extract: the purslane extract, namely the purslane (PORTULACA OLERACEA) extract (CAS number: 90083-07-1) in INCI, is a common cosmetic raw material. It has broad-spectrum antibacterial and antiinflammatory effects, and can be used for preventing and treating dermatoses such as skin eczema, allergic dermatitis, contact dermatitis, etc.; has good oxygen free radical scavenging ability and obvious antioxidation effect; has good skin moisture retention; and has the capability of helping the acne muscles to diminish inflammation and sterilize and accelerating wound healing.

Example 1 preparation of Artemisia annua volatile oil (1)

Extracting oleum Artemisiae Annuae with petroleum ether mother liquor left after artemisinin production, placing 3L of petroleum ether extraction mother liquor in a 10L round bottom flask, placing in a microwave heater, and respectively connecting a temperature probe, a condenser, a collector and a circulating water pump to start experiments, wherein the experimental steps are as follows:

firstly, turning on a microwave heater to adjust the output power to 600w to heat mother liquor, decompressing and collecting and recovering petroleum ether, turning off the microwave heater when the temperature suddenly jumps to more than 90 ℃, and cooling to room temperature to obtain sweet wormwood oil concentrate;

secondly, adding 3L of ethanol into a round-bottom flask, inserting an electric stirring slurry, setting the rotating speed to be 200 revolutions per minute, stirring for 10 minutes, standing for half an hour, and removing wax floating on the surface of the ethanol to obtain an initial oil ethanol solution of sweet wormwood;

Thirdly, placing the round bottom flask filled with the sweet wormwood primary oil ethanol solution in a heater, opening the microwave heater to adjust the output power to 600w, heating the mother liquor, decompressing, collecting and recovering ethanol, and when the temperature is suddenly changed to be more than 75 ℃, closing the microwave heater to obtain about 1.5L of sweet wormwood primary oil;

fourthly, 500ml of sweet wormwood primary oil is taken and placed in a 5L round bottom flask, distilled water with the concentration of 5 times (V/V) is added, the sweet wormwood primary oil is placed in a microwave heater, the microwave heater is turned on to adjust the output power to 600w and heated to boiling, the water vapor containing oil is collected, and when the temperature is suddenly changed to be more than 90 ℃, the microwave heater is turned off; adding distilled water with the concentration of 5 times (V/V) again, repeating the distillation process for 2 times, and collecting 7.5L of oily water solution; continuously heating to 110+/-5 ℃, maintaining for 30 minutes, collecting distillate oil under reduced pressure, and combining with an oily water solution;

fifthly, standing the oily water solution overnight for layering, and discarding a water layer to obtain 200ml of sweet wormwood oil;

and step six, transferring 200ml of sweet wormwood oil to a temperature of 80 ℃, adding 4g of silica gel while the sweet wormwood oil is hot, stirring for 30 minutes, standing for 30 minutes, and filtering while the sweet wormwood oil is hot to obtain 189ml of sweet wormwood essential oil finished product.

Example 2 preparation of Artemisia annua volatile oil (2)

The experiment was continued starting from the fourth step of the above procedure with the crude sweet wormwood oil prepared in example 1:

Fourthly, 500ml of sweet wormwood primary oil is taken and placed in a 5L round bottom flask, 3 times (V/V) distilled water is added, the sweet wormwood primary oil is placed in a microwave heater, the microwave heater is turned on to adjust the output power to 1600w and heat the sweet wormwood primary oil to boiling, the oily vapor is collected, and when the temperature is suddenly changed to be more than 90 ℃, the microwave heater is turned off; adding distilled water 3 times (V/V) again, repeating the above distillation process for 2 times, and collecting 4.5L oily water solution; continuously heating to 115+/-5 ℃, maintaining for 30 minutes, collecting distillate oil under reduced pressure, and combining with an oily water solution;

fifthly, standing the oily water solution overnight for layering, and discarding a water layer to obtain 181ml of sweet wormwood oil;

and sixthly, transferring 180ml of sweet wormwood oil to a temperature of 70 ℃, adding 3g of silica gel while the sweet wormwood oil is hot, stirring for 30 minutes, standing for 30 minutes, and filtering while the sweet wormwood oil is hot to obtain 175ml of sweet wormwood essential oil finished product.

Example 3 preparation of Artemisia annua volatile oil (3)

The experiment was continued starting from the fourth step of the above procedure with the crude sweet wormwood oil prepared in example 1:

fourthly, 500ml of sweet wormwood primary oil is taken and placed in a 10L round bottom flask, distilled water with the volume of 10 times (V/V) is added, the sweet wormwood primary oil is placed in a microwave heater, the microwave heater is turned on to adjust the output power to 1000w and heated to boiling, the water vapor containing oil is collected, and when the temperature is suddenly changed to be more than 90 ℃, the microwave heater is turned off; adding 10 times (V/V) distilled water again, repeating the distillation process, and collecting 10L oily water solution; continuously heating to 115+/-5 ℃, maintaining for 30 minutes, collecting distillate oil under reduced pressure, and combining with an oily water solution;

Fifthly, standing the oily water solution overnight for layering, and discarding a water layer to obtain 205ml of sweet wormwood oil;

and step six, transferring 200ml of sweet wormwood oil to a temperature of 60 ℃, adding 5g of silica gel while the sweet wormwood oil is hot, stirring for 30 minutes, standing for 30 minutes, and filtering while the sweet wormwood oil is hot to obtain 190ml of sweet wormwood essential oil finished product.

