CN111388535B - Application of osmanthus flower extract in preparation of medicine for preventing and treating sunburn - Google Patents

Abstract

The invention discloses application of an Osmanthus fragrans (Osmanthus fragrans flower) (Thunb.) Lour.) flower extract in preparation of a medicine for preventing and treating sunburn, wherein an active ingredient in the medicine is an Osmanthus fragrans flower extract, and the Osmanthus fragrans flower extract is Osmanthus fragrans absolute disclosed in the Chinese invention patent application with the application number of CN 201710420375.7. The osmanthus flower extract is used as an active substance for preventing and treating sunburn skin diseases, and the osmanthus flower extract is used as an active ingredient to be absorbed through skin, so that the immunity of a patient is improved, sunburn can be treated, and particularly various symptoms in an acute stage can be relieved, and the skin repair time can be shortened.

Description

Application of osmanthus flower extract in preparation of medicine for preventing and treating sunburn

Technical Field

The invention belongs to the field of medicine preparation, and particularly relates to application of an Osmanthus fragrans (Thunb.) fragrans extract in preparation of a medicine for preventing and treating sunburn.

Background

Sunburn (sunburn), also known as Solar Dermatitis (Solar Dermatitis) or Solar Dermatitis, is an acute inflammatory reaction generated after normal skin is exposed to sunlight, i.e., a phototoxic reaction generated after the skin is over-irradiated by medium-wave ultraviolet rays (mainly UVB) in sunlight. After normal skin is irradiated by ultraviolet rays, epithelial cells release inflammatory mediators such as histamine, 5-hydroxytryptamine, kinin and the like, so that intradermal blood vessels are expanded and have increased permeability, erythema, edema or blisters with clear boundaries appear on the surface of the skin, the skin feels burning and pain, and pigmentation spots are left after healing. Sunburn is often caused by hot summer in midsummer, and often occurs on exposed parts of face, neck, arms, back of hand, etc. Epidemiological investigations have shown that sunburn is common in children, women and people with tender skin.

Sunburn is a disease which can be self-healed, and the self-healing time is 7-10 days. The disease course can be divided into three stages of an acute stage (1-2 days, a remission stage (3-4 days) and a recovery stage (3-4 days). The acute stage is characterized in that erythema, edema or blisters with clear boundaries appear on the surface of the skin, are accompanied by burning and pain and are the most serious period of symptoms, then the remission stage and the recovery stage are carried out, the erythema, the edema or the blisters and the accompanied burning and pain are gradually reduced until the fever and the pain are recovered, but pigmentation spots generally remain.

Osmanthus fragrans is a commonly known name of Osmanthus fragrans (Osmanthus fragrans) of the genus Olea of the family Oleaceae (Oleaceae), also known as Cinnamomum japonicum, Murraya paniculata and the like, and is evergreen tree or shrub, with a small shape due to the petal-like division. Because of its remarkable sweet and fragrant smell and taste, osmanthus has been widely used and has a long history as an additive ingredient in the field of food processing.

Osmanthus fragrans (Thunb.) Lour.) flower contains a certain amount of absolute oil components, is an oily substance with non-volatile fat-soluble components as the main components, and is an important secondary metabolite. The chemical components of the absolute oil are generally complex and can be classified into terpenoids, aromatic compounds, aliphatic compounds and sulfur-containing and nitrogen-containing heterocyclic compounds according to the structures of the absolute oil. Part of the components of the osmanthus absolute are also components forming the characteristic smell of the osmanthus. The existing research shows that although the current sweet osmanthus varieties are various, at least the compositions of the characteristic components of the sweet osmanthus are not substantially different.

The Chinese invention patent application with the application number of CN201710420375.7 discloses an osmanthus flower extract-osmanthus flower absolute oil, and at present, the osmanthus flower extract is mainly used as spice or common skin care, and the application of the osmanthus flower extract in the preparation of the sun burn prevention and treatment medicine is not reported.

Disclosure of Invention

In order to solve the problems, the invention provides a new application of an osmanthus flower extract, namely an application of the osmanthus flower extract in preparing a medicine for preventing and treating sunburn. Based on the application, the osmanthus flower extract can be used as an active ingredient for treating sunburn skin diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the osmanthus flower extract in preparing the medicine for preventing and treating sunburn comprises the active component of the osmanthus flower extract, wherein the osmanthus flower extract is the osmanthus flower absolute disclosed in the Chinese invention patent application with the application number of CN 201710420375.7.

The medicine is medicinal product, medical apparatus or cosmetic.

The mass percentage concentration range of the osmanthus flower extract is 0.01-2%.

The administration mode of the medicine is external administration.

The dosage form of the medicine is gel, condensation, spray, application, ointment, cream, emulsion and aqua.

The medicine also comprises other auxiliary materials, and the auxiliary materials are auxiliary materials of corresponding formulations allowed to be used for preparing medicines, medical instruments or cosmetics.

Compared with the prior art, the invention has the beneficial effects that:

the osmanthus flower extract is used as an active substance for preventing and treating sunburn skin diseases, and the osmanthus flower extract is used as an active ingredient to be absorbed by skin, so that the immunity of a patient is improved, sunburn can be treated, various symptoms of sunburn in an acute stage can be obviously relieved, and the self-healing period is shortened. The osmanthus flower extract has a good immunoregulation effect, is used as an active ingredient of a medicine for treating sunburn, and achieves the purpose of treating sunburn by utilizing the immunoregulation effect after being absorbed by skin.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate only some, but not all, of the embodiments of the present invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.

