CN117257852A - Centella asiatica total glycoside liposome gel, preparation method and application thereof in resisting skin cancer - Google Patents
Centella asiatica total glycoside liposome gel, preparation method and application thereof in resisting skin cancer Download PDFInfo
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- CN117257852A CN117257852A CN202311273663.6A CN202311273663A CN117257852A CN 117257852 A CN117257852 A CN 117257852A CN 202311273663 A CN202311273663 A CN 202311273663A CN 117257852 A CN117257852 A CN 117257852A
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- Prior art keywords
- centella asiatica
- percent
- asiaticoside
- total glycosides
- gel
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Abstract
The invention discloses centella asiatica total glycoside liposome gel, a preparation method thereof and application thereof in resisting skin cancer. The invention firstly provides application of asiaticoside including asiaticoside and madecassoside in preparing anti-skin cancer medicine, and provides the asiaticoside liposome gel comprising the following components: 1 to 5 percent of centella asiatica total glycosides, 0.1 to 0.5 percent of lecithin, 0.02 to 0.1 percent of cholesterol, 1 to 5 percent of carbomer 940 or sodium alginate, 10 to 20 percent of glycerol, 2 to 4 percent of triethanolamine, 5 to 20 percent of absolute ethyl alcohol and 50 to 75 percent of purified water. The asiaticoside liposome gel has the medicine encapsulation rate of 90.80%, good percutaneous permeation and absorption performance, can inhibit the proliferation of mouse skin melanoma cells B16F10, and has an IC50 value on B16F10 mouse melanocytes which is obviously lower than that of common asiaticoside gel.
Description
Technical Field
The invention belongs to the field of biological medicine. More particularly, relates to a centella asiatica total glycoside liposome gel, a preparation method thereof and application thereof in resisting skin cancer.
Background
Centella asiatica is a dry whole herb of centella asiatica Centella asiatica (L.) Urb belonging to the family Umbelliferae, has the effects of clearing heat and promoting diuresis, and removing toxic substances and detumescence, and can be used for treating jaundice due to damp-heat, heatstroke diarrhea, stranguria with blood, carbuncle swelling sore and traumatic injury. Centella asiatica has been used for treating various clinical diseases from ancient times, some ancient medical books in China have relevant records of centella asiatica, and in modern times, centella asiatica is widely used for treating chronic kidney diseases, diabetic nephropathy, malignant intestinal obstruction, wound healing, limited scleroderma and the like clinically. Through analysis and research, the chemical components in centella mainly comprise: amino acids and derivatives thereof, alkaloids, lipid compounds, saccharides and glycosides compounds, triterpene compounds, flavonoids and steroids, etc., wherein triterpene compounds are main effective components. Pharmacological studies prove that the centella asiatica extract has the activities of resisting tumor, resisting inflammation, protecting nervous system functions, promoting wound healing, inhibiting scar hyperplasia, repairing skin injury and the like.
Centella asiatica total glycosides are the general term for saponins extracted from centella asiatica (including asiaticoside and madecassoside), and are mainly used clinically for treating skin wounds and repairing scars.
In addition, the common transdermal preparation of the centella asiatica total glycosides is asiaticoside cream ointment, such as the treatment of localized scleroderma by the combination of the centella asiatica total glycosides and tranilast, can obviously improve the immune function of the organism and improve the related symptoms; the asiaticoside cream ointment and the asiaticoside tablet are used for treating patients after anorectal diseases operation, so that the wound healing of the patients can be obviously promoted; the strong pulse light of 650nm is combined with asiaticoside cream ointment to treat atrophic scar of acne; the asiaticoside cream ointment for treating cobra bite has relatively high clinical effect and capacity of improving systemic inflammatory response. However, asiaticoside and madecassoside have large molecular weight, small solubility in water, large logP (oil-water partition coefficient) value, and are not easy to penetrate skin barrier, and may have poor transdermal absorption performance in vivo. The stratum corneum is the main skin barrier that hinders transdermal absorption of drugs, and in the development of transdermal drug delivery formulations, the absorption of drugs is often promoted by various physical methods (microneedle method, iontophoresis method), chemical methods (addition of transdermal absorption promoters) and pharmacological methods (development of novel transdermal drug delivery formulations), increasing the retention of drugs in the skin and alleviating toxic and side effects. Since both physical and chemical methods inevitably have a certain irritation to the skin or damage the stratum corneum to a different extent, the development of a novel transdermal drug delivery preparation (e.g., microemulsion, liposome, etc.) to improve the transdermal osmotic absorption performance of the drug is an important research direction in the current pharmacy.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the prior art, expand the application of asiaticoside, solve the problems of poor therapeutic effect caused by large molecular weight, small solubility in water, difficult penetration of skin barrier and possibly poor transdermal absorption performance in vivo of the asiaticoside, and provide the asiaticoside liposome gel, and the preparation method and the application thereof.
