CN113041286B - Plant anti-allergic agent for skin - Google Patents
Plant anti-allergic agent for skin Download PDFInfo
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- CN113041286B CN113041286B CN201911373890.XA CN201911373890A CN113041286B CN 113041286 B CN113041286 B CN 113041286B CN 201911373890 A CN201911373890 A CN 201911373890A CN 113041286 B CN113041286 B CN 113041286B
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Abstract
The invention provides a composition of plant extracts for resisting skin allergy, which contains extracts of gentian, tetrandra root and centella. The invention also provides a method for preparing the plant extract composition. The plant extract composition of the present invention can be used for skin care, especially for preventing and relieving skin inflammation, allergy and itching pain. The invention also relates to a pharmaceutical or cosmetic composition containing the plant extract composition as an anti-allergy agent.
Description
Technical Field
The invention relates to a composition of plant extracts with skin allergy resistance. The plant extract composition of the present invention contains extracts of gentian, tetrandra and centella asiatica, and is useful for skin care, particularly for preventing and relieving skin inflammation, allergy and itching pain. The invention also relates to a preparation method of the composition of the plant extract and application of the composition in medicines or cosmetics, and further relates to the medicines or cosmetics containing the composition of the plant extract.
Background
With the increasingly poor environment, the excessive use of poor cosmetics and the like, the skin proportion of problems is higher and higher, such as inflammation, red swelling, allergy, chapping, itching and pain and the like, and how to solve the problems of the skin has great practical significance. The current research begins to focus on the field of safer traditional Chinese medicines, but many commercially available anti-skin allergy agents (particularly cosmetics) have the problems that the efficacy does not achieve the expected purpose, the product is unstable, and great troubles are brought to users. Natural anti-skin allergy agents with definite effects, safety and no adverse reactions are highly needed.
Disclosure of Invention
The invention provides a powerful composition of plant extracts for resisting skin allergy, which contains extracts of gentian, tetrandra and centella, wherein the extracts can be prepared by extracting gentian, tetrandra and centella as raw materials together, or by extracting gentian, tetrandra and centella respectively and then mixing the extracts.
The plant extract composition of the present invention can be used for skin care, especially for preventing and relieving skin inflammation, allergy and itching pain. The invention also provides the application of the composition in medicines or cosmetics, and medicines or cosmetics containing the plant extract composition as an anti-allergy agent. The product prepared according to the invention has strong synergistic anti-inflammatory, antiallergic and anti-irritant effects, and is stable, safe and free of side effects.
In a further preferred embodiment, the present invention provides a method of preparing a composition comprising a plant extract as an anti-sensitiser. Preferably, the composition prepared by the method has the characteristics of high content of effective components, stable effect, high product yield and the like.
Detailed Description
In an embodiment according to the present invention, the present invention provides a composition of anti-skin allergy plant extracts with potent synergistic effect, the composition comprising the plant extracts of gentian, tetrandra root and centella asiatica, preferably the content of each plant extract is: 30-70% of gentian, 10-40% of radix stephaniae tetrandrae and 10-40% of centella asiatica; more preferably, 40-65% of gentian, 15-30% of radix stephaniae tetrandrae and 15-30% of centella asiatica. The compositions of the present invention have a significant synergistic effect compared to the same concentration of extracts of one or both plants.
The invention also provides a method for preparing the plant extract composition. The composition can be prepared by extracting the gentian, the tetrandra root and the centella asiatica plant materials together according to the proportion, or by extracting the gentian, the tetrandra root and the centella asiatica plant materials respectively and then mixing the extracts, wherein the proportion of each plant material is about: 30-70% of gentian, 10-40% of radix stephaniae tetrandrae and 10-40% of centella asiatica; preferably, 40-65% of gentian, 15-30% of radix stephaniae tetrandrae and 15-30% of centella asiatica. Or wherein the weight ratio between the plant materials is about: 3-7 parts of gentian: 1-4 of radix stephaniae tetrandrae: 1-4 parts of centella asiatica; preferably, gentian 4-6: 1-3 of radix stephaniae tetrandrae: 1-3 parts of centella asiatica. It should be noted that the percentage of each plant extract in the present invention may be based on the dry weight of the composition, but solutions containing the extract are often used in the preparation process.
In an embodiment according to the invention, the extract of the invention is a water and/or C2-6 alcohol extract and the solvent for extraction is water, C2-6 alcohol or a combination thereof. The C2-6 alcohol is preferably ethanol, propanol, propylene glycol, n-butanol, or a combination thereof, more preferably propylene glycol and ethanol. Preferably, the extract composition is obtained by extracting the pulverized plant material with water, extracting with 20% -70% aqueous solution of C2-6 alcohol, and mixing the two extracts. In a further preferred embodiment, the extract composition is obtained by first extracting comminuted plant material with water, then with a 20% to 50% aqueous solution of a C2-6 alcohol, further with a 50% to 70% aqueous solution of a C2-6 alcohol, and mixing the three extracts.
More preferably, the extract composition is obtained by extracting the comminuted plant material with water, then with a 20% to 70% aqueous solution of propylene glycol and/or ethanol, and then mixing the two extracts. In a further preferred embodiment, the extract composition is obtained by first extracting the comminuted plant material with water, then with an aqueous solution of 20% to 50% propylene glycol and/or ethanol, and further with an aqueous solution of 50% to 70% propylene glycol and/or ethanol, and mixing the three extraction liquids.