Example 4 detection of the composition of Artemisia annua volatile oil

The volatile oil of Artemisia annua prepared in example 1 was taken for GC-MS analysis.

Chromatographic conditions: HP-5 capillary chromatographic column (60 m 0.25mm 0.5 μm); carrier gas He, flow rate 1.0ml/min; the split ratio is 10:1; the temperature of the sample inlet is 260 ℃, the initial temperature is 60 ℃, the temperature is kept for 5min, the temperature is increased to 80 ℃ at 5 ℃/min, the temperature is kept for 5min, and the temperature is increased to 260 ℃ at 10 ℃/min; the sample loading was 0.2. Mu.L.

Mass spectrometry conditions: adopting EI mode, electron energy is 70eV; the ion source is 200 ℃; the scanning range is 30-600amu, spaced 0.5s apart.

Qualitative analysis was performed on more than 2.0% of the components, with the following results:

TABLE 1 GC-MS detection results of Artemisia annua volatile oil

Name of the name	Molecular formula	Molecular weight	Relative content (%)
				Santalia triene	C 10 H 16	136.2	2.48
Artemisinin triene	C 10 H 16	136.2	15.42
				Myrcene	C 10 H 16	136.2	3.15
Arteol	C 10 H 18 O	154.2	5.11
				Artemisia ketone	C 10 H 16 O	152.2	33.24
(±) -camphor	C 10 H 16 O	152.2	5.25
				Peppermint-1, 8-dien-3-ols	C 10 H 16 O	152.3	3.82

In addition, the volatile oil of sweet wormwood herb in examples 2 and 3 is taken and detected in the same way, and the detection results are similar and are not described in detail herein.

EXAMPLE 5 cell experiments of Artemisia annua volatile oil

1. Human primary skin keratinocyte viability experiment

Human primary skin keratinocytes (purchased from Dojindo, cat#C04)) were cultured under culture conditions containing 5% carbon dioxide at 37℃until the cells grew to 80% density, and they were cultured according to 1X10 ⁵ Inoculating/ml density into 96-well plate, adhering for 48 hr, adding 0.05%, 0.1%, 0.25%, 0.5%, 1% herba Artemisiae Annuae volatile oil (oleum Artemisiae Annuae) respectively, treating with no oleum Artemisiae Annuae (normal group) as control for 72 hr, adding CCK reagent (purchased from Dojindo, cat#C04), and incubatingAbsorbance was measured at 450nm for 2 h. As shown in FIG. 1, the volatile oil of herba Artemisiae Annuae has obvious cytotoxicity at 0.25% and above, and no toxicity at 0.1% and below on primary skin keratinocytes.

2. Experiment of Effect on human Primary skin Keratin Barrier protein Gene expression

Human primary skin keratinocytes were cultured at 37℃with 5% CO in medium ₂ Culturing in an incubator with a humidity of 95%. After the cells grow to more than 80% of fusion, the plates are plated. After further cultivation, the test actives, IL-4 and IL-13 (15 ng/ml) were added separately and the supernatant was aspirated after 3 days of stimulation.

Filaggrin (FLG) is an important skin barrier protein expressed by keratinocytes. After the cells were induced by IL-4/IL-13 stimulation in this experiment, the expression of the skin barrier protein FLG was significantly decreased, suggesting that the activation of Th2 signaling pathway had some damage to the skin barrier (FIG. 2). After cells are treated by the sweet wormwood volatile oil with different concentrations, as shown in fig. 2, the experimental results show that 0.1% and 0.05% sweet wormwood volatile oil can promote FLG gene expression and have a certain dose-dependent relationship, so that the sweet wormwood volatile oil has a certain repairing effect (P is less than 0.01) on IL-4/IL-13 stimulation to induce skin barrier protein expression reduction.

3. Effect on human primary skin keratinocyte IL-8 expression

Human primary skin keratinocytes were cultured at 37℃with 5% CO in medium ₂ Culturing in an incubator with a humidity of 95%. After the cells grow to more than 80% of fusion, the plates are plated. After further incubation, the test actives, IL-4 and IL-13 (15 ng/ml), were added separately and the supernatant was aspirated for 3 days and the amount of IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA).