A first object of the present invention is to provide the use of total asiaticoside, including asiaticoside and madecassoside, in the preparation of an anti-skin cancer drug.
The second object of the invention is to provide a centella asiatica total glycosides liposome gel.
The third object of the invention is to provide the application of the centella asiatica total glycoside liposome gel in resisting skin cancer.
The above object of the present invention is achieved by the following technical scheme:
the studies of the present invention show that the inhibition of skin cancer (melanoma) by centella asiatica total glycosides, including asiaticoside and madecassoside, is significantly better than asiaticoside.
Thus, the present invention claims the use of total asiaticoside, including asiaticoside and madecassoside, in the preparation of an anti-skin cancer drug.
Preferably, the mass ratio of asiaticoside to madecassoside is 1:1.5-2.
Preferably, the total content of asiaticoside and madecassoside in the centella asiatica total glycosides is 55% -95%.
The invention also develops the centella asiatica total glycoside liposome gel for resisting skin cancer, and the formula and the process of the centella asiatica total glycoside liposome gel are researched, so that the transdermal absorption performance of the finally obtained centella asiatica total glycoside liposome gel is obviously improved, and the effect of resisting skin cancer (melanoma) is obviously improved.
The centella asiatica total glycoside liposome gel of the invention comprises the following components in percentage by weight: 1 to 5 percent of centella asiatica total glycosides, 0.1 to 0.5 percent of lecithin, 0.02 to 0.1 percent of cholesterol, 1 to 5 percent of carbomer 940 or sodium alginate, 10 to 20 percent of glycerol, 2 to 4 percent of triethanolamine, 5 to 20 percent of absolute ethyl alcohol and 50 to 75 percent of purified water.
Preferably, the total content of asiaticoside and madecassoside in the centella asiatica total glycosides is 55% -95%; the lecithin is from soybean or egg yolk, wherein the PC (phosphatidylcholine) content is 20% -90%.
Preferably, the centella asiatica total glycoside liposome gel comprises the following components in percentage by weight: centella asiatica total glycosides 2%, egg yolk lecithin 0.15%, cholesterol 0.03%, carbomer 940.5%, glycerin 17.5%, triethanolamine 4%, absolute ethyl alcohol 5% and purified water 69.82%.
More preferably, the total content of asiaticoside and madecassoside in the centella asiatica total glycosides is 85% and the PC content in the egg yolk lecithin is 80%.
The invention provides a method for preparing the centella asiatica total glycosides liposome gel, which comprises the following steps:
s1, adding absolute ethyl alcohol into centella asiatica total glycosides, lecithin and cholesterol to dissolve the centella asiatica total glycosides, lecithin and cholesterol, and obtaining a clear solution;
s2, dropwise adding the solution into purified water in a stirring state, and stirring to obtain centella asiatica liposome solution;
s3, adding glycerol into carbomer 940 or sodium alginate to moisten the carbomer, and adding absolute ethyl alcohol and purified water to swell the carbomer or sodium alginate to obtain a space Bai Ningjiao;
s4, adding the centella asiatica liposome solution into blank gel, uniformly stirring, adding triethanolamine, and uniformly stirring to obtain centella asiatica total glycosides liposome gel.
