CN111643488A

CN111643488A - Application of bakuchiol in improving anti-tumor effect of chemotherapeutic drugs

Info

Publication number: CN111643488A
Application number: CN202010678806.1A
Authority: CN
Inventors: 周昆; 张盼阳; 于英莉; 张玥; 宋蕾; 陈梦颖
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2020-09-11

Abstract

The invention discloses application of bakuchiol in improving the anti-tumor effect of chemotherapeutic drugs, wherein the bakuchiol increases the anti-tumor effect of the chemotherapeutic drugs and simultaneously reduces the immune injury of the chemotherapeutic drugs, the synergistic effect of the bakuchiol and the chemotherapeutic drugs promotes the related expression of apoptosis factors of tumor cells and promotes the apoptosis of the tumor cells, and simultaneously the bakuchiol reduces the damage of the chemotherapeutic drugs to white blood cells. Provides a basis for clinical experiments and also provides a new way for the research and development of new drugs.

Description

Application of bakuchiol in improving anti-tumor effect of chemotherapeutic drugs

Technical Field

The invention relates to the technical field of medicines, in particular to application of a bakuchiol self-microemulsion in improving the anti-tumor effect of cyclophosphamide.

Background

Malignant tumors are one of the three major causes of death in humans, and are a major health problem facing all countries in the world today. In recent years, anti-cancer drugs have been developed rapidly, and it has become a research target to search for new drugs or drug combinations against malignant tumors.

The chemotherapeutic drug can act on different links of tumor cell growth and reproduction to inhibit or kill tumor cells, and is one of the main means for treating tumors at present. The chemotherapy drugs commonly used in clinic at present are cyclophosphamide, cisplatin, mitomycin, vincristine and the like. However, the anti-tumor chemotherapeutic drugs have toxic and side effects of different degrees, and some serious toxic and side effects are direct reasons for limiting the dosage or use of the drugs. They kill tumor cells and cells of normal tissues, especially blood and lymph tissue cells which grow vigorously in human body, and these cells and tissues are important immune defense system of human body, and destroy immune system of human body, so that cancer can develop rapidly, and serious consequences are caused.

Disclosure of Invention

The invention aims to provide application of bakuchiol in improving the anti-tumor effect of chemotherapeutic drugs aiming at the technical problem that the chemotherapeutic drugs have toxic and side effects such as bone marrow suppression and the like in the prior art.

The technical scheme adopted for realizing the purpose of the invention is as follows:

application of bakuchiol in improving antitumor effect of chemotherapeutic drugs is provided.

In the technical scheme, the bakuchiol increases the anti-tumor effect of the chemotherapeutic drug and simultaneously reduces the immune injury of the chemotherapeutic drug.

In the technical scheme, the psoralen and the chemotherapeutic drug have synergistic effect to promote the relative expression of apoptosis factors of tumor cells and promote the apoptosis of the tumor cells, and meanwhile, the psoralen reduces the damage of the chemotherapeutic drug to white blood cells.

In the technical scheme, the psoralen and the chemotherapeutic drugs act synergistically to up-regulate the expression of Caspase 3, Caspase8, JNK and Bax in tumor tissues and down-regulate the expression of Bcl-2.

In the above technical scheme, the chemotherapeutic drug is cyclophosphamide, cisplatin, mitomycin or vincristine.

In the technical scheme, the mass ratio of the bakuchiol to the chemotherapeutic drug is (75-240): (10-40).

In the technical scheme, the bakuchiol is a bakuchiol self-microemulsion, and the mass ratio of the bakuchiol self-microemulsion to the chemotherapeutic drug is (250-800): (10-40).

In the technical scheme, the bakuchiol self-microemulsion is prepared by the following method: selecting bakuchiol as oil phase, adding emulsifier and co-emulsifier, placing in water bath at 37 + -2 deg.C and stirring at 100 + -5 rpm.

In the technical scheme, the emulsifier is Cremophor RH40 or Cremophor EL 35, and the auxiliary emulsifier is Labrasol.

In the technical scheme, the mass ratio of the bakuchiol to the emulsifier to the co-emulsifier is (1-4): (3-5): (2-4).