Example 1

A medicine for preventing and treating sunburn is a spray, and is prepared from flos Osmanthi Fragrantis extract 0.01 wt%, PEG-40 hydrogenated castor oil 0.006 wt%, Tremella extract 0.05 wt%, phenoxyethanol 0.3 wt%, and pure water in balance. Adopting a conventional method, firstly taking the osmanthus flower extract and PEG-40 hydrogenated castor oil, and heating to 70-80 ℃ in a water bath to obtain a phase A; adding pure water into Tremella extract, swelling, adding phenoxyethanol, mixing, and heating in water bath to 70-80 deg.C to obtain phase B. Slowly adding phase B into phase A under stirring at 70-80 deg.C, stirring, standing, cooling to room temperature, and packaging into spray bottle to obtain spray. In application, the medicine is sprayed on affected parts.

Example 2

A medicine for preventing and treating sunburn, in particular to a dressing, the raw materials of the medicine are 0.05 percent of sweet osmanthus flower extract by mass, 0.006 percent of PEG-40 hydrogenated castor oil by mass, 5 percent of glycerin by mass, 0.2 percent of hyaluronic acid by mass, 0.3 percent of phenoxyethanol by mass and the balance of pure water. Adopting a conventional method, firstly taking the osmanthus flower extract and PEG-40 hydrogenated castor oil, and heating to 70-80 ℃ in a water bath to obtain a phase A; adding hyaluronic acid into pure water, swelling, adding glycerol and phenoxyethanol, mixing, and heating in water bath to 70-80 deg.C to obtain phase B. Slowly adding phase B into phase A under stirring at 70-80 deg.C, stirring, standing, and cooling to room temperature to obtain dressing solution. And (3) immersing the cut non-woven fabric pieces into the dressing liquid, and packaging the non-woven fabric pieces in pieces after the non-woven fabric pieces fully absorb the dressing liquid to obtain the dressing. The dressing can be applied to the affected part.

Example 3

A medicine for preventing and treating sunburn is specifically emulsion, wherein the mass percentage concentration of a sweet osmanthus flower extract is 0.1%, the mass percentage concentration of allantoin is 0.5%, the mass percentage concentration of glycerin is 5%, the mass percentage concentration of stearyl alcohol is 5%, the mass percentage concentration of squalane is 5%, the mass percentage concentration of self-emulsifying monoglycoside is 2%, the mass percentage concentration of phenoxyethanol is 0.4%, and the balance is pure water. Mixing water, allantoin and glycerol, dissolving, and heating to 70-80 deg.C to obtain phase A; mixing flos Osmanthi Fragrantis extract, stearyl alcohol, squalane and self-emulsifying monoglyceride, and heating to 70-80 deg.C for melting to obtain phase B; slowly adding phase A into phase B under stirring at 70-80 deg.C, emulsifying, homogenizing, and cooling to room temperature under stirring to obtain emulsion. The lotion can be applied to affected part.

Example 4

A medicine for preventing and treating sunburn is a water aqua, wherein the mass percentage concentration of a sweet osmanthus flower extract is 0.5%, the mass percentage concentration of glycerin is 5%, the mass percentage concentration of 1, 3-propylene glycol is 5%, the mass percentage concentration of D-panthenol is 0.5%, the mass percentage concentration of squalane is 5%, the mass percentage concentration of phenoxyethanol is 0.27%, the mass percentage concentration of ethylhexyl glycerin is 0.03%, the mass percentage concentration of self-emulsifying monoglyceride is 1%, the mass percentage concentration of tocopherol is 2%, and the balance is pure water. Heating flos Osmanthi Fragrantis extract, squalane, tocopherol, and self-emulsifying monoglycoside to 70-80 deg.C for melting to obtain phase A; heating glycerol, 1, 3-propylene glycol, D-panthenol, phenoxyethanol, ethylhexyl glycerol and pure water to 70-80 deg.C to obtain phase B. Slowly adding phase B into phase A under stirring at 70-80 deg.C, emulsifying, homogenizing, and cooling to room temperature under stirring to obtain aqua. The liquid is applied to the affected part.

Example 5

A medicine for preventing and treating sunburn, in particular to a gel, wherein the mass percentage concentration of an osmanthus flower extract is 1.0%, the mass percentage concentration of glycerin is 5%, the mass percentage concentration of 1, 3-propylene glycol is 5%, the mass percentage concentration of squalane is 5%, the mass percentage concentration of phenoxyethanol is 0.3%, the mass percentage concentration of carbomer is 0.2%, the mass percentage concentration of potassium hydroxide is 0.05%, the mass percentage concentration of potassium sorbate is 0.3%, and the balance is pure water. Mixing flos Osmanthi Fragrantis extract and squalane by conventional preparation method of gel, heating to 70-80 deg.C, melting, and mixing to obtain phase A; adding water into carbomer, swelling, adding glycerol, 1, 3-propylene glycol, and phenoxyethanol, mixing, and heating to 70-80 deg.C to obtain phase B; dissolving potassium hydroxide and potassium sorbate in water, and heating to 70-80 deg.C to obtain phase C. Adding phase A into phase B, mixing, maintaining at 70-80 deg.C under stirring, slowly adding phase C into phase AB until condensation is formed, and cooling to room temperature under slow stirring to obtain condensation. The gel can be applied to the affected part.