Preferably, ultrasonic dissolution is used in step S1.
Preferably, the stirring conditions in step S2 are: stirring for 2-6 hours at 25-50 ℃.
More preferably, the stirring conditions in step S2 are: stirred at 40℃for 4 hours.
The invention also provides application of the centella asiatica total glycoside liposome gel in resisting skin cancer.
Preferably, the skin cancer is melanoma.
More preferably, the melanoma is skin melanoma cell B16F10.
The invention has the following beneficial effects:
the invention uses asiaticoside including asiaticoside and madecassoside to prepare asiaticoside liposome gel for resisting skin cancer (melanoma), has good proliferation inhibiting effect on melanoma cells, and the liposome is a microvesicle with phospholipid bilayer structure, has similar composition and structure to biological membrane, has amphipathy and membrane fluidity, and can promote percutaneous absorption of medicine by increasing humidification and hydration of horny layer and changing permeability of horny layer. The phospholipid in the liposome has high compatibility with the lipid of the skin horny layer, can be mutually fused, on one hand, plays a role in reducing the barrier of the skin horny layer, and on the other hand, the phospholipid is fused with the lipid to carry out biotransformation, so that the medicine taking the liposome as a carrier is easy to accumulate in the skin horny layer to form a medicine reservoir, the medicine wrapped by the liposome can be released continuously, and better penetrates through the horny layer, thereby playing a role in treating a lesion part and greatly improving the treatment effect. The liposome gel is a novel gel which disperses liposome suspension in a polymer gel matrix, the gel can reduce the fluidity of a liposome phospholipid bilayer, reduce the loss of a drug to a certain extent, stabilize the liposome and increase the stability of the drug, and the gel is combined with the liposome, so that the detention performance of the drug on the skin can be enhanced, the bioavailability of the drug can be improved, the administration frequency can be reduced, the permeation of the drug on the skin can be enhanced, the side effect related to dosage can be reduced, and the treatment effect can be improved.
The centella asiatica total glycoside liposome gel has the advantages that the encapsulation rate is 90.80 percent, and the transdermal penetration and absorption performance is good and superior to that of the common gel as determined by an in vitro transdermal experiment; and proved by an in vitro MTT (thiazole blue) colorimetric test, the effect of inhibiting the growth of melanoma cells in vitro can be achieved, and the IC50 value (the minimum concentration of the drug required for killing 50% of host cells) of the B16F10 mouse melanin cytotoxicity is obviously lower than that of common gel of centella asiatica total glycosides. The asiaticoside total glycoside liposome gel of the invention widens the application range of asiaticoside.
Drawings
FIG. 1 fluorescent imaging of centella asiatica total glycosides liposome gels and centella asiatica total glycosides common gels skin permeation test. The graph A shows common gel of centella asiatica total glycosides, and the graph B shows liposome gel of centella asiatica total glycosides.
FIG. 2 Effect of centella asiatica Total glycoside liposome gel and centella asiatica Total glycoside common gel on B16F10 cell viability
FIG. 3 IC50 value of centella asiatica total glycosides liposome gel for B16F10 cells
FIG. 4 IC50 value of common gelata of total glycosides of centella asiatica against B16F10 cells
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The total asiaticoside used in the following examples was purchased from Shaanxi's Ann natural raw material sink, production lot number 20200728.
EXAMPLE 1 preparation of Total glycosides Liposome gel of centella asiatica
(1) Proportioning materials
The ingredients of centella asiatica total glycosides (total asiaticoside and madecassoside content 85%) 2%, egg yolk lecithin (PC content 80%) 0.15%, cholesterol 0.03%, carbomer 940.5%, glycerin 17.5%, triethanolamine 4%, absolute ethyl alcohol 5% and purified water 69.82% are taken according to weight percentage. The total weight of the prescription is 200g.