IL-8 inflammatory factor secretion was significantly increased after induction of cells by IL-4/IL-13 stimulation, suggesting activation of the Th2 signaling pathway (FIG. 3). After cells are treated by the sweet wormwood volatile oil with different concentrations, as shown in figure 3, 0.1% and 0.05% sweet wormwood volatile oil can reduce IL-8 inflammatory factor secretion and have a certain dose dependency, so the sweet wormwood volatile oil has a certain inhibition effect (P is less than 0.01) on IL-4/IL-13 stimulation and induction of skin inflammation.

4. Experiment of Effect on expression of human Primary skin keratinocyte TLSP Gene

Human primary skin keratinocytes were cultured at 37℃with 5% CO in medium ₂ Culturing in an incubator with a humidity of 95%. After the cells grow to more than 80% of fusion, the plates are plated. After further incubation, the test actives were added separately and incubated for 1 hour. Subsequently, the treatment medium is replaced with fresh with test materialLive staphylococcus aureus (ATCCO 29213 a) in the medium and cultured for 24 hours under standard culture conditions. After the culture, culture supernatant was collected, and cytokine levels were measured by ELISA. As shown in the experimental result in FIG. 4, with doxycycline hydrochloride as a positive control, the expression level of TSLP after treatment of sample groups with different concentrations is observed, which shows that 100 mug/ml sweet wormwood volatile oil has obvious inhibition effect (P < 0.01) on the expression level of TSLP.

5. Experimental experiment on influence of macrophage Raw264.7 Activity

Raw264.7 mouse macrophage cell line containing 5% CO at 37deg.C ₂ The cells were cultured normally in DMEM medium containing 10% FBS. When the cells grow to 90% density, they are grown according to 8X10 ⁵ After inoculating to 96-well plate with density/ml for 8h, adding samples with different concentrations, respectively treating for 24h, adding CCK reagent according to the instruction, incubating for 2h, and measuring absorbance at 450 nm. As shown in the figure 5, the effect on cell viability is obvious in the dose-dependent effect after the treatment of samples with different concentrations, wherein 1.25% or more of the sweet wormwood volatile oil has obvious cytotoxicity effect; the sweet wormwood volatile oil has no obvious toxic effect on macrophage Raw264.7 when the sweet wormwood volatile oil is lower than 0.63 percent.

6. Experiment of Effect on macrophage secretion of inflammatory factor NO expression

Raw264.7 mouse macrophage cell line containing 5% CO at 37deg.C ₂ The cells were cultured normally in DMEM medium containing 10% FBS. When the cells grow to 90% density, inoculating the cells into a 96-well plate according to the density of 8x105/ml, attaching the cells for 8 hours, adding samples with different concentrations, respectively treating for 24 hours, and thenThe machine is divided into a blank control group, an LPS, an LPS+positive control group and an LPS+sample group, corresponding test samples are added, and the temperature is 37 ℃ and the mixture contains 5% CO ₂ Culturing for 24h under saturated humidity, and collecting supernatant for detection. As shown in FIG. 6, the secretion of inflammatory factor NO by cells treated with LPS was significantly up-regulated, and the NO expression level was inhibited after the positive control dexamethasone treatment. The secretion of NO after the sample group treatment is observed, and the obvious inhibition effect (P is less than 0.01) of the volatile oil of sweet wormwood herb on NO is found.

7. Experiment of Effect on macrophage secretion of inflammatory factor IL-6 expression

Raw264.7 mouse macrophage cell line (from cell bank of the Chinese sciences, accession number TCM 13) at 37℃with 5% CO ₂ The cells were cultured normally in DMEM medium containing 10% FBS. When the cells grow to 90% density, they are grown according to 8X10 ⁵ Inoculating/ml density into 96-well plate, adhering for 8 hr, adding samples with different concentrations, treating for 24 hr, randomly dividing into blank control group, LPS, LPS+ positive control group, LPS+ sample group, adding corresponding test sample, and adding 5% CO at 37deg.C ₂ Culturing for 24h under saturated humidity, and collecting supernatant for detection. As shown in FIG. 7, the secretion of inflammatory factor IL-6 by Raw264.7 cells after LPS treatment is obviously up-regulated, and the amount of IL-6 is inhibited after positive control dexamethasone treatment. The secretion of IL-6 after treatment of sample groups with different concentrations is observed, and obvious inhibition effect (P < 0.01) on IL-6 is found in each concentration, and the dose-dependent effect is obvious.

Example 6 Artemisia annua volatile oil, portulaca oleracea extract, and lactobacillus/soymilk fermentation product filtrate composition and cell experiments thereof

The sweet wormwood essential oil, the purslane (PORTULACA OLERACEA) extract (purchased from Tianjin Heng Kangtai technology Co., ltd.) and the lactobacillus/soybean milk fermentation product filtrate (purchased from AXIALYS INNOVATIONS) of example 1 were prepared into compositions according to the following proportions, respectively, and dissolved in water.