In a further embodiment, the above-obtained extract composition is subjected to membrane filtration for purification, and a concentrated solution is obtained. Preferably, a 5000-; optionally, a nano-filtration membrane of 100-.
In a preferred embodiment, the composition of plant extracts of the present invention is prepared according to the following method:
1. weighing plant materials, wherein the plant materials comprise gentian, tetrandra and centella asiatica, and the ratio of each plant material is as follows in percentage by weight: 30-70% of gentian, 10-40% of radix stephaniae tetrandrae and 10-40% of centella asiatica; preferably, 40-65% of gentian, 15-30% of radix stephaniae tetrandrae and 15-30% of centella asiatica are subjected to crushing; or wherein the weight ratio between the plant materials or plant extracts is about: 3-7 parts of gentian: 1-4 of radix stephaniae tetrandrae: 1-4 parts of centella asiatica; preferably, gentian 4-6: 1-3 of radix stephaniae tetrandrae: 1-3 parts of centella asiatica;
2. extracting with water at a temperature of preferably about 50-100 deg.C, more preferably about 60-80 deg.C; the extraction time is preferably 0.5-2 h; removing the residue to obtain extractive solution A1;
3. further extracting the residue of step 2 with about 20% -50% aqueous solution of C2-6 alcohol or a combination thereof as solvent; the extraction temperature is preferably about 40-85 deg.C, more preferably about 50-75 deg.C; extracting for about 0.5-2 hr, removing residue to obtain extractive solution A2;
4. optionally, further extracting the residue of step 3 with about 50% -70% aqueous solution of C2-6 alcohol or a combination thereof as solvent; the extraction temperature is preferably about 40-85 deg.C; extracting for about 0.5-1.5 hr, removing residue to obtain extractive solution A3;
5. combining extracts a1 and a2, and, optionally, extract A3;
6. filtering with a filter membrane, preferably adopting an ultrafiltration membrane with the concentration of about 5000-;
7. optionally, the concentrated solution is obtained by further filtering with a nanofiltration membrane of about 100 and 1000 daltons and recovering the solvent.
In a further preferred embodiment, the present invention relates to a process for the preparation of a composition of plant extracts comprising the steps of:
1. weighing plant materials, pulverizing the plant materials into 10-50 mesh coarse powder, preferably 10-20 mesh coarse powder;
2. extracting with water, preferably about 3-8 times the amount of water, more preferably about 5-6 times the amount of water; the extraction temperature is preferably about 50-100 deg.C, more preferably about 60-80 deg.C; the extraction time is preferably 0.5-2h, more preferably 0.5-1 h;
preferably, an external circulation extraction method is adopted, an online spectrophotometer is adopted for monitoring, the end point is judged according to the absorbance, the specific range is that the absorbance value of a sample solution after ten times of dilution at the wavelength of 240nm is more than or equal to 1.3, the absorbance at the wavelength of 550nm is less than or equal to 1.6, and residues are removed at the end point to obtain an extracting solution A1;
3. further extracting the residue of step 2 with about 20% -50% aqueous solution of C2-6 alcohol or a combination thereof as solvent; preferably about 2-5 times the amount of solvent is added, the extraction temperature is preferably 40-85 deg.C, preferably 50-75 deg.C; the extraction temperature can be adjusted according to the boiling point of the solvent, and if the boiling point of the solvent is higher, the extraction temperature can be properly increased; the extraction time is preferably about 0.5 to 2 hours, more preferably about 1 to 1.5 hours; preferably about 30% to 45% aqueous solution of a C2-6 alcohol or a combination thereof; in a still further preferred embodiment, about 30-45% propylene glycol, ethanol, or a combination thereof is used for extraction; removing the residue to obtain extractive solution A3;
preferably, an external circulation extraction method is adopted, an online spectrophotometer is adopted for monitoring, the end point is judged according to the absorbance, the specific range is that the absorbance value of a sample solution after ten times of dilution at the wavelength of 280nm is more than or equal to 1.5, the absorbance at the wavelength of 550nm is less than or equal to 1.4, residues are removed at the end point, and an extracting solution A2 is obtained;
4. optionally, further extracting the residue of step 3 with 50% -70% C2-6 alcohol aqueous solution or their combination as solvent; preferably, 2-5 times the amount of solvent is added, preferably at about 40-85 deg.C, more preferably about 50-75 deg.C; the extraction temperature can be adjusted according to the boiling point of the solvent, and if the boiling point of the solvent is higher, the extraction temperature can be properly increased; the extraction time is preferably about 0.5-1.5 h; preferably about 50% to 70% of an aqueous solution of a C2-6 alcohol or a combination thereof; in a still further preferred embodiment, about 50-70% propylene glycol, ethanol, or a combination thereof is used for extraction; removing the residue to obtain extractive solution A3;
preferably, an external circulation extraction method is adopted, an online spectrophotometer is adopted for monitoring, the end point is judged according to the absorbance, the specific range is that the absorbance value of the sample liquid after ten times of dilution at the wavelength of 280nm is more than or equal to 1.0, the absorbance at the wavelength of 550nm is less than or equal to 1.2, and the residue is removed at that time to obtain the extracting solution A3.