(2) Preparation of centella asiatica total glycosides liposome solution
Placing centella asiatica total glycosides, egg yolk lecithin and cholesterol in a 50mL beaker, adding absolute ethyl alcohol, and performing ultrasonic treatment to dissolve the centella asiatica total glycosides, egg yolk lecithin and cholesterol into a clear ethanol solution; another 250mL beaker is taken, put into a rotor and purified water, put on a magnetic stirrer, and simultaneously stirred, and the ethanol solution is sucked and dripped by a disposable dropper; after the dripping is completed, stirring for 4 hours at 40 ℃ to volatilize ethanol in the solution, thus obtaining centella liposome solution.
(3) Preparation of centella asiatica total glycosides liposome gel
Adding glycerol to wet carbomer 940, adding absolute ethanol and residual purified water to swell, and obtaining a space Bai Ningjiao; adding the centella asiatica liposome solution into blank gel, stirring uniformly, adding triethanolamine, and stirring uniformly to obtain centella asiatica total glycosides liposome gel.
EXAMPLE 2 preparation of Total glycosides Liposome gel of centella asiatica
(1) Proportioning materials
The ingredients of centella asiatica total glycosides (total content of asiaticoside and madecassoside is 85%) 2%, soybean lecithin (PC content is 80%) 0.15%, cholesterol 0.03%, sodium alginate 1.5%, glycerin 17.5%, triethanolamine 4%, absolute ethyl alcohol 5% and purified water 69.82% are taken according to weight percentage. The total weight of the prescription is 200g.
(2) Preparation of centella asiatica total glycosides liposome solution
Placing centella asiatica total glycosides, soybean lecithin and cholesterol in a 50mL beaker, adding absolute ethyl alcohol, and carrying out ultrasonic treatment to dissolve the centella asiatica total glycosides, soybean lecithin and cholesterol into a clear ethanol solution; another 250mL beaker is taken, put into a rotor and purified water, put on a magnetic stirrer, and simultaneously stirred, and the ethanol solution is sucked and dripped by a disposable dropper; after the dripping is completed, stirring for 4 hours at 40 ℃ to volatilize ethanol in the solution, thus obtaining centella liposome solution.
(3) Preparation of centella asiatica total glycosides liposome gel
Adding glycerol into sodium alginate to moisten the sodium alginate, and adding absolute ethyl alcohol and residual purified water to swell the sodium alginate to obtain a space Bai Ningjiao; adding the centella asiatica liposome solution into blank gel, stirring uniformly, adding triethanolamine, and stirring uniformly to obtain centella asiatica total glycosides liposome gel.
EXAMPLE 3 preparation of Total glycosides Liposome gel of centella asiatica
(1) Proportioning materials
The ingredients of centella asiatica total glycosides (total content of asiaticoside and madecassoside is 85%) 2%, egg yolk lecithin (PC content is 80%) 0.15%, cholesterol 0.03%, sodium alginate 1.5%, glycerin 17.5%, triethanolamine 4%, absolute ethyl alcohol 5% and purified water 69.82% are taken according to weight percentage. The total weight of the prescription is 200g.
(2) Preparation of centella liposome solution
Adding asiaticoside, egg yolk lecithin and cholesterol into 50mL beaker, adding absolute ethanol, and ultrasonic treating to dissolve into clear ethanol solution; another 250mL beaker is taken, put into a rotor and purified water, put on a magnetic stirrer, and simultaneously stirred, and the ethanol solution is sucked and dripped by a disposable dropper; after the dripping is completed, stirring for 4 hours at 40 ℃ to volatilize ethanol in the solution, thus obtaining centella liposome solution.
(3) Preparation of centella asiatica total glycosides liposome gel
Adding glycerol into sodium alginate to moisten the sodium alginate, and adding absolute ethyl alcohol and residual purified water to swell the sodium alginate to obtain a space Bai Ningjiao; adding the centella asiatica liposome solution into blank gel, stirring uniformly, adding triethanolamine, and stirring uniformly to obtain centella asiatica total glycosides liposome gel.
Comparative example 1 preparation of general gel of total glycosides of centella asiatica
The ingredients of centella asiatica total glycosides (total content of asiaticoside and madecassoside 85%) 2%, carbomer 940 1.5%, glycerin 17.5%, triethanolamine 4%, absolute ethanol 5% and purified water 70.0% are taken according to weight percentage. The total weight of the prescription is 200g.