TABLE 2 Artemisia annua volatile oil, portulaca oleracea extract and lactobacillus/soymilk fermentation product filtrate composition

The results of the primary skin keratinocyte viability assay, the effect assay on the expression of the human primary skin keratinocyte barrier protein gene, the effect assay on the expression of the macrophage-secreted inflammatory factor NO, and the effect assay on the expression of the macrophage-secreted inflammatory factor IL-6 were performed as in example 5, using the compositions shown in Table 2 are shown in FIGS. 8-11. From the results, it can be seen that:

(1) After the sweet wormwood volatile oil is combined with the purslane extract, compared with the sweet wormwood volatile oil alone, the sweet wormwood volatile oil has obviously increased effects of promoting FLG gene expression and inhibiting inflammatory factors NO and IL-6, which indicates that the potential anti-inflammatory and skin barrier repairing effects are obviously enhanced.

(2) After lactobacillus/soybean milk fermentation product filtrate is added to sweet wormwood volatile oil and purslane extract, the influence of the composition on cell viability is obviously reduced, in addition, compared with the sweet wormwood volatile oil and the purslane extract, the effect of promoting FLG gene expression and inhibiting inflammatory factors NO and IL-6 is not obviously different, in addition, compared with the FLG relative expression amount of composition 4 of composition 5, the effect of promoting FLG gene expression of composition 5 is slightly lower than that of composition 4. The results show that the combination of the sweet wormwood volatile oil, the purslane extract and the lactobacillus/soybean milk fermentation product filtrate has higher safety and potential anti-inflammatory and skin barrier repairing effects.

(3) With the increase of the content of the sweet wormwood volatile oil and the purslane extract, the potential anti-inflammatory and skin barrier repairing effects tend to be increased, but the activity change of the composition is not obvious when the sweet wormwood volatile oil is increased from 0.1% to 0.25% and the purslane extract is increased from 3.0% to 5.0%. Considering effects, safety and costs comprehensively, the preferred combinations are: a combination of 0.1% sweet wormwood essential oil, 3.0% purslane extract and 1.0% lactobacillus/soymilk fermentation product filtrate (composition 2).

Example 7A essence containing sweet wormwood volatile oil

Essence is formulated as follows.

Table 3 essential liquid composition and mass percent

Example 8 human efficacy clinical trial of essence

Subjects with sensitive skin (slightly reddish face, conscious skin sensitivity, positive results of lactic acid stinging test) aged 18-55 were selected and the essence (i.e., test product) of example 7 was used continuously for four weeks (28 days). The product was applied to the face with 2-3 pumps each time. The use frequency is 2 times daily (each time in the morning and evening), and the use is continued for 28 days. Skin condition detection was performed before the product was used, after 14 days and after 28 days. The method comprises the following steps: the dermatologist assessed safety, performed a lactic acid stinging experiment with 10% lactic acid solution at the nasolabial folds, detected facial images with Visia-CR, analyzed Visia-CR pictures with IPP software to determine skin red color a values, utilizedMX18 skin heme detection and skin moisture meter +.>CM825 detecting skin moisture content and measuring instrument for skin moisture loss>TW300 detects skin transepidermal water loss, skin elasticity and firmness analysis using Revismeter RVM600, facial objective signs by clinical assessment by dermatologists, clinical assessment by dermatologists Pore grade score, subject experience, and self-assessment analysis.

Statistical analysis of the test data was performed using SPSS 17.0 software. The data obeys normal distribution, represented by mean value +/-standard deviation, and the paired samples are selected for T test to obtain an F value; the data were not normally distributed and Mann-Whitney U test was chosen. The difference of P < 0.05 is statistically significant.

Specific evaluation indexes, test items and results are as follows:

1. security assessment

Evaluation criteria: the product is suggested to be likely to cause adverse reactions to human skin when more than 2 cases (excluding 2 cases, the same applies below) of the number of cases of the adverse reactions of the skin of class 1, or more than 1 case of the adverse reactions of the skin of class 2, or any adverse reactions of the skin of class 1, class 3 or more than class 3 occur in 30 subjects.

After 4 weeks of application of the essence, 32 subjects did not develop symptoms of itching, stinging, burning, tightening, flushing. The subjects were strictly following the test product instructions and did not find unexpected safety events. Meanwhile, the results of vital signs (body temperature, respiration, pulse and blood pressure) of the subjects are normal during the test, and no other signs are changed. The product has good use comfort, and the satisfaction degree of the subject is 100% (32/32). In conclusion, the essence is judged to have good safety.

2. Lactic acid stinging experiment

The differences in the subject's lactic acid stinging test scores at various time points before and after use of the test product are shown in figure 12.

The difference of the scores of the lactic acid stinging experiments of the subjects at different time points before and after the essence is used has statistical significance (P is less than 0.05), and the scores of the lactic acid stinging are lower than D0 (P is less than 0.05) after the essence is used for 2 weeks and 4 weeks.

After the essence is used, the lactic acid stinging score is obviously reduced (P is less than 0.05) after the D14 (14 days) and the D28 (28 days) are used compared with the D0 (0 days). It is stated that the lactic acid stinging test score tends to decrease with increasing use time and the test product improves skin sensitivity.