Example 6

A medicine for preventing and treating sunburn is a cream, wherein the mass percent concentration of a sweet osmanthus flower extract is 1.5%, the mass percent concentration of phenoxyethanol is 0.5%, the mass percent concentration of glycerin is 5%, the mass percent concentration of cetearyl alcohol is 6%, the mass percent concentration of carbomer is 0.2%, the mass percent concentration of lanolin is 5%, the mass percent concentration of self-emulsifying monoglyceride is 4%, the mass percent concentration of potassium hydroxide is 0.04%, and the balance is pure water. Mixing flos Osmanthi Fragrantis extract, cetostearyl alcohol, lanolin and self-emulsifying monoglyceride, and heating to 70-80 deg.C for melting to obtain phase A. Adding carbomer into pure water, swelling, adding phenoxyethanol and glycerol, mixing, and heating to 70-80 deg.C to obtain phase B; dissolving potassium hydroxide in appropriate amount of pure water, and heating to 70-80 deg.C to obtain phase C. Slowly adding phase B into phase A under stirring at 70-80 deg.C, emulsifying, homogenizing, adding phase C, stirring, and cooling to room temperature to obtain cream. The cream can be applied to affected part.

Example 7

A medicine for preventing and treating sunburn is a gel, wherein the mass percentage concentration of a sweet osmanthus flower extract is 2.0%, the mass percentage concentration of glycerin is 8%, the mass percentage concentration of carbomer is 6%, the mass percentage concentration of sodium hydroxide is 0.5%, the mass percentage concentration of potassium sorbate is 0.5%, and the balance is pure water. Dissolving flos Osmanthi Fragrantis extract in glycerol and pure water with same amount, and ultrasonically shaking to obtain glycerol phase; and additionally, dispersing carbomer into pure water, fully swelling and uniformly stirring to obtain a carbomer solution. And adding the glycerol phase dissolved with the sweet osmanthus flower extract into the carbomer solution, and fully and uniformly stirring to obtain the glycerol carbomer solution. Sodium hydroxide and potassium sorbate are dissolved in a suitable amount of water to obtain an alkaline liquid phase. Adding the alkali liquid phase into the glycerol carbomer solution under stirring, and continuously stirring until gel is formed. The gel preparation can be applied to affected part.

The function experiment is carried out on the medicine prepared by taking the osmanthus flower extract as the active ingredient.

First, the osmanthus flower extract is used for treating sunburn.

Subject: 40 cases of sunburn patients, the age of 3-50 years.

Diagnostic criteria: the sun is exposed to the sun, and the local skin after the sun is exposed to the sun has erythema, blister or edema, is conscious of burning and pain, and is in an acute stage.

Inclusion criteria were: patients who meet the diagnosis standard of sunburn, do not use any external medicine and have no other internal and external diseases and skin diseases.

Exclusion criteria: patients with exudation at the skin lesion; patients who cannot take medication regularly; patients who use other internal or external drugs during treatment.

The selected patients are randomly divided into osmanthus fragrans extract treatment groups and calamine lotion control groups, wherein each group comprises 20 cases. The patients in each group were comparable in age, sex, course of disease, and condition.

Methods of treatment.

Osmanthus flower extract treatment group: the emulsion with the mass percentage concentration of the osmanthus flower extract of 0.1 percent is used.

Calamine control group: calamine lotion (national drug standard H31022790, Shanghai good Huangpu pharmaceutical Co., Ltd.).

The using method comprises the following steps: the lotion and calamine lotion are applied to affected part for 4 times a day and 1 time every 3 hours.

Other requirements and evaluation time nodes: each group was simultaneously applied to the affected area by cold compress, and the efficacy was evaluated at 12 hours, 24 hours, and 48 hours after the first treatment.

And (5) judging the curative effect.

And (3) healing: the erythema, blister or edema completely subsides, and the burning and pain sensations disappear;

the effect is shown: the fading of erythema, blister or edema is more than or equal to 70 percent, and the burning feeling and the pain feeling are obviously relieved;

the method has the following advantages: the fading of erythema, blister or edema is more than or equal to 40 percent, and the burning feeling and the pain feeling are relieved;

and (4) invalidation: the erythema, blister or edema subsides by less than 40%, and burning and pain are not reduced.

The total significant efficiency (%) + the recovery rate (%) + the significant efficiency (%), while observing adverse reactions and tolerance.

Statistical treatment: the data were processed by Excel, using F-test, P < 0.05: the difference is significant; p is less than 0.01: there was a very significant difference.

And (5) experimental results.

A. The results of the comparison of the efficacy of the two groups of treatments are shown in Table 1.

TABLE 1 comparison of two groups of efficacy (after 48 hours, number of examples)

As can be seen from Table 1, the total significant efficiency of the patients after 48 hours of treatment with the Osmanthus fragrans extract reaches 90% (16 cases are cured, and 2 cases are significant). And only adopts the calamine lotion for treatment, the total effective rate is only 35 percent (2 cases are healed, 5 cases are effective). Therefore, the treatment effect of the preparation containing the osmanthus fragrans extract on sunburn is remarkably higher than that of a control group (p is less than 0.05).