Collecting asiaticoside, adding absolute ethanol, and ultrasonic treating to dissolve into clear ethanol solution; adding glycerol to wet carbomer 940, and adding purified water to swell to obtain empty Bai Ningjiao; adding the ethanol solution into blank gel, stirring, adding triethanolamine, and stirring to obtain common gel of herba Centellae total glycosides.
Example 4 liposome entrapment assay
Measuring a sample: centella asiatica liposome solution prepared in example 1
Chromatographic conditions: chromatograph: an ACQUITY high performance liquid chromatograph; chromatographic column: moon Xup-C18 column (25 cm. Times.4.6mm. Times.5 μm); mobile phase: acetonitrile-water (25.5:74.5); flow rate: 0.8mL/min; isocratic elution; column temperature: 25 ℃; the sample injection amount is 10 mu L; ultraviolet detector detection wavelength: 220nm.
Preparation of a control solution: precisely weighing asiaticoside reference substance and madecassoside reference substance, respectively, and adding methanol to obtain solutions with concentration of 1.0 mg/mL.
Drawing a standard curve: taking 10 μl of asiaticoside control solution and 10 μl of madecassoside control solution respectively, and loading into liquid chromatograph for measurement. The standard curve is drawn by taking the peak area as the ordinate and the concentration of asiaticoside and madecassoside as the abscissa, and linear regression analysis is carried out, and as a result, the concentration of asiaticoside is in good linear relation with the peak area within the range of 0.025-0.800 mg/mL, and the standard curve equation is Y=8.47 e +02 X-2.35e +03 R= 0.99996 (Y is the peak area integral value, X is the concentration mg/mL), the concentration of madecassoside is in good linear relation with the peak area within the range of 0.030-0.820 mg/mL, and the standard curve equation is Y=1.66 e +02 X-0.39e +03 R= 0.99997 (Y is peak area integrated value, X is concentration mg/mL).
Determination of the total amount of drug in centella asiatica total glycosides liposomes (W total, mg): precisely measuring 1g of centella asiatica total glycosides liposome solution, placing in 10mL test tube with plug scale, adding 10 drop of 10% calcium chloride solution, shaking, adding methanol to 10mL, ultrasonic treating for 10 min, filtering with 0.22 μm microporous membrane, collecting 10 μl of filtrate, and adding into liquid chromatograph. Substituting the measured asiaticoside peak area value A1 and madecassoside peak area value A2 into a standard curve equation respectively, and calculating W total:
w total= (a1+2.35e) +03 )/8.47e +02 ×10+(A2+0.39e +03 )/1.66e +02 ×10
Determination of the amount of unencapsulated free drug (W stream) in centella asiatica total glycosides liposomes: precisely measuring 1g of centella liposome solution in an ultrafiltration centrifuge tube of 3000 daltons, centrifuging for 30 minutes at 15000r/min, taking an outer tube solution, placing in a 10mL test tube with a plug scale, adding methanol to 10mL, shaking uniformly, filtering with a microporous filter membrane of 0.22 mu m, taking 10 mu L of the subsequent filtrate, and injecting into a liquid chromatograph for measurement. Substituting the measured asiaticoside peak area value A3 and madecassoside peak area value A4 into a standard curve equation respectively, and calculating W stream:
wrun= (A3+2.35e) +03 )/8.47e +02 ×10+(A4+0.39e +03 )/1.66e +02 ×10
Encapsulation efficiency (EE%) =1-W stream/W total x 100%
Results: the encapsulation efficiency of centella asiatica total glycosides in centella asiatica total glycosides liposome solution was measured to be 90.80%.