3. Skin heme detection

(1) VISIA-CR value comparison (skin red region a value)

The red skin area a value indicates the quantitative analysis of the red skin area (the lower the score, the better) see fig. 13, 14.

After the essence is used, the VISIA-CR erythema value is significantly reduced (P < 0.05) at the D28 return visit compared to D0. The test product was shown to improve skin redness and soothing efficacy, and in addition, the photograph of fig. 14 also shows that the subject had significantly improved facial redness.

(2) Comparison of skin heme content before and after

Skin heme content was tested with multiple probes and the results are shown in figure 15.

After the essence is used, the subject's heme value is significantly reduced (P < 0.05) at the D28 return visit compared to D0. Indicating that the skin has reduced heme content after use of the test product, the results further demonstrate that the test product has improved skin redness and soothing efficacy.

4. Skin barrier improvement

Percutaneous moisture loss value (TEWL): depending on the integrity of the skin barrier function, TEWL increases when the skin barrier function is impaired, whereas a decrease in TEWL indicates repair of the barrier. The smaller the value, the better (fig. 16).

Stratum corneum moisture content: the principal plastic substance of the stratum corneum, when hydrated, makes the skin appear smooth and elastic. Is the most commonly used indicator of reactive skin barrier function. The larger the value, the better (fig. 16).

After the essence is used, the percutaneous moisture loss value of the subject is significantly reduced (P < 0.05) at the D28 return visit compared with D0. The water content of the stratum corneum of the subject increased significantly (P < 0.05) at both D14 and D28 visits.

From the above results, it is clear that the test product can reduce the percutaneous moisture loss value, increase the moisture content of the horny layer, and the trend of reduction or increase becomes larger with the increase of the use time, indicating that the test product can have the effect of improving the skin barrier.

5. Skin elasticity and firmness improving condition

The skin elasticity parameters R2, R5, the compactness parameter F4, the closer the value is to 1, the better the skin elasticity (fig. 17).

After the essence is used, the F4 value is obviously reduced (P is less than 0.05) at the time of D28 return visit compared with D0. The test product is shown to have an effect of improving skin firmness.

6. The dermatologist evaluates the extent of erythema, edema, and desquamation

The degree of erythema, edema and desquamation of the product part is evaluated by a professional dermatologist, and the scoring result adopts 0-3 scores (the larger the score is, the heavier the index degree is). The non-expression is 0 point, and the light, medium and heavy expressions are 1, 2 and 3 points respectively. The extent of erythema, edema, desquamation before and after use of the test product is shown in figure 18.

The erythema degree is lower than before the use (P < 0.05) at 2 weeks and 4 weeks after the essence is used, and the desquamation degree and the edema are unchanged at 4 weeks after the essence is used. The test product is proved to have the effect of improving skin erythema. In addition, no edema exists before and after the test product is used, which indicates that the test product can not cause edema, namely skin allergy.

7. Subject self-evaluation

(1) Subjective symptom scoring

The subjects scored the presence of itching, tingling, burning, tightening, flushing, springiness, sensitivity on the parts where the essence was used, and the scoring results were scored from 0 to 10 points (the larger the branches indicated the greater the degree of the index), as shown in fig. 19.

After the essence is used, compared with D0, the itching, tingling, burning, tightening, flushing and sensitivity scores of the subjects are obviously reduced (P is less than 0.05) on the 14 th day and the 28 th day of return visit; the elasticity score increased significantly (P < 0.05) at the 28 th day return visit compared to D0. The test shows that the test subjects can well evaluate the essence, and the essence can not have allergic symptoms such as itching, tingling, burning, tightening, flushing, sensitivity and the like after being used, and the skin elasticity is increased.

(2) Use feeling score

A total of 32 subjects evaluated the use experience of the test product at the D14/D28 return visit. At each return visit time point, subjects were asked to evaluate according to subjective feelings and use experiences after use of the test product and with reference to scoring criteria, the scoring results were scored 1 to 7 points, the greater the score, the higher the degree of identity (7 points very identity, 6 points very identity, 5 points somewhat identity, 4 points neither identity nor objection, 3 points somewhat different identity, 2 points relatively different, 1 point very different identity) (table 4).

Table 4 subject use experience score

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Each item counts the identity of more than or equal to 5 minutes: 75% of the subjects considered the overall appearance of the test product good, 84% considered the consistency appropriate, 93.8% considered the texture of the product better. All test items have more than 50% of subject identification degree of more than or equal to 5 minutes in the return visit of D14 and/or D28, which indicates that the test product has corresponding effects in the aspects of moisture preservation, compactness, comfort and repair. In improving skin relaxation, elasticity and firmness, subjects D14 had a lower acceptance of about 40% and D28 had a significant increase in acceptance of 65.6%, 75% and 90.6%, respectively, indicating that the test product improved skin relaxation, elasticity and firmness over time. The data above demonstrate that the longer the product is used, the more the subject is able to tolerate the product and the higher the acceptance.