5. Combining extracts a1 and a2, and, optionally, extract A3;
6. filtering with a filter membrane, preferably adopting an ultrafiltration membrane with the temperature of about 5000-; collecting the filtrate;
7. optionally, the concentrated solution is obtained by further filtering with a nanofiltration membrane of about 100 and 1000 daltons and recovering the solvent. Optionally, adjusting the labeled concentration of the extract to 100% with water, namely: the weight of the extract was adjusted to correspond to the weight of the plant material originally used.
Marked concentration of extractive solution is 100% of weight of plant material/weight of final extractive solution
In a preferred embodiment, the preparation method adopts an external circulation extraction method, improves the extraction rate of the effective components through gradient extraction, and reduces the use of solvents and the dissolution of impurities; in addition, the on-line spectrophotometer is adopted to monitor the dissolution of the effective components and impurities in real time, so that the end point of the extraction step is controlled, the component composition and the content of the extract are more accurately controlled, and the stability of the product is further controlled.
The plant extract composition prepared by the method contains a plurality of compound plant active ingredients, mainly comprising gentiopicroside, tetrandrine and asiaticoside.
The invention also relates to a plant extract composition which comprises gentiopicroside, tetrandrine and asiaticoside; preferably, the weight ratio of the components is about: 3-7 parts of gentiopicroside: 1-4 parts of hanfangchin A: 1-4 parts of asiaticoside; preferably, gentiopicroside 4-6: 1-3 of sweatsiangchinoline: 1-3 parts of total asiaticoside.
The composition of extracts containing three plants of gentian, tetrandra and centella asiatica and the medicine or cosmetic containing the composition according to the present invention have unexpected effects, especially have strong synergistic anti-inflammatory, antiallergic and anti-irritant effects, and the composition containing three plant extracts of the present invention has significantly improved effects and better synergistic effects compared with the composition containing only one or two plant extracts.
Alternatively, individual plant materials may be separately extracted to obtain extracts, and the resulting extracts may then be combined to obtain the extract composition of the present invention. The plant material may be extracted using known extraction methods, but preferably, the extraction is performed using the methods described in the present specification. The product prepared by the method is more stable and safe and has no side effect.
The invention also relates to the use of a composition containing extracts of three plants, namely gentian, tetrandra and centella asiatica, for preparing a therapeutic or prophylactic medicament or cosmetic product which can be used for antiallergic, anti-inflammatory, redness-relieving, itching-relieving, anti-irritant or skin-repairing purposes. Tests prove that the composition of the plant extracts has excellent antiallergic, anti-inflammatory, redness relieving, itching relieving, irritation resisting and repairing effects on skin, and can be used for preparing cosmetics for skin care and therapeutic or preventive medicines; the composition has definite effect, good safety, no side effect and stability in cosmetics and pharmaceutical preparations; compared with the existing like products, the effect is more obvious, and the safety is better.
The composition containing the plant extract can be widely used for preparing medicines or cosmetics, and the medicines or cosmetics can be creams, lotions, pastes, ointments, facial masks, gels or lotions and the like. Can be prepared by mixing the above materials according to conventional method. The content of the plant extract composition in the pharmaceutical or cosmetic may be, for example, about 0.1 to 20%, preferably 2 to 15%, more preferably 5 to 10%, by weight, of the plant extract solution having a concentration of 100% as indicated in the pharmaceutical or cosmetic. The concentration can be adjusted by those skilled in the art according to the actual situation and need. The pharmaceutical or cosmetic of the invention may also contain one or more other ingredients such as other plant extracts, nutritional additives, surfactants, fragrances and perfumes, pigments, preservatives, antioxidants, humectants, ultraviolet absorbers, astringents, penetration aids, pH regulators, and the like. The skilled person will be able to select them on the basis of their general knowledge and specific needs.
Drawings
FIG. 1: shows the experimental results of various test samples for inhibiting the inflammatory factor IL-8;
FIG. 2: shows the experimental results of various test samples for inhibiting the inflammatory factor NF-kB;
FIG. 3: shows the inhibition effect of various test samples on mouse itching caused by dextran;
FIG. 4: the efficacy of the various test samples in soothing the degree of redness is shown.
Detailed description of the preferred embodiments
For simplicity, in the following specific examples of the invention, the external circulation extraction method is used and the endpoint is determined based on-line spectrophotometric monitoring as described above.
Example 1
Taking 1000g of plant materials, wherein 50% of gentian, 25% of stephania tetrandra and 25% of centella asiatica are crushed into 10-mesh coarse powder, adding about 6 times of water for external circulation extraction, and extracting at the temperature of about 60 ℃ to obtain an extracting solution A1; adding about 3 times of about 40% propylene glycol into the residue, and performing external circulation extraction at about 80 deg.C to obtain extractive solution A2; adding about 2 times of about 70% propylene glycol into the residue, and performing external circulation extraction at about 80 deg.C to obtain extractive solution A3. Mixing A1, A2 and A3, filtering with 10000 Dalton ultrafiltration membrane, and collecting filtrate; filtering with 100 dalton nanometer filter membrane, recovering solvent to obtain concentrated solution, and adjusting with water to 1000ml to obtain 100% extractive solution.