Example 5 ex vivo transdermal assay
1. Measuring a sample: centella asiatica total glycosides liposome gel prepared in example 1 and centella asiatica total glycosides general gel prepared in comparative example 1
2. Skin treatment: several Kunming mice were taken, and the long hairs on the abdomen were cut short 24 hours before the experiment, and the abdominal villi were removed with depilatory cream. During experiments, the mice are anesthetized by isoflurane, superfluous villus is removed by a shaver to enable the belly skin to be in a state close to the naked skin, then the mice are killed by removing the neck, the belly naked skin is immediately wiped by normal saline, the naked skin is sheared off by surgical scissors, subcutaneous fat and adhesion are sheared off, the mice are repeatedly washed by the normal saline, 10% glycerol is soaked and then placed in a plastic bag, and the mice are frozen and stored at the temperature of minus 20 ℃ for no more than 1 week.
3. In vitro transdermal test: taking out experimental skin, naturally thawing before experiment, soaking in physiological saline for 30 min, sucking with filter paper, and cutting into 2.35cm 2 The dermis layer side faces the receiving cell side and is fixed to the diffusion cell. 6.5mL of PBS buffer solution containing 30% ethanol is injected into the receiving chamber as a receiving medium, the magnetic stirring speed is 600r/min, and the water bath temperature is 3731 ℃. 2.0g of centella asiatica total glycosides liposome gel and 2.0mL of centella asiatica common gel are respectively weighed and evenly coated on the skin, 2.0mL of centella asiatica total glycosides liposome gel and centella asiatica common gel are respectively sampled at 1,2,4,8, 12 and 24 hours, and receiving mediums with the same amount of 3731 ℃ are timely supplemented. Samples were taken at each time point, evaporated to dryness under reduced pressure using a rotary evaporator (60 ℃ C.), the residue was dissolved in a suitable amount of methanol, the volume was set to 2mL, and after filtration through a 0.22 μm microporous membrane, the filtrate was poured into an HPLC apparatus and subjected to measurement, and the chromatographic conditions and measurement method were the same as in example 4. The measured peak area values of asiaticoside and madecassoside are substituted into the standard curve equation of example 4, and the total amount of asiaticoside is calculated, and the calculation formula of the accumulated amount of the drug per unit area and the accumulated transmittance percentage at each time point is as follows:
P＝(Qn×A/B)×100％
wherein,
qn is the cumulative drug permeation amount per unit area;
cn, ci are the drug concentrations measured at the nth and ith sampling points;
v0 is the receiving cell volume;
vi is the volume of each sample;
a is the diffusion area;
b is the total glycosides of centella asiatica in the measured sample;
p is the cumulative transmittance percentage.
4. Skin permeation test
100 mu L of rhodamine 123 solution (1 mg/mL) is taken, 10g of centella asiatica total glycosides liposome gel and 10g of centella asiatica total glycosides common gel are respectively added, after magnetic stirring for 4 hours, the gel is tightly combined with rhodamine, and the rest rhodamine is removed by centrifugation (10000 r/min). Taking rhodamine marked centella asiatica total glycosides liposome gel and centella asiatica total glycosides common gel 1.0g respectively, uniformly coating on skin, performing transdermal test, processing skin samples after 24 hours in an automatic tissue processor, longitudinally cutting into 15 μm thickness by using a frozen microtome, fixing the slices on a glass slide, observing fluorescence (asiaticoside shows green fluorescence) by using a fluorescence microscope, and analyzing distribution and aggregation conditions of asiaticoside in stratum corneum, epidermis and dermis of the skin.
5. Measurement of intradermal retention
Taking a skin sample after the skin permeation test is finished, cleaning by PBS solution, sucking water by filter paper, and cutting off 0.4cm 2 The small pieces were then frozen and sectioned by a microtome (slicing temperature-23 ℃ C.), and the stratum corneum (25 μm), epidermis (75 μm) and dermis (1.5 mm) tissues were placed in EP tubes, respectively, and immersed in 1mL of methanol for 12 hours, and filtered through a 0.22 μm microporous filter membrane, and the filtrate was measured by HPLC, and the chromatographic conditions and the measurement method were the same as in example 4. The measured peak area values of asiaticoside and madecassoside are substituted into the standard curve equation of example 4, and the total asiaticoside amount of each skin tissue layer is calculated, namely the intradermal retention.