In summary, the clinical sensitive skin safety and efficacy evaluation test is carried out by using the test products for 28 days continuously for 32 sensitive skin subjects, and the results show that:

(1) During the course of the test, 32 subjects had no adverse reactions. The test product is proved to have higher safety in sensitive skin population.

(2) The overall score of the subject's skin sensitivity in the lactic acid stinging test tended to decrease after use of the test product, and both D14 and D28 scores differed by a statistical significance (P < 0.05) compared to the baseline value. The test product has the effects of improving skin sensitivity and relieving in the test.

(3) Skin heme detection shows that the VISIA-CR and skin heme content of the subject are decreasing after the test product is used, indicating that the test product can be suitable for improving the redness of the skin of the sensitive muscle.

(4) The skin barrier improvement test shows that after the test product is used, the percutaneous moisture loss value of the subject is reduced, the moisture content of the horny layer is increased, and the test product has the effects of moisturizing and repairing the skin barrier.

(5) The skin elasticity and compactness test result shows that after the test product is used, the compactness parameter F4 of the test subject is reduced, which indicates that the test product has the effect of compacting the skin.

(6) From subjective evaluation of the subjects, 32 subjects in the test have higher satisfaction and acceptance in terms of relieving skin discomfort of the tested products, which shows that the products have a certain effect on improving skin sensitivity. Has higher acceptance in the aspects of relieving, moisturizing, repairing and tightening.

(7) After use of the test product, the subject had a reduced score for itch, tingling, burning, tightness, flushing, sensitivity as compared to the baseline value. The test product was shown to improve subjective symptoms in subjects and increase skin elasticity.

In conclusion, the test product (essence) is used for sensitive muscle groups, has high safety, and has the effects of improving skin sensitivity, relieving, reddening, increasing skin elasticity, moisturizing and repairing skin barriers.

Example 9 skin microecology experiment

Subjects as in example 8, facial skin flora samples (62 samples total) from 31 subjects were collected before the application of the serum (day 0, D0) and on day 7 of the application of the serum (D7), respectively, and whether the test samples could maintain and improve facial micro-ecology of sensitive skin population was evaluated by 16S rRNA gene sequencing technology.

1. Sample collection method

The subject sits still in the sampling room for 30min, the sampling personnel wears a mask and sterile gloves, a sterile cotton swab is dipped in a microecological sampling liquid (0.15M NaCl and 0.1% Tween20 solution), the sterile cotton swab is completely immersed in the solution, then the sample is gently taken out, the sample is tightly adhered to the surface of the skin within the range of 2cm multiplied by 2cm, rubbed back and forth for 40 times, finally the collected sample is transferred into a sterile freezing tube and marked, and is transferred into a refrigerator with the temperature of minus 80 ℃ for 30min for preservation.

2. Test method

(1) The DNA extraction method comprises the following steps: total genomic DNA was extracted from the samples using CTAB method. The DNA concentration and purity were measured on a 1% agarose gel, and based on the concentration, the DNA was diluted 1. Mu.g/. Mu.L with sterile water for use.

(2) PCR amplification and purification method: the diluted genomic DNA was subjected to PCR amplification, and 30ng of DNA sample and the fusion primer were taken for PCR reaction.

Forward primer (V3 region 341F): 5 '-CCTAYGGRBGCASCAG-3';

reverse primer (V4 region 806R): 5'-GGACTACNNGGGTATCTAAT-3'.

Build 30 μl PCR reaction system: 10ng of DNA, 15. Mu.l of 2X Phusion Master Mix buffer, 1. Mu.l of forward and reverse primers, and 2. Mu.l of double distilled water. And PCR is performed using high-efficiency and high-fidelity enzyme (New England Biolabs), ensuring amplification efficiency and accuracy. PCR amplification conditions: pre-denaturation at 98℃for 1min; denaturation at 98℃for 10s, annealing at 50℃for 30s, elongation at 72℃for 45s,30 cycles; extending at 72℃for 5min. The PCR product was stored at-20 ℃. The PCR products were mixed at equal concentration according to the concentration of the PCR products, and after the mixture was thoroughly mixed, the PCR products were purified by electrophoresis using agarose gel having a concentration of 1×TAE of 2%.

(3) Library construction and sequencing methods: library construction is carried out by using a TruSeq DNA PCR-Free Library Preparation Kit library construction kit of Illumina company, and after the constructed library is qualified by Qubit quantification and library detection, the library is subjected to on-machine sequencing by using NovaSeq 6000.

(4) Bioinformatics analysis method: and (3) splitting the obtained sequencing data through a barcode joint sequence to obtain an effective sequence, and then carrying out clustering denoising, splicing and de-embedding on the original sequence by using a DADA2 plug-in Qiame (1.9.1) software to generate an amplified characteristic sequence (operational taxonomic units, OTU). The method comprises the steps of clustering all Effective Tags of all samples by using an Upsse algorithm (Upsse v7.0.1001), clustering sequences into OTUs (Operational Taxonomic Units) by using 100% consistency (Identity), selecting a representative sequence of OTUs, and screening a sequence with the highest occurrence frequency in the OTUs as the representative sequence of the OTUs according to the algorithm principle. Alpha diversity and beta diversity analysis was performed mainly with QIIME (1.9.1) inserts.