Example 2
Taking 1000g of plant materials, wherein 45% of gentian, 30% of stephania tetrandra and 25% of centella asiatica are crushed into 20-mesh coarse powder, adding about 5 times of water for external circulation extraction, and extracting at the temperature of about 80 ℃ to obtain an extracting solution A1; adding about 3 times of about 40% ethanol into the residue, and performing external circulation extraction at about 60 deg.C to obtain extractive solution A2; adding about 2 times of about 70% ethanol into the residue, and performing external circulation extraction at about 60 deg.C to obtain extractive solution A3. Mixing A1, A2 and A3, filtering with 5000 dalton ultrafiltration membrane, and collecting filtrate; filtering with 1000 Dalton nanofiltration membrane, recovering solvent to obtain concentrated solution, and adjusting with water to 1000ml to obtain 100% extractive solution.
Example 3
Taking 1000g of plant materials, wherein the gentian 60%, the tetrandra root 25% and the centella asiatica 15% are crushed into 20-mesh coarse powder, adding about 5 times of water for external circulation extraction, and extracting at the temperature of about 70 ℃ to obtain an extracting solution A1; adding about 4 times of mixed solvent of 25% ethanol and 25% propylene glycol at a ratio of 1:1 into the residue, and performing external circulation extraction at 70 deg.C to obtain extractive solution A2; adding about 3 times of mixed solvent of 70% ethanol and 70% propylene glycol at a ratio of 1:1 into the residue, and performing external circulation extraction at 50 deg.C to obtain extractive solution A3. Mixing A1, A2 and A3, filtering with 8000 Dalton ultrafiltration membrane, and collecting filtrate; filtering with 500 Dalton nanometer filter membrane, recovering solvent to obtain concentrated solution, and adjusting with water to 1000ml to obtain 100% extractive solution.
Comparative example 1
1000g of the plant material, of which 60% of gentian and 40% of fangji, was extracted by a method similar to that of example 3.
Comparative example 2
1000g of the plant material, including 60% Gentiana scabra and 40% centella asiatica, was extracted in a similar manner to comparative example 1.
Comparative example 3
1000g of the plant material, wherein 50% of Fangji root and 50% of Centella asiatica were extracted by the method similar to that of comparative example 1.
Comparative example 4
1000g of gentian plant material was taken and extracted in a similar manner to comparative example 1.
Comparative example 5
1000g of the plant material of Stephania tetrandra was taken and extracted by a method similar to that of comparative example 1.
Comparative example 6
Centella asiatica plant material 1000g was taken and extracted in a similar manner to comparative example 1.
Comparison of physicochemical experiments
| Stability at 48 ℃ | Stability at 4 deg.C | Colour(s) | |
| Example 1 | 3 months old | 6 areMoon cake | Light brown |
| Example 2 | 3 months old | 6 months old | Light brown |
| Example 3 | 3 months old | 6 months old | Light brown |
| Comparative example 1 | 3 months old | 6 months old | Light brown |
| Comparative example 2 | 1 |
2 months old | Brown colour |
| Comparative example 3 | 1 |
2 months old | Brown colour |
| Comparative example 4 | 1 |
2 months old | Light brown |
| Comparative example 5 | 1 |
2 months old | Light brown |
| Comparative example 6 | 1 |
2 months old | Light brown |
As can be seen from the comparison in the table, the product obtained by the extraction method of the invention has high stability, high consistency of the content of the extract and lighter color.
The total active ingredient content is the total content of gentiopicroside, tetrandrine and asiaticoside.
The content testing method of gentiopicroside, tetrandrine and asiaticoside is carried out according to the first part of pharmacopoeia 2015 of the people's republic of China.
Second, efficacy test comparative experiment 1
2.1. Preparing a reagent:
5% of each sample tested in the examples: 5% (w/w) of each sample to be tested in examples were prepared by adding 5g of the 100% labeled extract prepared in examples 1 to 3 to 95g of physiological saline.
5% of each sample tested in comparative example: 5% (w/w) of each sample to be tested in comparative example was prepared by adding 5g of the extract solution having the concentration indicated as 100% prepared in comparative examples 1 to 6 to 95g of physiological saline.
2.2. Test method
2.2.1 anti-irritation test
5% sodium dodecyl sulfate was used as a model group, hydrocortisone cream (specification: 100g/10mg) was used as a positive control group, and the dose was 0.1 g/mouse. Physiological saline was used as a negative control sample (negative control modeling group), and the experimental sample group was 5% of the test samples of each example or comparative example. Samples of an experimental group, a positive control group and a negative control group are respectively mixed with 5% of sodium dodecyl sulfate, and the mixture is smeared and observed according to a multiple skin irritation test method of technical Specification for cosmetic safety 2015, wherein each group comprises 6 guinea pigs and male and female halves. The red and swollen state of the applied part is observed. The red and swollen are 4-5 points, the slight red and swollen are 1-3 points, and the unchanged point is 0 point.