6. Results
(1) In vitro transdermal tests determine the cumulative release of drug per unit area of sample over 24 hours. The cumulative drug release amount per unit area Qn (μg/cm) is taken as the abscissa of the sampling time t (hours) 2 ) And (3) carrying out linear regression on the Qn to t to obtain a cumulative permeation linear equation of the centella asiatica total glycosides liposome gel, wherein the cumulative permeation linear equation of the centella asiatica total glycosides liposome gel is Qn= 185.65t-25.7 (R2=0.9998), the cumulative permeation linear equation of the centella asiatica total glycosides common gel is Qn= 74.259t-10.28 (R2=0.9998), and the cumulative permeation linear equation and the slope of the centella asiatica total glycosides common gel are the permeation rate. Therefore, the transdermal absorption performance of the centella asiatica total glycoside liposome gel is better than that of the common gel, and the transdermal steady-state permeation rate of the medicine is 2.5 times that of the common gel.
(2) The skin permeation test results are shown in figure 1, the centella asiatica total glycosides of the two gels are distributed and aggregated on the cuticle layer, the epidermis layer and the dermis layer of the skin, and the fluorescence intensity of the centella asiatica total glycosides liposome gel is stronger than that of the common gel, which indicates that the percutaneous permeation absorption performance of the centella asiatica total glycosides liposome gel is better.
(3) The measurement result of the intradermal retention shows that the retention of the centella asiatica total glycosides of centella asiatica total glycosides liposome gel in each tissue layer of the skin is 113.7437.65 mug/cm respectively 2 248.0834.67 μg/cm of skin layer 2 Dermis 422.42310.16 μg/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The retention of centella asiatica total glycosides in each tissue layer of skin is 374.13312.94 μg/cm 2 168.5236.72 μg/cm of skin layer 2 Dermis 110.8938.33 μg/cm 2 . Therefore, the total quantity of the total asiaticoside liposome gel is 1.2 times of that of the common gel, and the total asiaticoside liposome gel mainly gathers in the dermis layer to exert the drug effect, and the retention quantity in the dermis layer is 3.8 times of that of the common gel.
EXAMPLE 6MTT colorimetric assay for in vitro inhibition of B16F10 mouse melanoma cells
Measuring a sample: centella asiatica total glycosides liposome gel prepared in example 1 and centella asiatica total glycosides general gel prepared in comparative example 1
(1) Cell culture:
cell culture was performed on 10cm Hyclone cell culture dishes, and the cells B16F10 were cultured on 10cm Hyclone cell culture dishes 6 The cells were passaged 2-3 times (with space inside the flask or dish as a ratio) when the cell density reached 80% by inoculating/mL to 10mL of DMEM cell culture medium containing 10% fetal bovine serum.
(2) Cell transfection:
B16F10 cell density was adjusted to 1X 10 4 After 24 hours of incubation, 5 concentrations (200 mg/mL, 100mg/mL, 50mg/mL, 25mg/mL, 12.5 mg/mL) of centella asiatica total glycosides liposome gel and 5 concentrations (200 mg/mL, 100mg/mL, 50mg/mL, 25mg/mL, 12.5 mg/mL) of centella asiatica total glycosides ordinary gel were added, each 0.1mL.
(3) MTT colorimetric assay for B16F10 cell viability:
after 48 hours of incubation, 20. Mu.L MTT solution (5 mg/mL) was added to each well, after 4 hours of incubation in an incubator, 150. Mu.L isopropanol was added to each well, the 96-well plate was slowly shaken well to evenly distribute the reagents, and the OD value was measured by a full-automatic microplate reader at 570 nm. After the centella asiatica total glycosides liposome gel and the centella asiatica total glycosides common gel with different concentrations are acted on B16F10 cells for 48 hours, the cell activity of the B16F10 cells and the IC50 value of the medicine are calculated.