(5) The data analysis method comprises the following steps: statistical analysis of the test data was performed using SPSS 17.0 software. The data obeys normal distribution, represented by mean value +/-standard deviation, and the paired samples are selected for T test to obtain an F value; the data were not normally distributed and Mann-Whitney U test was chosen. The difference of P < 0.05 is statistically significant.

3. Test results and analysis

(1) Alpha diversity analysis

Both the Chao1 and Shannon values were relatively elevated in the D7 group compared to the D0 group, with significant differences in the Chao1 index change (FIG. 20, letters a, b above the box indicate P < 0.05).

(2) Microbial classification and inter-group differential analysis

The five microbial statistics of the D0 and D7 groups before the relative abundance of phylum, class, order, family, genus levels are shown in the following table.

TABLE 4 statistical (pre-abundance 5) differential analysis of group D0 and group D7 flora levels

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* Represents P < 0.05 compared to group D0.

A. Horizontal analysis of door class

A total of 29 bacterial phyla were obtained, with both groups of samples being actinomycetes (Actinobacteria) with the highest relative abundance, followed by Proteus (Proteus), firmides (Firmides), bacteroides (Bactoides), exceptiococcus-Thermus (Deinococcus). Differential analysis was performed on two groups of sample mycoplasmas, D0 and D7, and found that the abundance of bacteroides was 3.16±3.80 and 3.76±2.44, respectively, and the difference between the two groups was statistically significant (p=0.043).

B. Class level analysis

The total obtained 52 bacterial species were actinomycetes (actinomycetes) with higher relative abundance, and then beta-proteobacteria (Betaproteobacteria), gamma-proteobacteria (gammaproteins), bacillus (bacilis) and clostridium (clostridium), and the difference between two groups of sample bacterial species D0 and D7 was analyzed, and the difference of the abundance of the bacterial species was not statistically significant (P is equal to 0.05).

C. Mesh level analysis

A total of 96 bacterial orders were obtained, of which the relative abundance was higher, propionibacterium (Propionibacterium), followed by Neisseriales (Neisseriales), bacillus (Bacilles), pseudomonas (Pseudomonas), corynebacterium (Corynebacterium). The difference analysis is carried out on two groups of sample bacterial meshes of D0 and D7, and the bacterial mesh abundance difference has no statistical significance (P is more than 0.05).

D. Department level analysis

178 bacterial families were obtained, with relatively high abundance of Propionibacterium (Propionibacterium), neisseriaceae (Neisseriaceae), staphylococcus (Staphylococcus), corynebacterium (Corynebacterium), pseudomonas (Pseudomonas). Differential analysis was performed on two groups of sample bacterial families D0 and D7, where the abundance of pseudomonas in D0 and D7 was 4.63±5.37 and 1.48±0.93, respectively, and the differences were statistically significant (p=0.002).

E. Generic level analysis:

487 bacteria were obtained in total from 62 samples, including Propionibacterium (Propionibacterium), staphylococcus (Staphylococcus), pseudomonas (Pseudomonas), streptococcus (Streptococcus), and Methyloversatilis, which were relatively abundant. Differential analysis was performed on two groups of sample genera D0 and D7, where the abundance of pseudomonas at D0 and D7 was 4.60±5.36 and 1.47±0.93, respectively, with a statistical significance (p=0.002).

The results show that the skin micro-ecology of the subject changes after the essence is used.

(1) The significant increase in the alpha-diversity Chao1 index of the D7 microflora compared to the D0 group indicates a significant change in the abundance of facial skin microflora composition 7 days after use of the product. The human healthy skin microbiome generally exhibits high diversity and stability, skin microbial imbalance, reduced diversity, reduced expression of antimicrobial polypeptides, reduced pathogenic bacteria killing, and causes severe skin inflammation. The increase of alpha diversity can reduce the occurrence of skin diseases such as atopic dermatitis, psoriasis and the like to a certain extent, so that the skin enters a healthier state, and the test product can improve the micro-ecological environment of the face of a subject.

(2) The D7 group was different in the classification level of phylum, family and genus as compared with the D0 group. The bacteroides abundance was significantly increased in group D7. Previous studies have shown that many species of bacteroides are found in the human or animal gut, which bacteria confer a variety of benefits to the host, including fermenting indigestible polysaccharides of the host to provide energy, and reintroducing the bacteria upon imbalance of the host gut flora can promote the gut micro-ecology to return to equilibrium as soon as possible. However, the relation between the bacteroides and the sensitive skin is less studied at present, and the test shows that the bacteroides is raised after using a test product, which indicates that the bacteroides can improve the state of the sensitive skin.