2.2.2 anti-allergy test: dextran method
Healthy mice with the weight of 18-22 g are divided into halves and males at random according to the sex and the weight, and each group contains 10 mice. The animal was depilated with 1% sodium sulfide, the area of the depilated skin region was 1.5cm by 1.5cm, and used for the experiment after 2 days. The hair-removed area of the back of each group of animals is respectively and locally coated with a sample to be tested (1 ml/kg). Hydrocortisone cream (specification: 100g/10mg) was used as a positive control group at a dose of 0.1 g/mouse. Saline was used as a negative control sample (negative control model), and the experimental sample group was 5% of the extract prepared in example or comparative example. For 5 consecutive days, each mouse was injected with dextran 0.95mg/kg in the tail vein 30min after the last administration. Taking the head of the mouse scratched with the front paw, the trunk of the mouse with the back paw and the whole body bitten by the mouth as itching indications, and recording the itching times of the mouse within 30 minutes.
The pruritus inhibition rate is (pruritus times of the negative control group-pruritus times of the sample group) ÷ pruritus times of the negative control group 100%.
2.2.3 anti-inflammatory assay: experiment for mouse auricle swelling caused by xylene
Healthy mice with the weight of 18-22 g are divided into halves and males at random according to the sex and the weight, and each group contains 10 mice. The right ear of the mouse was smeared with each corresponding group of samples to be tested, 0.2 ml/mouse, once a day for 3 consecutive days. Hydrocortisone cream (specification: 100g/10mg) was used as a positive control group at a dose of 0.1 g/mouse. Saline was used as a negative control sample (negative control model), and the experimental sample group was 5% of the extract prepared in example or comparative example. 30min after the last application, 0.02 ml/mouse left ear was left without any treatment, the animals were sacrificed 4 hours later, both ears were immediately cut off, the same part of the ear was taken with an 8mm punch, and the ear was weighed on an analytical balance, and the swelling degree was determined by subtracting the weight of the left ear from the weight of the right ear, and the swelling rate was determined by (weight of right ear-weight of left ear)/weight of left ear × 100%.
Results of the experiment
| Group of | Irritation of Guinea pig | The pruritus inhibition rate% | The rate of ear swelling% |
| Hydrocortisone cream group | 0.3 | 76.5 | 7.8 |
| Negative control group | 4.5 | / | 6.9 |
| Example 1 | 0.3 | 75.4 | 12.1 |
| Example 2 | 0.4 | 75.5 | 13.5 |
| Example 3 | 0.3 | 74.8 | 11.6 |
| Comparative example 1 | 0.8 | 44.9 | 16.9 |
| Comparative example 2 | 1.0 | 57.6 | 17.4 |
| Comparative example 3 | 1.5 | 40.3 | 18.9 |
| Comparative example 4 | 1.7 | 27.1 | 19.2 |
| Comparative example 5 | 2.2 | 11.3 | 19.4 |
| Comparative example 6 | 2.9 | 24.7 | 20.7 |
From the results in the table, the technical scheme of the invention has obvious improvement on the anti-irritation, anti-allergy and anti-inflammatory effects.
Third, efficacy test contrast experiment 2
3.1. Experimental report of the plant extract for inhibiting the inflammatory factor IL-8
3.1.1. Experiment reagent and equipment
N-Hacat cells, DMEM culture Solution (SIGMA), serum, an incubator (Sammerfei), a fluorescence microplate reader (MEUG MD), an IL-8 detection kit (SIGMA), cell lysate, DPBS, PBS, pancreatin (SIGMA) and a 96-well plate.
3.1.2. Sample to be tested
Test samples of example 3: the sample concentration was 0.01%, 0.05%, 0.1%, 0.5% (v/v). An appropriate amount of the extract solution having the concentration indicated as 100% prepared in example 3 was added to a DMEM culture solution to prepare a sample having the above volume concentration.
The following comparative examples were prepared in the same manner as the test samples. Comparative example 5 (stephania tetrandra): the sample concentration is 0.01%, 0.05%, 0.1%, 0.5%
Comparative example 6 (centella asiatica): the sample concentration is 0.01%, 0.05%, 0.1%, 0.5%
Reagent: epigallocatechin gallate (EGCG, 50uM), TNF-alpha (10ng/ml)
3.1.3. Experimental methods
Taking out cultured N-Hacat cells from the incubator, digesting and centrifuging to obtain suspension, and adjusting cell density to 1 × 105One/ml to 5 x 105And (4) inoculating 135 mu l of the culture medium into a 96-well plate per ml, and continuing to culture for 12-24 h. After the culture, 15. mu.l of each prepared sample to be tested (the mixed extract, the tetrandra root extract, the centella asiatica extract and the EGCG in example 3, and phosphate buffered saline PBS is used as a negative control) with each concentration is added into the pore plate, after the culture is continued for 12 to 24 hours, 16.5. mu.l of TNF-alpha is added into each pore of the pore plate (16.5. mu.l of PBS is added into each pore of the control group). Culturing for 12-24h, collecting culture solution after culturing, detecting with IL-8 detection kit (purchased from SIGMA), and placing into fluorescence enzyme labeling instrument to read at OD450 nm.
3.1.4. Results of the experiment
The experimental results show that the centella asiatica extract, the tetrandra root extract and the three plant mixed extracts (compound solutions) in the embodiment 3 can reduce the concentration of the inflammatory factor IL-8, wherein the compound solutions have enhanced inhibition effect on the inflammatory factor IL-8 along with the increase of the solution concentration and show dose dependence, and the inhibition effect on the inflammatory factor IL-8 is stronger than that of the centella asiatica extract and the tetrandra root extract under the highest concentration of the current determination, which shows that the compound solutions have stronger anti-inflammatory effect.