B. Comparison of the effects of treatment at different time nodes after treatment (table 2).

TABLE 2 comparison of the treatment Effect at different time nodes after treatment (number of cases, percentage)

As can be seen from Table 2, the total significant efficiency of the patients at 12 hours after the first treatment of the osmanthus fragrans extract reaches 40% (3 patients are cured, 5 patients are significantly effective). And the total significant efficiency is only 15% at 12 hours after the first treatment of the sunburn by only adopting the calamine lotion. The total significant efficiency of the patient is 70% 24 hours after the first treatment of the osmanthus fragrans extract (10 cases are cured, and 4 cases are significant). And the total significant efficiency of only 25% at 24 hours after the first treatment of the sunburn by only adopting the calamine lotion (1 case is cured, and 4 cases are significant). It can be seen that the treatment effect of the preparation containing the osmanthus fragrans extract on sunburn is significantly higher than that of the control group at different time points (p is less than 0.05).

C. Adverse reaction

No serious adverse reactions associated and possibly associated with the inventive drug were found during the treatment period.

Second, experiment for improving organism immunity by osmanthus flower extract

In order to explore the principle of the osmanthus flower extract for treating sunburn, the invention also provides an experiment of regulating the organism immunity by the osmanthus flower extract, and specifically provides the experiment.

1 materials and methods.

1.1 sample: osmanthus fragrans (Thunb.) Lour.) flower extract is processed by the inventor and provided as yellow brown to brown semisolid at normal temperature.

1.2 Experimental reagents and instruments.

1.2.1 Primary reagents.

The main reagents used in this discovery are shown in table 3.

TABLE 3 Primary reagents

1.2.2 Main instruments.

The main instruments used in this finding are shown in Table 4.

TABLE 4 Main Instrument

Main instrument	Manufacturer of the product
		Electronic balance	Sartorius
High-speed table centrifuge	Changshan Xiang instrument centrifuge Instrument Co
		Low-temperature high-speed centrifuge	Thermo Scientific
723 Spectrophotometer	Sartorius
		Carbon dioxide incubator	Thermo Fisher science and technology, Inc., USA
Automatic enzyme label reading instrument	Thermo Fisher science and technology, Inc., USA
		Slide measure (precision 0.02mm)	Jiangxi tool works
BME microscope	Leica
		Vertical refrigerator	Chinese Haier
Flow cytometer	BIO-RAD
		24-hole culture plate	Nunc
96-well culture plate	Nunc
		Micro-syringe	Nunc
Micropore sample adding gun (device)	Eppendorf Co, Germany
		SW-CJ-IF type superclean bench	Suzhou Antai air technology of Jiangsu Sujing group Ltd

1.3 Experimental animals.

160 Kunming mice provided by the laboratory animal center of the Chinese medicinal academy of sciences of Sichuan province are full-female, have the weight of 18g-22g, and have the production license number as follows: SCXK 2018-19, SPF grade. The feed is sourced from Xiaohe science and technology limited company in the city of Bizhou, and the production license number is as follows: threo feed (2014) 03058. The experimental animal room is a barrier system, and the license number used by the experimental animal is as follows: SYXK 2016-. The temperature is 20-25 ℃, the relative humidity is 40-70%, and the light and shade alternating period is 12 h. During the feeding period, the animals can freely and fully take food and drink without interference, and the drinking water and the feed are in a sterile state.

2 experimental methods.

2.1 dose design and grouping.

Animals were acclimatized for 3 days and randomized into first, second, third and fourth groups of 40 animals each (see table 5), wherein: the first group was used for ConA-induced splenic lymphocyte transformation experiments in mice; the second group was used for antibody-producing cell experiments, serum hemolysin assay and delayed allergy experiments; the third group was used for mouse carbon clearance experiments; the fourth group was used for the analysis of the organ/body weight ratio and the Treg/Th17 ratio of splenocytes. Each group of mice is randomly divided into a solvent control group (Jinlongyu edible blend oil) and low, medium and high dose groups (50mg/kg BW, 150mg/kg BW and 450mg/kg BW) of osmanthus flower extracts according to the weight, the mice are orally gavaged once per day according to 10mL/kg BW for 30 days continuously, the weight is weighed once per week, and the gavage volume is adjusted according to the weight change.

TABLE 5 Experimental dose design and grouping of animals

2.2 organ/body weight ratio.

The day after the last gavage, the fourth group of mice were weighed, sacrificed by removing the cervical vertebrae, the spleen and thymus were rapidly removed under aseptic conditions, the external stains of the viscera were blotted with filter paper, and weighed with an electronic analytical balance. The corresponding organ coefficients are calculated as follows. The organ coefficient of the Osthin group of the Osmanthus fragrans extract is higher than that of the solvent control group, so that the experimental result can be judged to be positive.

Organ coefficient is organ weight (g)/body weight (g).

2.3 ConA-induced mouse lymphocyte transformation experiments (MTT method).

2.3.1 reagent preparation.

RPMI1640 complete medium: the RPMI1640 culture solution is sterilized by filtration, 10% calf serum, 1% glutamine (200mmol/L), penicillin (100U/mL), streptomycin (100. mu.g/L) and 5X 10-5 mol/L2-mercaptoethanol are added before use, and the pH is adjusted to 7.0-7.2 by using sterile 1mol/L HCl or 1mol/L NaOH, thus obtaining the complete culture solution.