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

1. the bakuchiol is a natural medicinal component extracted from natural medicament fructus psoraleae seeds, has no serious toxic or side effect like a chemotherapeutic medicament, improves the dosage form, and has relatively higher oral bioavailability compared with the original bakuchiol. Experiments prove that the combination of cyclophosphamide and bakuchiol self-microemulsion has stronger anti-tumor effect.

2. The cell in-vitro experiment adopts tumor cells S180, bakuchiol is used as a therapeutic drug, the cell activity of bakuchiol is measured through cck8, and the anti-tumor effect of bakuchiol is verified. The tumor generation of mice is induced by an S180 cell line, then grouping and respectively administering (CTX, CTX + BAK self-microemulsion and BAK self-microemulsion) for treatment, and the determination by blood routine detection of the number of leucocytes, tumor tissue HE staining, Tunel tissue staining and Western blotting shows that the bakuchiol self-microemulsion type has the function of increasing cyclophosphamide anti-tumor effect. The invention increases the anti-tumor effect of cyclophosphamide by researching the bakuchiol self-microemulsion type, provides basis for clinical experiments and provides a new way for the research and development of new drugs.

3. The combination of the psoralen self-microemulsion and cyclophosphamide can reduce the damage of CTX to the immune system, and the effect is very obvious.

4. The combination of the psoralen self-microemulsion and cyclophosphamide strengthens the relative expression of apoptosis factors of tumor cells of mice, and can promote the apoptosis of the tumor cells.

Drawings

FIG. 1 shows the growth of tumors in M-, CTX-and CM-group mice (measured on day 14 after modeling).

Fig. 2 shows tumor weights (measured on day 14 after modeling) of the M group, CTX group, and CM group.

FIG. 3 shows tumor size changes in the M, CTX and CM groups.

FIG. 4 shows the numbers of leukocytes in M group, CTX group and CM group.

Fig. 5 is the HE staining pathology results (measured 14 days after modeling).

Figure 6 is TUNEL staining results (measured 14 days after modeling).

FIG. 7 is the expression of protein in mouse tumor tissue (measured 14 days after modeling).

FIG. 8 is a graph of cell viability of NVB, NVB in combination with different concentrations of BAK.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Reagents and manufacturers used in the following examples:

the psoralen self-microemulsion is prepared by the following method:

bakuchiol (bakuchiol) is selected as an oil phase, cremophor RH40 is selected as an emulsifier, Labrasol is selected as an auxiliary emulsifier, bakuchiol, RH40, Labrasol, 3:4:3 are selected according to the proportion (by mass), and the bakuchiol, the RH40, the Labrasol and the Labrasol are uniformly stirred at 100rpm under the condition of 37 ℃ water bath.

Bakuchiol is available from Doppel Biotechnology, Inc.

Cyclophosphamide is available from Shanghai Michelin Biotechnology, Inc.

Polyoxyethylene (40) hydrogenated Castor oil (26126575L0), BASF, Germany

Caprylic capric acid polyethylene glycol glycerides (126715), Effia

Sesame oil, Shandong Lu Hua group Co Ltd

Physiological saline (190610011), Shandong Kelun pharmaceutical Co., Ltd

DMEM high-glucose medium (06-1055-57-1ACS), BI Inc

Fetal bovine serum (04-001-1ACS), BI Ltd

Cyan/streptomycin (MA0110), Dalian Melam Biotechnology Ltd

Pancreatin (03-050-1A), BI Ltd

Rabbit anti-mouse monoclonal antibody Bax (2772), Cell signalling Technology

Rabbit anti-mouse monoclonal antibody Bcl-2(3498), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody Caspase-3(9662), ABCAM

Rabbit anti-mouse monoclonal antibody Caspase-8(4790), Cell Signaling Technology

Rabbit anti-mouse monoclonal antibody JNK (9252), Cell signalling Technology

Rabbit anti-mouse monoclonal antibody beta-actin (4970), Cell Signaling Technology