For specific experimental data, see fig. 1.
3.2. Experimental report for inhibiting inflammatory factors NF-kB by using plant extract
3.2.1. Experiment reagent and equipment
N-Hacat cells, DMEM culture fluid (SIGMA), serum, incubator (sermer fly), luciferase reader (metgu MD), luciferase assay kit (purchased from shanghai yiying biotechnology limited), cell lysate (obtained from luciferase assay kit), DPBS, PBS, pancreatin (SIGMA), 96-well plate.
3.2.2. Sample to be tested
Test samples of example 3: the sample concentration was 0.01%, 0.05%, 0.1%, 0.5% (v/v). An appropriate amount of the extract solution labeled with 100% concentration prepared in example 3 was added to a DMEM culture solution to prepare a sample to be measured having the above volume concentration.
The following comparative examples were prepared in the same manner as the test samples.
| Sample (I) | Composition (I) | Test concentration |
| Example 3 | Compound solution | 0.1％，0.5％，1％ |
| Comparative example 1 | Radix Gentianae and radix Stephaniae Tetrandrae | 0.1％，0.5％，1％ |
| Comparative example 2 | Radix Gentianae and herba Centellae | 0.1％，0.5％，1％ |
| Comparative example 3 | Radix Stephaniae Tetrandrae and herba Centellae | 0.1％，0.5％，1％ |
| Comparative example 4 | Radix Gentianae | 0.1％，0.5％，1％ |
| Comparative example 5 | Root of fangji | 0.1％，0.5％，1％ |
| Comparative example 6 | Centella asiatica | 0.1％，0.5％，1％ |
Reagent: epigallocatechin gallate (EGCG, 50uM), TNF-alpha (10ng/ml)
3.2.3. Experimental methods
Taking out cultured N-Hacat cells from the incubator, digesting and centrifuging to obtain suspension, and adjusting cell density to 1 × 105One/ml to 5 x 105One/ml, 1 per well35 μ l, inoculated into 96-well plates, and cultured for 12-24 h. After the culture, 15 μ l of pre-prepared samples to be tested (the mixed extract, the tetrandra root extract, the centella asiatica extract and the EGCG in example 3, and phosphate buffered saline PBS is used as negative control) with various concentrations are added into the pore plate, after the culture is continued for 12-24h, TNF-alpha is added into each pore of the pore plate. The culture is continued for 12-24h, after the culture is finished, 100 μ l of lysate (obtained from luciferase assay kit, see kit instructions) is added into each well of the well plate, the operation is carried out according to the instructions of the luciferase assay kit (obtained from Shanghai Ying Biotechnology Co., Ltd.), and the reading is carried out in a Lum mode of a fluorescence microplate reader.
3.2.4. Results of the experiment
The experimental results show that various test solutions can reduce the NF-kB signal of the inflammatory factor, wherein the inhibition effect of the three plant extracts (compound solutions) in the example 3 on the NF-kB signal is dose-dependent, and the inhibition effect on the NF-kB is obviously stronger than that of other sample solutions under the highest concentration of the current measurement, which indicates that the plant combined with the gentiana scabra bunge in the compound solution can play a synergistic role with the gentiana scabra bunge and play an anti-inflammatory role in inhibiting the inflammatory factor together.
See fig. 2 for specific experimental data.
Fourth, efficacy test contrast experiment 3
4.1 materials and methods
4.1.1 Experimental animals
ICR mice, weight 18 ~ 22g, male and female half.
4.1.2 Experimental samples
Blank emulsion sample:
dissolving the oil phase in the above components, heating to 80 deg.C, keeping the temperature, adding emulsifying wax, stirring, adding water phase, stirring at 3000 rpm for 10 min, and cooling to obtain blank emulsion.
Positive control sample: diphenhydramine hydrochloride (Shanghai Tantan Technique Co., Ltd.)
Negative control molding sample: dextran 40 (Shanghai Tata Techni Co., Ltd.)
Depilatory cream: weiting depilatory cream (Lijieshi group)
Test samples of the compound solution of example 3: the sample concentration was 2%, 5%, 8% (w/w). An appropriate amount of the extract solution with the labeled concentration of 100% prepared in example 3 was added to the blank emulsion to prepare a sample to be tested with the above-mentioned weight concentration.
The following comparative examples were prepared in the same manner as the test samples.
Test sample of gentian extract of comparative example 4: concentration 5%, 8% (w/w).
4.2 Experimental methods
4.2.1 inhibitory Effect of each test drug on mouse itching caused by dextran
Taking 80 ICR mice with the weight of 18-22 g and half of each sex, dividing the ICR mice into 8 groups according to the weight of the sex: blank, dextran 40 negative control building block, diphenhydramine hydrochloride positive control, 2% example 3 sample set, 5% example 3 sample set, 8% example 3 sample set, 5% comparative example 4 sample set, 8% comparative example 4 sample set. The animal's back hair was removed with depilatory cream, and the area of the depilated skin area was 2cm × 2cm, and used for the experiment after 2 days. During the experiment, the hair removal area on the back of each group of animals is locally coated with corresponding experimental samples (0.2 ml/animal). For 5 consecutive days, diphenhydramine hydrochloride (2mg/ml) was administered by intraperitoneal injection (administered at 0.1ml/10 g) on the last day. 30min after the last administration of all groups, 0.02% low molecular dextran 40 is injected into tail vein, and the dosage is 0.05ml/10 g. Taking the positions of the front paw scratching head, the back paw scratching trunk and the whole body bitten by mouth of the mouse as itching indications, recording the itching times of the mouse within 30 minutes, and comparing the difference between groups.