ConA liquid: preparing 100 μ g/mL solution with double distilled water, filtering for sterilization, and storing in a low temperature refrigerator (-20 deg.C).

Sterile Hank's solution: sterile NaHCO 3.5% before use₃Adjusting pH to 7.2-7.4.

MTT solution: 5mg of MTT was dissolved in 1mL of PBS (pH7.2) and used as it is.

Acidic isopropanol solution: to 96mL of isopropanol was added 4mL of 1mol/L HCl and the mixture was prepared just before use.

2.3.2 spleen cell suspension preparation.

The day after the last gavage, the first group of mice were aseptically splenomed, ground with forceps in a dish containing sterile Hank's solution to make a cell suspension, filtered through a 200 mesh screen, washed 2 times with Hank's solution, centrifuged 10min (1000r/min) each time, and the pellet was suspended in 1mL of complete medium and counted by staining with talofen blue (above 95%). The cell concentration was adjusted to 3X 106 cells/mL.

2.3.3 lymphocyte proliferation response.

Adding each spleen cell suspension into 24-well culture plate in two wells, each well containing 1mL of solution, one well containing 75 μ of LCona solution, the other well as control, and adding 5% CO₂Incubate at 37 ℃ for 72 h. 4h before the culture is finished, gently sucking 0.7mL of supernatant in each hole, adding 0.7mL of RPMI1640 culture solution without calf serum, simultaneously adding 50 muL of MTT (5mg/mL) in each hole, continuing to culture for 4h, after the culture is finished, adding 1mL of acidic isopropanol in each hole to completely dissolve purple crystals, subpackaging the purple crystals to 96-hole culture plates, making 3 parallel holes in each hole, and measuring the optical density value at the wavelength of 570nm by using a 2010-type microplate reader.

2.3.4 determination of results.

The difference between the optical density of the ConA-added wells minus the optical density of the non-added wells represents the proliferative capacity of lymphocytes. The optical density difference value of the osmanthus flower extract group is higher than that of the solvent control group, and the result of the experiment can be judged to be positive.

2.4 delayed-type allergy (DTH, plantar thickening).

2.4.1 Sheep Red Blood Cell (SRBC) cell suspension preparation.

Sheep blood was collected and washed 3 times with physiological saline, and centrifuged (2000r/min) for 10min each time. The packed SRBC were made into 2% (v/v) cell suspension with physiological saline.

2.4.2 sensitization.

Mice were immunized intraperitoneally with 2% (v/v) SRBC, and each mouse was injected with 0.2mL (approximately 1X 108 SRBC).

2.4.2 Generation and measurement of DTH.

After 4 days of immunization, the thickness of the left hind foot plantar region was measured, followed by subcutaneous injection of 20% (v/v) SRBC at the measurement site, 20 μ L per mouse (about 1 × 108 SRBC), and the thickness of the left hind foot plantar region was measured at 24h after injection, three times at the same site, and the average value was taken.

2.4.3 determination of results.

The extent of DTH is expressed as the difference in plantar thickness (degree of swelling of the plantar surface) before and after the challenge. The thickness difference of the osmanthus flower extract group is higher than that of the solvent control group, and the result of the experiment can be judged to be positive.

2.5 detection of antibody-producing cells (Jerne modified slide method).

2.5.1 preparation of complement.

Collecting guinea pig blood, separating serum (at least 5 guinea pig mixed serum), adding 1mL packed SRBC into 5mL guinea pig serum, standing in refrigerator at 4 deg.C for 30min, shaking frequently, centrifuging to obtain supernatant, packaging, and storing at-70 deg.C; when used, the solution is diluted by SA buffer according to the ratio of 1: 15.

2.5.2 slide coating.

A thin layer of agarose (0.5g agarose in 100mL double distilled water) was brushed on the clean slide and dried for future use.

2.5.3 immunization of animals.

Each mouse was intraperitoneally injected with 0.2mL 2% (v/v) SRBC cell suspension, approximately 2X 108 SRBC.

2.5.4 spleen cell suspension preparation.

The cervical vertebra of a mouse immunized for 4-5 days is dislocated and killed, the spleen is taken out and placed in a dish containing Hank's liquid, the spleen is ground to prepare cell suspension, the cell suspension is filtered through a 200-mesh screen, centrifuged (1000r/min) for 10min, washed for 2 times by the Hank's liquid, finally the cells are suspended in 5mL of RPMI1640 culture solution, the staining count of the Taifenlan is higher than 95 percent, and the cell concentration is adjusted to be 5 multiplied by 106/mL to prepare spleen cell suspension.

2.5.5 determination of plaques.

Agarose is prepared into 1% aqueous solution, water bath is carried out, the temperature is 45 ℃, the aqueous solution is mixed with equivalent double concentration Hank's solution, the mixture is subpackaged into small test tubes, each tube is 0.5mL, 10% (prepared by SA buffer solution) of packed SRBC 50 muL is added into the tube, each 20 muL of spleen cell suspension is prepared into two parallel samples, after the two parallel samples are rapidly and uniformly mixed, the two parallel samples are poured onto agarose thin-layer glass slides, after agar is solidified, the glass slides are horizontally buckled on a slide rack and put into a carbon dioxide incubator for incubation for 1.5h, then complement is added into grooves of the slide rack, and the incubation is continued.