B16f10 cell viability = 1- [ (Ac-As)/(Ac-Ab) ]x100%
As: experimental well absorbance (cell, medium, MTT solution and drug solution);
ac: control well absorbance (with cells, medium, MTT solution, without drug);
ab: blank wells absorbance (medium, MTT solution, cell, drug free).
Results:
as shown in FIG. 2, the cell activities of common gels of asiaticoside with concentrations of 200, 100, 50, 25, 12.5mg/mL after 48h of the common gels were 14.81%, 21.52%, 61.11%, 72.89%, 93.07% respectively. The cell activities of centella asiatica total glycoside liposome gel with the concentrations of 200, 100, 50, 25 and 12.5mg/mL after acting on B16F10 melanoma cells for 48 hours are 13.55%, 19.23%, 34.09%, 62.89% and 71.92% respectively. Compared with common asiaticoside gel, the asiaticoside liposome gel has stronger effect of inhibiting the growth of melanoma cells in vitro and has larger influence on the cell viability of B16F10 melanoma cells.
After the cell viability data are imported into Graphpad Prism, the statistical analysis shows that the IC50 value of the centella asiatica total glycosides liposome gel is 32.43mg/mL (shown in figure 3), the IC50 value of the centella asiatica total glycosides common gel is 55.43mg/mL (shown in figure 4), the IC50 value of the centella asiatica total glycosides liposome gel on B16F10 melanocytes is obviously lower than that of the common gel, and the inhibition effect of the centella asiatica total glycosides liposome gel on B16F10 melanocytes is obviously better than that of the centella asiatica total glycosides common gel.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. Use of centella asiatica total glycosides, including asiaticoside and madecassoside, in the manufacture of a medicament for treating skin cancer.
2. Use according to claim 1, characterized in that the mass ratio of asiaticoside to madecassoside is 1:1.5-2.
3. Use according to claim 1 or 2, characterized in that the total content of asiaticoside and madecassoside in the centella asiatica total glycosides is 55% to 95%.
4. The centella asiatica total glycoside liposome gel for resisting skin cancer is characterized by comprising the following components in percentage by weight: 1 to 5 percent of centella asiatica total glycosides, 0.1 to 0.5 percent of lecithin, 0.02 to 0.1 percent of cholesterol, 1 to 5 percent of carbomer 940 or sodium alginate, 10 to 20 percent of glycerol, 2 to 4 percent of triethanolamine, 5 to 20 percent of absolute ethyl alcohol and 50 to 75 percent of purified water; wherein the total content of asiaticoside and madecassoside in the centella asiatica total glycosides is 55% -95%, and the mass ratio of the asiaticoside to the madecassoside is 1:1.5-2.
5. The centella total glycosides liposome gel of claim 4, wherein the lecithin is soybean lecithin or egg yolk lecithin, and the PC (phosphatidylcholine) content in the lecithin is 20% -90%.
6. The centella total glycosides liposome gel according to any one of claims 4 to 5, which is prepared by a method comprising the following steps:
s1, adding absolute ethyl alcohol into centella asiatica total glycosides, lecithin and cholesterol to dissolve the centella asiatica total glycosides, lecithin and cholesterol, and obtaining a clear solution;
s2, dropwise adding the solution into purified water in a stirring state, and stirring to obtain centella asiatica liposome solution;
s3, adding glycerol into carbomer 940 or sodium alginate to moisten the carbomer, and adding absolute ethyl alcohol and purified water to swell the carbomer or sodium alginate to obtain a space Bai Ningjiao;
s4, adding the centella asiatica liposome solution into blank gel, uniformly stirring, adding triethanolamine, and uniformly stirring to obtain centella asiatica total glycosides liposome gel.
7. The centella asiatica total glycosides liposome gel according to claim 6, wherein the stirring condition in step S2 is: stirring for 2-6 hours at 25-50 ℃.
8. Use of the centella asiatica total glycosides liposome gel of any one of claims 4-7 for anti-skin cancer.
9. The use according to any one of claims 1 to 3, 8, wherein the skin cancer is melanoma.
10. The use according to claim 9, wherein the melanoma is skin melanoma cells B16F10.
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