4.3 results of the experiment
After tail vein injection of 0.02% low molecular dextran, the duration of scratching was observed for all mice within 30min and counted.
The results show that the duration of scratching is significantly different between the dextran molding group and the blank group (p < 0.001), indicating that the molding is successful.
Wherein the sample groups of example 3 at different concentrations are dose-dependent on the antipruritic effect of the mice. In particular, 8% of the sample set of example 3 significantly differed from the duration of scratching by the dextran-forming module (p < 0.001), indicating that 8% of the sample set of example 3 had a significant relief from the mouse itching caused by dextran.
The 8% of the sample group of comparative example 4 has a certain difference (p < 0.01) from the dextran model group in the duration of scratching, which indicates that the 8% of the sample group of comparative example 4 has a certain alleviating effect on mouse itching caused by dextran. The other comparative example 4 sample group has no obvious difference in the mouse itching duration time compared with the dextran model group.
The 8% sample group in the example 3 has an obvious relieving effect on mouse itching caused by dextran, which is obviously stronger than that of the 8% sample group in the comparative example 4, and shows that the plant combined with the gentian plays a certain synergistic effect and jointly plays an antipruritic effect on mice.
See fig. 3 for specific experimental data.
Fifth, efficacy test comparative experiment 4
5.1. Purpose of testing
The ability of the solution of the compound composition of example 3 to soothe redness was evaluated by a closed patch-induced red model and the soothing efficacy of the gentian extract (comparative example 4), tetrandra root extract (comparative example 5) and centella asiatica extract (comparative example 6) alone at the same concentration was compared in order to demonstrate the synergistic efficacy of the compound composition of example 3.
5.2. Materials and methods
Test subject
The Chinese healthy people of 19-60 years old meet the selection of 5 subjects in the skin patch test of human body of 2015 edition of technical Specification for cosmetic safety.
Test sample
Red-causing mold making medicine: sodium dodecyl Sulfate (SIGMA)
A plaque chamber device: finn Chamber 8mm
Test samples of example 3: 5% of the samples to be tested of example 3: the sample concentration was 2%, 5%, 8% (w/w). An appropriate amount of the extract solution with the labeled concentration of 100% prepared in example 3 was added to the blank emulsion to prepare a sample to be tested with the above-mentioned weight concentration.
The following comparative examples were prepared in the same manner as the test samples.
Test sample of comparative example 4: 5%, 8% Gentiana scabra Bunge extract
Test sample of comparative example 5: 5% and 8% of radix Stephaniae Japonicae extract
Test sample of comparative example 6: 5% and 8% of centella asiatica extract
5.3. Experimental methods
Subjects were subjected to a skin screen (BL) of the back test area by staff. And selecting a skin area without influence evaluation factors for spot pasting. A red-forming agent was added to each of the applicator wells and applied to the back for 24 hours. In order to prevent the spot tester from falling off, the periphery of the spot tester can be fixed by medical adhesive tapes.
After 24 hours, the reddening mold drug was removed, and the clinical dermatologist evaluated according to "skin reaction grading standard in skin closed patch experiment of table 1" in "method 6" in "cosmetic safety technical specification 2015 edition" in human skin patch test "(after reddening-T0.5). Samples were divided into 8 groups: a blank control (water) group, a 5% compound component of example 3, a gentian extract (the concentration of gentian in the 5% compound component of example 3), a gentian extract (the concentration of gentian in the 8% compound component of example 3), a tetrandra extract (the concentration of tetrandra in the 5% compound component of example 3), a tetrandra extract (the concentration of tetrandra in the 8% compound component), a centella asiatica extract (the concentration of centella asiatica in the 5% compound component), and a centella asiatica extract (the concentration of centella asiatica in the 8% compound component). Equal amounts of samples were added to the different chamber chambers and were removed 24 hours after application to the reddish area and again evaluated by the clinical dermatologist (post-relief-T24).
5.4. Results of the experiment
The establishment of a erythropoiesis model and the efficacy evaluation of the degree of remission redness of 8 groups of samples are effectively completed, and the results show that:
the evaluation value of the redness of the skin after the treatment of 5% of the sample to be tested in example 3 is obviously reduced, and a significant difference exists (p is less than 0.05), which indicates that 5% of the sample to be tested in example 3 has obvious relieving effect on the symptoms of the redness of the skin. The evaluation of redness in the skin after treatment with gentian extract (8% of the sample of comparative example 4) was significantly lower with a significant difference (p < 0.05), indicating that gentian extract alone (8% of the sample of comparative example 4) had a significant relief from the symptoms of skin redness.
The individual extracts of 5% gentian, 5% tetrandra, 5% centella asiatica, 8% tetrandra, 8% centella asiatica showed no significant difference in redness values before and after soothing (p > 0.05), indicating that the individual plant extracts had no relief effect on the symptoms of skin redness at these concentrations.