1.5h, counting the number of hemolytic plaques.

2.5.6 and judging the result.

Expressed as number of plaques/106 splenocytes. The number of the hemolytic plaques of the osmanthus flower extract group is higher than that of the solvent control group, so that the test result is judged to be positive.

2.6 serum hemolysin assay (hemagglutination).

2.6.1 immunization of animals and serum isolation.

Injecting 0.2mL SRBC (2% (v/v) into the abdominal cavity of each mouse for immunization, continuously performing intragastric administration for 5 days, removing eyeballs, taking blood from the eyeballs, placing the blood in a centrifugal tube, standing for 1h, centrifuging for 10min at 2000r/min, collecting serum, diluting the serum by using physiological saline in a multiple ratio manner, diluting 12 holes in each part, placing the serum with different dilutions in a blood coagulation plate with 100 mu L of each hole, adding 100 mu L SRBC suspension with 0.5% SRBC suspension, uniformly mixing, placing in a wet box at 37 ℃ for 3h, observing the result, and recording the coagulation degree of each hole.

2.6.2 determination of results.

The degree of serum aggregation is generally classified into 5-stage (0-IV) records, and the antibody product is calculated according to the following formula. The number of antibody products of the osmanthus flower extract group is higher than that of the solvent control group, and the experimental result can be judged to be positive.

Antibody level (S1+2S2+3S3 … … nSn)

In the formula, 1, 2 and 3 … … n represent indexes of double dilution, S represents the grade of agglutination degree, and the larger the antibody volume number, the higher the serum antibody.

Level 0: the red blood cells sink completely and are concentrated at the bottom of the hole to form a compact round point shape, and the liquid around the hole is clear;

stage I: most of the red blood cells are deposited at the bottom of the hole to form a round point shape, and a small amount of agglutinated red blood cells are arranged at the periphery of the hole;

II stage: the agglutinated red blood cells form a thin layer at the bottom of the hole, and a loose red spot can be obviously seen in the center;

grade III: uniformly spreading the agglutinated red blood cells at the bottom of the hole to form a thin layer, wherein a small red spot is invisible in the center;

stage IV: the agglutinated red blood cells spread evenly in a thin layer at the bottom of the well, the clumps sometimes being rolled up. And (5) calculating the volume of the antibody.

2.7 mouse carbon clearance test.

2.7.1 solution preparation.

Ink for injection: diluting India ink stock solution with physiological saline by 5 times;

Na₂CO₃solution: 0.1g of Na is taken₂CO₃Distilled water was added to 100 mL.

2.7.2 inject ink.

Diluted indian ink (at 0.1mL/10g) was injected intravenously from the mouse tail by body weight and the time was immediately counted after ink injection.

2.7.3.

20 μ L of blood was collected from the angular venous plexus at 2 and 10min after the injection of ink, and added to 2mL of Na₂CO₃In solution with Na₂CO₃The solution was used as a blank and the optical density value (OD) was determined at 600nm using 723 spectrophotometer.

After blood collection, mice were sacrificed, and livers and spleens were collected, blotted with filter paper to remove blood stains on the surfaces of the organs, and weighed separately.

2.7.4 and judging the result.

The ability of the mice to clear carbon is expressed as a phagocytic index. The phagocytic index a was calculated as follows. The number of antibody products of the osmanthus flower extract group is higher than that of the solvent control group, and the experimental result can be judged to be positive.

K＝(lgOD1－lgOD2)/(t2－t1)。

Phagocytosis index a ═ body weight/(liver weight + spleen weight) × K1/3.

2.8 data processing and result judgment.

The results of the experimental data are expressed as means ± standard deviation (x ± s), statistical analysis is performed using SPSS 25.0 software, and the mean test of multiple independent samples is performed using one-way ANOVA. Carrying out homogeneity of variance test before analysis, if the variances are uniform, calculating an F value, and when P is more than 0.05, indicating that the mean difference of each sample has no statistical significance; when P is less than 0.05, the difference of the mean values of all samples is statistically significant, and the Dunnett-t test is adopted to carry out pairwise comparison of each dosage group of the osmanthus flower extract and a solvent control group. If the variance is not uniform or the data is not normally distributed, the analysis is performed by a rank sum test.

The results of two experiments in the cellular immunity and the humoral immunity tests are positive, or the results of two dosage groups in any experiment are positive, so that the positive results of the corresponding immunity tests can be judged; the osmanthus flower extract has positive results in any two aspects of cellular immune function, humoral immune function and mononuclear-macrophage system carbon clearance function, and the osmanthus flower extract can be judged to have the function of enhancing the immunity.

3, experimental results.

3.1 Effect of Osmanthus flower extract on mouse body weight.

As can be seen from table 6, the initial weight balance of the first, second, third, and fourth groups of animals (P > 0.05); the initial body weights of animals in the first, second, third and fourth dose groups were substantially the same. The body weight of mice in each group tended to increase during the feeding period, but the body weight of mice in each group was not statistically increased in each dose during the entire experiment (P > 0.05). Therefore, the body weight of the experimental mice was not affected by the osmanthus flower extract.