Phosphatase inhibitor cocktail (14E15B83, Technology Ltd of Tianjin Berrett

RIPA tissue lysate (R0020), Beijing Solebao Tech Co., Ltd

Anti-Rabbit Detection Module (DM001), ProteinSimple, USA

Standard Pack 1(PS-ST01-8), ProteinSimple, USA

PBS buffer (G0002), Google organism

Proteinase K, Roche

Membrane-breaking liquid (G1204), Google organism

Xylene (10023418), national pharmaceutical group chemical reagents Limited

Absolute ethanol (100092683), national pharmaceutical group chemical reagents, Inc

HE dye liquor set (G1003), Servicebio

Tunel kit (11684817910), Roche

The experimental animals and tumor strains used in the following examples:

healthy male Kunming mice, 3-4 weeks, weight 17-19g, 70, SPF grade (license number: SCXK Jing-2016-0011), purchased from Beijing Wintolite laboratory animals technologies, Inc. The animals were all housed in the institute of radiology, institute of Chinese medical sciences. The temperature of the room is 20-25 ℃, the relative humidity is 40-60%, and standard feeding modes are adopted.

S180 mouse ascites tumor cells, purchased from Wuhan Punuoist Biotech, Inc.

S180 cell culture method:

① S180 sarcoma cells, DMEM complete medium containing 10% FBS + 1% double antibody, at 37 deg.C and 5% CO₂Culturing in an incubator.

Adding 2-3ml pancreatin for digestion when the cell concentration in the culture bottle reaches 80-90%, placing in an incubator, and digesting for 3 min; observing under microscope, adding 2ml complete culture medium when cell connection is loose, blowing, stopping digestion, and transferring into centrifuge tube.

③ centrifuging at 4 deg.C and 1000rpm for 5min, removing supernatant, resuspending cells in 5ml PBS, sucking 10ul of the cell onto a cell counting plate with a pipette gun, counting cells under an inverted microscope, diluting with precooled PBS, adjusting cell count to 6 × 10⁷Cell suspension/mL.

The Kunming mouse modeling method comprises the following steps:

taking one mouse of Kunming species for 3-4 weeks, applying depilatory cream to the axilla of mouse 9cm²And (4) removing hair. The next day, the exposed skin was sterilized with alcohol cotton ball for 2-3 times, 0.1mL of the above tumor cell suspension was aspirated with a 1mL sterile syringe, and the mouse was fixed with the left hand and injected 3cm below the mouse's axilla with the right hand held with the syringe.

Example 1

Selecting 40 male mice with the weight of 18-19g, randomly dividing into 4 groups, namely a control group, a model group, a cyclophosphamide group (CTX) and a combined group (CM); except for the control group, each group of mice was tumor modeled according to the kunming mouse modeling method described above. The specific administration times and modes are as follows:

control group: the stomach was gavaged daily with 0.2ml of physiological saline.

Model group (M): the stomach was gavaged daily with 0.2ml of physiological saline.

③ cyclophosphamide group (CTX): the second day of modeling, 20mg/kg of cyclophosphamide is injected into the abdominal cavity of each mouse, and the administration is carried out once every three days.

(iv) Combination (CM): administering psoralen self-microemulsion 400mg/kg (containing psoralen 120mg/kg) by intragastric administration every day; each mouse was administered with 20mg/kg of cyclophosphamide intraperitoneally, once every three days.

Psoralen phenol group (BAK): the self-microemulsion containing bakuchiol is administered by intragastric administration at a dose of 400mg/kg per day (containing bakuchiol 120 mg/kg).

1.1 measurement of tumor diameter and tumor growth inhibition Rate

When the tumor grows to a certain volume, as shown in fig. 1, the tumor diameter, longest diameter (a) and shortest diameter (b) of the mouse are measured by a vernier caliper every other day, and the volume is calculated according to the following formula: volume is a²*b²The tumor volume was calculated from the formula (c), the day of material collection, the isolated tumor tissue was washed with saline and accurately weighed, tumor inhibition was calculated as (%) inhibition ratio (tumor weight of control mice-tumor weight of treated mice) × 100/tumor weight of control mice.