The blank control (water) showed no significant change in redness values before and after the effect, indicating that water as the sample diluent had no effect of slowing redness.
In conclusion, the 5% compound component solution can remarkably relieve the redness symptom of the skin, but the effect of the single extracts of the gentiana scabra bunge, the tetrandra and the centella asiatica at the same concentration is not obvious, which shows that the plants combined with the gentiana scabra bunge play a certain synergistic effect.
See fig. 4 for specific experimental data.
Claims (18)
1. A composition of plant extracts for resisting skin allergy comprises the plant extracts of gentian, tetrandra and centella asiatica, wherein the content of each plant extract is as follows by weight percentage: 30-70% of gentian, 10-40% of radix stephaniae tetrandrae and 10-40% of centella asiatica; wherein the plant extract is prepared by the following method: extracting pulverized plant material with water at 60-80 deg.C, extracting with 20-70% C2-6 alcohol water solution at 40-85 deg.C, and further extracting with 50-70% C2-6 alcohol water solution at 40-85 deg.C, wherein C2-6 alcohol water solution is propylene glycol or ethanol, and mixing the three extractive solutions to obtain the composition of plant extract.
2. The composition according to claim 1, wherein the content of each plant extract is 40-65% of gentian, 15-30% of tetrandra root, 15-30% of centella asiatica.
3. The composition according to claim 1 or 2, wherein the composition is prepared by extracting gentian, tetrandra root and centella asiatica together; wherein the weight ratio of each plant raw material or each plant extract is as follows: 3-7 parts of gentian: 1-4 of radix stephaniae tetrandrae: 1-4 parts of centella asiatica.
4. A composition according to claim 3, wherein the weight ratio between the individual plant materials or the individual plant extracts is: 4-6 of gentian: 1-3 of radix stephaniae tetrandrae: 1-3 parts of centella asiatica.
5. A method for preparing a composition of plant extracts, the method comprising: weighing plant materials, wherein the plant materials comprise gentian, radix stephaniae tetrandrae and centella asiatica, and the weight ratio of each plant material or each plant extract is as follows: 3-7 parts of gentian: 1-4 of radix stephaniae tetrandrae: 1-4 parts of centella asiatica; extracting pulverized plant material with water at 60-80 deg.C, extracting with 20-70% C2-6 alcohol water solution at 40-85 deg.C, and further extracting with 50-70% C2-6 alcohol water solution at 40-85 deg.C, wherein C2-6 alcohol water solution is propylene glycol or ethanol, and mixing the three extractive solutions to obtain the composition of plant extract.
6. The method according to claim 5, wherein the weight ratio between the individual plant materials or the individual plant extracts is: 4-6 of gentian: 1-3 of radix stephaniae tetrandrae: 1-3 parts of centella asiatica.
7. The method according to claim 5, wherein the extract composition is obtained by extracting the pulverized plant material with water, then extracting with 20% to 50% aqueous solution of C2-6 alcohol, further extracting with 50% to 70% aqueous solution of C2-6 alcohol, and mixing the three extracts.
8. The process according to claim 5, wherein the aqueous solution of the C2-6 alcohol has an extraction temperature of 50-75 ℃.
9. The method according to any one of claims 5 to 8, further comprising subjecting the obtained extract composition to membrane filtration for purification and obtaining a concentrate.
10. The method as claimed in claim 9, wherein the concentrated solution is obtained by filtering with 5000-10000 dalton ultrafiltration membrane, collecting the filtrate, filtering with 100-1000 dalton nanofiltration membrane, and recovering the solvent.
11. A method according to any one of claims 5 to 8, wherein the extract is obtained by extrinsic cycle extraction, monitored by an on-line spectrophotometer, and endpoint determined by absorbance, at which time the residue is removed.
12. A composition of plant extracts prepared according to the method of any one of claims 5 to 11.
13. The composition of plant extracts of claim 1, comprising gentiopicroside, tetrandrine and asiaticoside, wherein the weight ratio between the various components is: 3-7 parts of gentiopicroside: 1-4 parts of hanfangchin A: 1-4 parts of total asiaticoside.
14. The plant extract composition according to claim 13, wherein the weight ratio between the various components is: 4-6 parts of gentiopicroside: 1-3 of sweatsiangchinoline: 1-3 parts of total asiaticoside.
15. Use of a composition of plant extracts according to any one of claims 1 to 4 or 12 to 14 for the preparation of a therapeutic or prophylactic medicament or cosmetic for anti-allergic, anti-inflammatory, alleviating the degree of redness, relieving itching, anti-irritant or repairing the skin.
16. A medicament or cosmetic comprising a composition of a plant extract according to any one of claims 1 to 4 or 12 to 14, wherein the medicament or cosmetic is a cream, lotion, paste, ointment, mask, gel or lotion, the composition of plant extracts being present in the medicament or cosmetic in an amount by weight: contains 0.1-20% of plant extract with labeled concentration of 100%.
17. The medicament or cosmetic according to claim 16, wherein the content of the composition of plant extracts in the medicament or cosmetic is: contains plant extractive solution 2-15% of labeled concentration of 100%.
18. The medicament or cosmetic according to claim 16, wherein the content of the composition of plant extracts in the medicament or cosmetic is: contains 5-10% of plant extract with labeled concentration of 100%.
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