Table 6. effect of osmanthus flower extract on mouse body weight (x ± s, n ═ 10)

Group of

Initial body weight

Body weight at week 1

Body weight at week 2

Body weight at week 3

Body weight at end

Weight gain

1-A

20.80±1.03

25.40±0.97

29.90±2.69

31.90±2.81

33.90±2.08

13.10±2.38

1-B

20.90±1.29

25.20±1.14

29.30±1.89

30.90±1.79

32.60±1.71

11.70±1.64

1-C

21.10±1.37

26.50±1.72

29.60±2.46

31.50±2.72

33.80±1.87

12.70±1.70

1-D

21.30±0.95

25.70±1.89

28.90±1.85

31.10±2.03

33.40±2.59

12.10±2.89

2-A

20.50±0.85

24.50±1.51

28.30±1.89

29.60±2.37

32.50±1.90

12.00±2.21

2-B

20.70±1.16

25.50±1.27

28.90±1.29

31.40±1.17

33.20±1.23

12.50±1.78

2-C

20.80±1.03

24.20±1.14

29.20±1.32

31.10±1.52

33.40±1.51

12.60±2.22

2-D

20.40±1.17

25.60±1.51

28.90±1.45

31.60±1.08

34.20±1.23

13.80±1.81

3-A

20.60±1.27

24.30±1.16

27.50±1.43

29.50±1.51

32.50±1.65

11.90±1.85

3-B

20.40±0.97

24.90±1.52

28.60±1.71

30.60±1.58

33.30±1.49

12.90±1.45

3-C

20.20±0.79

24.30±0.82

28.90±1.20

31.00±1.05

33.20±1.14

13.00±1.05

3-D

21.10±1.20

26.30±1.49

29.00±1.41

30.70±1.25

32.90±0.88

11.80±1.14

4-A

20.90±0.99

25.10±0.99

28.60±2.41

30.60±2.22

32.40±2.68

11.50±2.88

4-B

21.40±0.97

25.70±1.57

29.70±1.49

32.00±1.41

34.10±2.08

12.70±2.54

4-C

20.60±1.27

26.10±1.66

30.30±2.54

32.20±2.44

34.00±2.31

13.40±2.72

4-D

20.80±0.92

25.60±1.43

28.90±1.52

31.90±1.85

33.50±1.96

12.70±1.64

Note: in table 6, 1, 2, 3 and 4 are respectively a first group, a second group, a third group and a fourth group; A. b, C, D respectively represent a solvent control group, a low-dose group (50mg/kg BW) of the osmanthus flower extract, a medium-dose group (150mg/kg BW) of the osmanthus flower extract and a high-dose group (450mg/kg BW) of the osmanthus flower extract.

3.2 Effect of Osmanthus flower extract on mouse organ coefficient.

The thymus coefficient of mice in each dose group was increased compared to the solvent control group (F-8.440, P < 0.01); wherein, the thymus coefficients of the low, medium and high dose groups of the osmanthus flower extract are all higher than that of the solvent control group (P is less than 0.05 or P is less than 0.01), and the thymus coefficient of the high dose group of the osmanthus flower extract is 0.50 +/-0.08 (P is less than 0.01). Spleen coefficients were increased in mice of each dose group compared to the solvent control group (F ═ 4.710, P < 0.01); wherein the spleen coefficient of the osmanthus flower extract high-dose group is higher than that of the solvent control group (P is less than 0.05), and the spleen coefficient of the osmanthus flower extract high-dose group is 0.44 +/-0.08 (shown in Table 7). Therefore, after the osmanthus fragrans flower extract is perfused into a mouse, the organ coefficient of the mouse in each dose group can be effectively increased, and the osmanthus fragrans flower extract is prompted to have a protective effect on thymus and spleen.

Table 7 mouse organ coefficients (x ± s, n ═ 10)

Group of	Thymus coefficient (%)	Spleen factor (%)
			Solvent control group	0.32±0.05	0.28±0.04
Low-dose group of osmanthus flower extract	0.43±0.09*	0.31±0.05
			Middle dose group of osmanthus flower extract	0.46±0.08**	0.39±0.05
High-dose set of flos Osmanthi Fragrantis extract	0.50±0.08**	0.44±0.08*

Note: p <0.05 (compared to solvent control); p <0.01 (compared to solvent control).

3.3 Effect of Osmanthus fragrans flower extract on ConA-induced mouse lymphocyte transformation experiments.

According to the results of table 8, the difference in optical density of the mice in each dose group was increased as compared with the solvent control group (F ═ 5.991, P < 0.01); wherein, the difference of the optical density difference of the high-dose group in the osmanthus flower extract and the solvent control group has statistical significance (P is less than 0.05 or P is less than 0.01), and a certain dose-effect relationship is presented, and the optical density difference value of the osmanthus flower extract high-dose group is 0.30 +/-0.06 (P is less than 0.01). The osmanthus flower extract can improve the lymphocyte proliferation capacity of mice.

Table 8. effect of osmanthus flower extract on ConA-induced mouse lymphocyte transformation experiments (x ± s, n ═ 10)

Group of	Difference in optical density
		Solvent control group	0.19±0.03
Low-dose group of osmanthus flower extract	0.25±0.05
		Middle dose group of osmanthus flower extract	0.27±0.08*
High-dose set of flos Osmanthi Fragrantis extract	0.30±0.06**

Note: p <0.05 (compared to solvent control); p <0.01 (compared to solvent control).