As shown in fig. 2, the weight of the tumor was significantly reduced in the CTX group mice compared to the M group. And tumor weights of mice in the CM group were reduced compared to those in the CTX group.

After modeling, tumor size was measured at

day

6, 8, 10, 12, and 14, respectively, as the tumor grew. As shown in table 1 below and fig. 3, it can be seen from the results that the tumors of the mice administered with CTX, the psoralen self-microemulsion alone or the CTX + psoralen self-microemulsion grew significantly less rapidly than the tumors of the mice not administered, and the tumors of the mice administered with CTX + psoralen self-microemulsion alone grew significantly more slowly than the tumors of the mice administered with CTX and psoralen self-microemulsion alone. The tumor weight is measured at 14 days, and the psoralen can promote the tumor growth inhibition effect of cyclophosphamide.

TABLE 1 measurement of tumor size on

days

6, 8, 10, 12, and 14

In the table, P <0.05, P <0.01, and P < 0.001.

1.2 leukocyte assay

After molding, 1ml of blood was collected from the fundus of the mouse on each of day 0, day 9 and day 15, and the blood was routinely analyzed in a MEK-6318K blood cell analyzer (Nippon Denshoku Co., Ltd.) for 2 hours, and the measurement index was the white blood cell count (WBC).

As shown in fig. 4, the number of conventional leukocytes in the model group tended to decrease as the tumor growth time increased after the molding, and the numbers of leukocytes in the CTX group also tended to decrease as compared with the model group in the four groups measured on the 9 th and 15 th days of tumor growth after the administration of the other groups, but the decrease in leukocytes was significantly alleviated as seen from the microemulsion group and the CM group using bakuchiol alone, and the number of leukocytes was significantly increased as compared with the CTX group. It can be seen that the use of the chemotherapeutic agent CTX has damage to the immune system, and the bakuchiol can reduce the damage, and the effect is very remarkable.

1.3 specimen Collection

Blood sample: on the 15 th day after model building, the mouse takes blood from the picked eyeballs, is placed in a 1.5mLEP tube and stands for 3 hours at normal temperature until the serum and the plasma are obviously layered. Centrifuging at 3500rpm for 10min, sucking supernatant, and storing in refrigerator at-20 deg.C.

Tissue sample: after the mice were sacrificed, the axillary tumor sites of the mice were cut off, placed on ice, and the tumor tissues were separated along the subcutaneous mucosa, half of the tumors were fixed in 4% paraformaldehyde, and the other half were stored in a refrigerator at-80 ℃ for use.

1.4 tumor histopathology H & E staining

(1) Paraffin sections were dewaxed to water, and the dehydration procedure was set as: putting the slices into xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II5 min-75% alcohol 5 min-distilled water washing in sequence.

(2) And (3) hematoxylin staining, namely staining the section in hematoxylin staining solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue with the blue returning solution, and washing with running water.

(3) And eosin staining, namely sequentially dehydrating the slices by 85 percent and 95 percent gradient alcohol for 5min respectively, and staining in eosin staining solution for 5 min.

(4) And (3) dehydrating and sealing, namely putting the slices into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, xylene I for 5min and xylene II for 5min in sequence, sealing the slices with neutral gum after finishing the reaction, and airing the slices in a cool place. Observed under a microscope and photographed.

As shown in FIG. 5, the tumor cells grew in clusters in the model group, and the nuclei were stained bluish purple, pink and dense tissue cells as seen from the color. The CTX group and the psoralen-only self-microemulsion group are lighter in color than the model group, and show red due to the fact that cells are gradually eosinophilic red and loose in arrangement compared with the model group, except the phenomenon, homogeneous red nucleus staining disappeared dead tissues without structural substances can be seen in the CM group, and chromatin can be seen in some cells to be concentrated into a mass without cytoplasm or to be in a hollow halo. The tumor tissue apoptosis of the combination of the bakuchiol self-microemulsion and the cyclophosphamide is stronger than that of the single cyclophosphamide and bakuchiol self-microemulsion.

1.5 TuNEL staining for tumor histopathology

(1) Paraffin section dewaxing to water: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing.