(3) The abundance of Pseudomonas and Pseudomonas in the D7 group was significantly reduced (P < 0.05) compared to D0. Pseudomonas is a skin surface-resident bacterium, associated with skin pathogenesis (especially Pseudomonas aeruginosa). The results demonstrate that the test products may act to improve skin micro-ecology and repair skin barriers by reducing the abundance of pseudomonas and pseudomonas.

In summary, after 7 days of application of the test product, the richness of the facial skin microflora composition of the human population tested was significantly changed and increased over that before application. The relative abundance of bacteroidetes bacteria is obviously increased compared with D0, and the relative abundance of pseudomonas and pseudomonas bacteria is obviously reduced compared with D0, which proves that the test product can improve the facial skin microecology of sensitive skin crowd.

Claims (10)

1. An essence containing sweet wormwood volatile oil, which is characterized by comprising the following components: the sweet wormwood volatile oil is prepared by using mother liquor after extracting sweet wormwood element.

2. The essence of claim 1, wherein the step of preparing sweet wormwood essential oil comprises the following steps:

s1, removing a solvent in a mother solution after artemisinin is extracted by adopting a microwave heating and reduced pressure distillation mode to obtain a concentrate;

s2, taking the concentrate in the step S1, adding an organic solvent, mixing and dissolving, and removing surface wax to obtain crude sweet wormwood oil solution;

S3, taking the crude sweet wormwood oil solution in the step S2, and removing the solvent by adopting a microwave heating and reduced pressure distillation mode to obtain a crude sweet wormwood oil product;

s4, adding distilled water into the crude sweet wormwood product in the step S3, distilling in a microwave heating and reduced pressure distillation mode, and collecting a first fraction at 70-100 ℃; adjusting the intensity of microwaves, raising the temperature to 110-120 ℃, and collecting a second fraction;

and S5, combining the first fraction and the second fraction collected in the step S4, standing for layering, and collecting an oil layer.

3. The essence of claim 2, wherein the step of preparing sweet wormwood essential oil further comprises:

s6: adding silica gel into the oil layer collected in the step S5, stirring, standing, filtering, and collecting filtrate.

4. The concentrate of claim 1, wherein the concentrate further comprises: purslane extract, lactobacillus/soybean milk fermentation product filtrate.

5. The essence of claim 4, wherein the sweet wormwood volatile oil is 0.01% -0.25%, the purslane extract is 0.001% -5.0%, and the lactobacillus/soybean milk fermentation product filtrate is 0.005% -2.0%.

6. The concentrate of claim 4, wherein the concentrate further comprises: butanediol, glycerol, panthenol, polydimethylsiloxane, 1, 2-pentanediol, 1, 2-hexanediol, fructooligosaccharides, p-hydroxyacetophenone, caprylic/capric triglyceride, C14-22 alcohol, shea butter (BUTYROSPERMUM PARKII) fruit fat, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, arginine, C12-20 alkyl glucosides, ceramide NP, dioctyl dodecanol lauroyl glutamate, dipotassium glycyrrhizinate, troxerutin, trehalose, white pool (LIMNANTHES ALBA) seed oil, hydrogenated lecithin, behenpolyether-20, carbomer, acrylic acid (esters) per C10-30 alkanol acrylate cross-linked polymer, xanthan gum, soybean (TAMARINDUS INDICA) seed polysaccharide, sunflower (HELIANTHUS ANNUUS) seed oil, phytosterol/octyldodecanol lauroyl glutamate, phytosphingosine.

7. The concentrate of claim 4, wherein the concentrate consists of butylene glycol, glycerin, panthenol, purslane extract, polydimethylsiloxane, 1, 2-pentanediol, 1, 2-hexanediol, fructooligosaccharides, p-hydroxyacetophenone, caprylic/capric triglyceride, C14-22 alcohol, shea (BUTYROSPERMUM PARKII) fruit fat, hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer, arginine, C12-20 alkyl glucoside, ceramide NP, dioctyl dodecanol lauroyl glutamate, dipotassium glycyrrhizinate, sweet wormwood volatile oil, troxerutin, trehalose, lactobacillus/soybean milk fermentation product filtrate, white pool (LIMNANTHES ALBA) seed oil, hydrogenated lecithin, behenpolyether-20, carbomer, acrylic acid (ester) type/C10-30 alkanol acrylate cross-linked polymer, xanthan gum, acid bean (TAMARINDUS INDICA) seed polysaccharide, sunflower (HELIANTHUS ANNUUS) seed oil, plant/octyldodecanol lauroyl glutamate, plant sphingosine and water.

8. Use of the concentrate according to any one of claims 1 to 7 for improving skin sensitivity.

9. Use of the essence according to one of claims 1 to 7 for skin soothing, moisturizing, repairing or tightening, characterized in that the skin is sensitive skin.

10. Use of the essence according to one of claims 1 to 7 for improving the micro-ecology of human skin, characterized in that it increases the relative abundance of bacteria of the phylum bacteroides dermalis, decreases the relative abundance of bacteria of the family and genus pseudomonas dermatitidae.