3.4 Effect of Osmanthus flower extract on delayed allergy (DTH) in mice.

As shown in table 9, the thickness difference (mm) of each dose group mouse increased with the increase of the dose compared to the solvent control group (F-4.213, P < 0.05); the difference between the thickness difference of the high-dose group of the osmanthus flower extract and the thickness difference of the solvent control group is statistically significant (P is less than 0.01), and the thickness difference of the high-dose group of the osmanthus flower extract is 1.25 +/-0.16, so that the osmanthus flower extract has an enhancing effect on the delayed allergy of mice caused by sheep red blood cell attack within 24 hours.

Table 9. effect of osmanthus flower extract on delayed allergy (DTH) in mice (x ± s, n ═ 10)

Group of	Thickness difference (mm)
		Solvent control group	0.95±0.11
Low-dose group of osmanthus flower extract	1.10±0.17
		Middle dose group of osmanthus flower extract	1.14±0.19
High-dose set of flos Osmanthi Fragrantis extract	1.25±0.16**

Note: p <0.01 (compared to solvent control).

3.5 Effect of Osmanthus flower extract on antibody-producing cells.

As shown in table 10, the number of hemolytic plaques in each dose group of mice was increased compared to the solvent control group (F ═ 5.192, P < 0.01); wherein, the ratio of the number of the hemolytic plaques of the high-dose group in the osmanthus fragrans extract to the number of the hemolytic plaques of the solvent control group is different (P is less than 0.05 or P is less than 0.01), and the number of the hemolytic plaques of the high-dose group of the osmanthus fragrans extract reaches 298.75 +/-43.40 (P is less than 0.01), which shows that the osmanthus fragrans extract can increase the number of antibody-producing cells.

TABLE 10 Effect of Osmanthus flower extract on antibody-producing cells (x. + -. s, n ═ 10)

Group of	Number of hemolytic plaques/106 splenocytes
		Solvent control group	240.63±27.70
Low-dose group of osmanthus flower extract	261.88±17.51
		Middle dose group of osmanthus flower extract	283.13±31.50*
High-dose set of flos Osmanthi Fragrantis extract	298.75±43.40**

Note: p <0.05 (compared to solvent control); p <0.01 (compared to solvent control).

3.6 Effect of Osmanthus flower extract on mouse serum hemolysin.

As shown in table 11, although the number of antibody pools was increased to some extent in mice in each dose group of the osmanthus fragrans extract compared with the solvent control group, the difference was not statistically significant ((F ═ 2.204, P > 0.05).

Table 11. effect of osmanthus flower extract on mouse serum hemolysin (x ± s, n ═ 10)

3.7 Effect of Osmanthus fragrans flower extract on mouse monocyte-macrophage carbon clearance function.

The osmanthus flower extract can enhance the capability of mononuclear-macrophages of mice to remove carbon in vivo and improve the phagocytic function (F is 3.901, P is less than 0.05). Compared with the solvent control group, the phagocytic index a of the high-dose group of the osmanthus fragrans flower extract is increased (P is less than 0.05), and the value is 6.80 +/-0.54, which indicates that the osmanthus fragrans flower extract has a promoting effect on the carbon clearance function of a mouse mononuclear-macrophage system. The results are shown in Table 12.

Table 12. influence of osmanthus flower extract on mouse monocyte-macrophage carbon clearance function (x ± s, n ═ 10)

Group of	Phagocytic index a
		Solvent control group	5.86±0.55
Low-dose group of osmanthus flower extract	6.01±0.73
		Middle dose group of osmanthus flower extract	6.65±0.82
High-dose set of flos Osmanthi Fragrantis extract	6.80±0.54*

Note: p <0.05 (compared to solvent control).

The above description is only a preferred embodiment of the present invention, and certainly should not be taken as limiting the scope of the present invention, which is therefore intended to be covered by the appended claims.

Claims (5)

1. The application of the osmanthus flower extract in preparing the medicine for preventing and treating sunburn is characterized in that the active ingredient of the medicine is the osmanthus flower extract, the osmanthus flower extract is osmanthus flower absolute, the sunburn, also called Solar Dermatitis (Solar Dermatitis) or Solar Dermatitis, is an acute inflammatory reaction generated after normal skin is exposed to the sun, and the administration mode of the medicine is external administration.

2. The use of the osmanthus flower extract according to claim 1 in the preparation of a medicament for preventing and treating sunburn, wherein the medicament can relieve various symptoms in the acute stage of sunburn and shorten the skin repair time.

3. The application of the osmanthus flower extract in preparing the medicine for preventing and treating sunburn according to claim 1, wherein the mass percentage concentration range of the osmanthus flower extract is 0.01-2%.

4. The application of the osmanthus flower extract in preparing the medicine for preventing and treating sunburn according to claim 1, wherein the dosage form of the medicine is gel, spray, application, ointment, cream, lotion or aqua.

5. The application of the osmanthus flower extract in preparing the medicine for preventing and treating sunburn according to claim 1, wherein the medicine further comprises an auxiliary material, and the auxiliary material is an auxiliary material of a corresponding dosage form allowed by preparing the medicine.