(2) Repairing: after the section is slightly dried, a circle is drawn around the tissue by a organizing pen (the liquid is prevented from flowing away), protease K working solution is dripped into the circle to cover the tissue, and the tissue is incubated in an incubator at 37 ℃ for 30 min. Slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

(3) Membrane breaking: after the section is slightly dried, the membrane-breaking working solution is dripped into the ring to cover the tissue, the incubation is carried out for 20min at normal temperature, and the slide is placed in PBS (PH7.4) and is shaken and washed on a decoloration shaking bed for 3 times, 5min each time.

(4) Adding reagents 1, 2: mixing a proper amount of reagent 1(TdT) and reagent 2(dUTP) in a Tunel reagent box according to the number of the slices and the size of the tissues at a ratio of 2:29, adding the mixture into a ring to cover the tissues, placing the slices in a wet box, incubating for 2 hours in a water bath kettle at 37 ℃, and adding a small amount of water in the wet box to keep the humidity.

(5) DAPI counterstained nuclei: sections were washed 3 times with PBS (pH7.4) for 5min each. After PBS was removed, DAPI staining solution was added dropwise to the circle, and incubated for 10min at room temperature in the dark.

(6) Sealing: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. The slices were slightly spun dry and mounted with an anti-fluorescent quenching mounting agent.

(7) And (5) microscopic examination and photographing: the slices were panoramically scanned under CaseViewer software and images were collected.

As shown in FIG. 6, the model group had a small green fraction and an overall bluish color, so that the number of apoptotic cells in the tissue was small. The apoptosis of the CTX group is stronger than that of the model group, and the color of the CM group is obviously green compared with that of the CTX group, so the apoptosis is more obvious. The bakuchiol self-microemulsion group alone has stronger fluorescence green intensity than the CM group, but has a larger blue area than the CM group.

1.6 protein expression assay

1.6.1 protein extraction

Taking out a tumor sample, transferring the tumor sample into liquid nitrogen, shearing 30-50mg of tumor tissue into a 1.5mL shell tube by using scissors soaked in non-enzyme water, adding 300-. 12000g of the lysed sample was centrifuged for 10min and the supernatant was taken.

1.6.2 protein concentration determination

(1) Preparing a BCA working solution: the reagent a and the reagent B in the BCA kit were mixed uniformly at a ratio of 50:1, and the solution was prepared as it is.

(2) Preparing a standard solution: protein standard gradient concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 0 mu g/mL are prepared according to gradient dilution.

(3) Adding 2 mu L of each sample into a 96-well plate, and then adding 18 mu L of lysate; adding 20 μ L of the above standard substances with different concentrations into other wells; 180. mu.L of BCA working solution was added to both the sample well and the standard well and incubated at 37 ℃ for 30 min.

(4) Measurement of absorbance: the OD value was measured at 562nm, and a standard curve was prepared based on the obtained value.

The standard protein with the concentration of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 and 0 mu g/mL is put into an enzyme labeling instrument according to the sample steps to measure the OD value at 562nm, and the OD value and the corresponding concentration are put into excel to prepare a standard curve. Then, the protein concentration of the sample is calculated according to the standard curve. The standard curve is a positive correlation straight line.

1.6.3 expression of related proteins in tumor tissues

(1) Preparing DTT: adding 40 mu L of deionized water into DTT powder, and uniformly blowing;

(2) prepare 5 × Master Mix (i.e. Loading Buffer): adding 20 mu L of DTT solution into the Master Mix powder, 20 mu L of 10 xSampleBuffer, and blowing and beating uniformly;

(3) preparing Ladder (i.e. marker, molecular weight standard): adding 16 mu L of ionized water, 2 mu L of LDTT solution and 2 mu L of 10 multiplied by Sample Buffer into the Ladder powder, uniformly blowing, sucking about 10 mu L of solution out, and putting the solution into an empty 0.6mL tube to be denatured;

(4) preparing a sample: assuming a sample concentration of y, the following volumes are required to formulate 1 well:

(4) the prepared Ladder and the sample are put together to denature for 5min at 95 ℃; after denaturation, the Ladder and sample were removed and cooled on ice for 5 min. Cooling, performing vortex oscillation, mixing, centrifuging for a short time, and placing on ice for later use;

(5) preparing a primary antibody: diluting the primary Antibody with Antibody Diluent II, and placing on ice for later use;

(6) preparing a luminescent liquid: taking 200 mu L of Lumino-S and Peroxide respectively, mixing by vortex oscillation, and placing on ice for later use;

(7) taking out the sample adding plate, sequentially adding the prepared reagents into the plate according to requirements, and centrifuging at 2500rpm for 5min after the addition is finished;

(8) and (4) putting the plate without bubbles into a machine to run, and carrying out processes such as automatic electrophoresis development and the like. Cleaved Caspase-3, Caspase-8, Bcl-2, Bax and JNK were analyzed for relative protein expression levels after correction with β -actin in the same samples based on peak intensity.

As shown in FIG. 7, the results of the experiments show that the combination of bakuchiol and cyclophosphamide can up-regulate the expression of Caspase 3, Caspase8, JNK and Bax in tumor tissues and down-regulate the expression of Bcl-2. The effect of the combination of the bakuchiol self-microemulsion and the cyclophosphamide is more obvious than that of the CTX group and the bakuchiol self-microemulsion group which are used singly. The psoralen self-microemulsion and cyclophosphamide are combined to enhance the relative expression of the apoptosis factor of the tumor cells of the mice.

When the bakuchiol is used in combination with other chemotherapeutic drugs, such as cisplatin, mitomycin and vincristine, similar experimental results are obtained, and the bakuchiol can improve the anti-tumor effects of the cisplatin, mitomycin and vincristine, promote the withering of tumor cells and reduce the inhibition effect of the chemotherapeutic drugs on the growth of leucocytes.

As shown in FIG. 8, NVB is vinorelbine with a concentration of 0.01umol/L, and the cells are A549 (human non-small cell lung cancer cells), and the cell viability is detected by CCK8 after the drug reaction. When 0.01umol/L NVB is combined with 0umol/L BAK (bakuchiol) respectively, 0.5umol/L and 1,5umol/L BAK, the results show that when the BAK concentration is more than 0.5umol/L, the activity of cancer cells can be obviously reduced.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of bakuchiol in improving antitumor effect of chemotherapeutic drugs is provided.

2. The use of claim 1, wherein bakuchiol increases the anti-tumor effect of the chemotherapeutic agent while decreasing the immunological damage of the chemotherapeutic agent.

3. The use as claimed in claim 1, wherein the bakuchiol acts synergistically with the chemotherapeutic to promote expression of apoptosis factor associated with tumor cells and promote apoptosis of tumor cells, and wherein the bakuchiol reduces damage to leukocytes by the chemotherapeutic.

4. The use as claimed in claim 1, wherein the psoralen and the chemotherapeutic agent act synergistically to up-regulate expression of Caspase 3, Caspase8, JNK, Bax and down-regulate expression of Bcl-2 in tumor tissues.

5. The use of claim 1, wherein the chemotherapeutic agent is cyclophosphamide, cisplatin, mitomycin or vincristine.

6. The use of claim 1, wherein the mass ratio of the bakuchiol to the chemotherapeutic agent is (75-240): (10-40).

7. The use as claimed in claim 1, wherein the bakuchiol is a bakuchiol self-microemulsion, and the mass ratio of the bakuchiol self-microemulsion to the chemotherapeutic agent is (250- & 800): (10-40).

8. The use of claim 7, wherein the bakuchiol self-microemulsion is prepared by: selecting bakuchiol as oil phase, adding emulsifier and co-emulsifier, placing in water bath at 37 + -2 deg.C and stirring at 100 + -5 rpm.

9. Use according to claim 8, wherein the emulsifier is Cremophor RH40 or Cremophor EL 35 and the co-emulsifier is Labrasol.

10. The use of claim 7, wherein the mass ratio of the bakuchiol to the emulsifier to the co-emulsifier is (1-4): (3-5): (2-4).