KR100860272B1 - Method for extraction and purification of effective component from agrimonia pilosa ledeb and crude drug composition containing extract thereof for prevention and treatment of pimple - Google Patents

Abstract

The present invention relates to a method for extracting and purifying an active ingredient from Seonhakcho, and to a composition for preventing and treating acne containing the extract, and more particularly, to a condition of finely pulverized Seonhakcho ( Agrimonia pilosa Ledeb ) powder at the level of nanoparticles. The present invention relates to a method for efficiently extracting and purifying an active ingredient having a specific antimicrobial effect against Propionibacterium acnes by extracting and purifying under the present invention, and a herbal composition for preventing and treating acne containing such sunchocho extract. .

Seolhakcho ( Agrimonia pilosa Ledeb ) extract according to the present invention has no side effects on the human body, and is very useful as a preventive and therapeutic agent for acne because of its excellent effect of inhibiting the growth of acne bacteria even at a small dose.

Seonhakcho, Acne Treatment, Propionibacterium Acnes Antibacterial Activity, Nanoparticles

Description

METHOD FOR EXTRACTION AND PURIFICATION OF EFFECTIVE COMPONENT FROM AGRIMONIA PILOSA LEDEB AND CRUDE DRUG COMPOSITION CONTAINING EXTRACT THEREOF FOR PREVENTION AND TREATMENT OF PIMPLE}

Figure 1 is a schematic diagram showing the process of extraction and purification of acne bacteria inhibitors from Sunhakcho.

Figure 2 compares the size of the growth inhibitory ring formed when the methanol extract of Seonchocho powder pulverized to 30 ~ 600 nanometers size and methanol extract of Seonchocho pulverized pulverized to 1 ~ 3 cm size dropwise to pre-cultured acne causative bacteria It is photograph to say.

Figure 3 is a photograph comparing the size of the growth inhibitory ring formed when the n-hexane fraction of Sunhakcho was first purified by silica gel column chromatography on the pre-cultured acne causative bacteria.

Figure 4 is a comparison of the size of the growth inhibitory ring formed when the purified products obtained by secondary purification of CH ₂ Cl ₂ : EtOAc = 4: 1 purified by Seonhakcho to silica gel column chromatography It is photograph to say.

The present invention relates to a method for extracting and purifying an active ingredient from Seonhakcho, and a herbal composition for preventing and treating acne containing the extract, and more particularly, 30 to 600 nanometers in diameter made by grinding Ahakoniacho ( Agrimonia pilosa Ledeb ). Extracting and purifying phosphorus powder under certain conditions to effectively extract and purify the active ingredient that strongly inhibits the growth of propionibacterium acnes , which is known as the main causative agent of worsening acne symptoms, and such sunchocho extract It relates to a pharmaceutical composition for preventing and treating acne containing.

In general, acne is an inflammatory disease occurring in the hair follicle sebaceous gland of the skin and is caused by the interaction of androgen (male hormone), skin keratinogenesis, and sebum and bacteria interaction, especially propionibacterium acnes , an anaerobic skin flora Known as the main causative organism.

That is, sebum secretion is promoted by testosterone which is one of the male hormones, and micro-comedones are formed as hyperkeratosis proceeds due to excessive secretion of sebum. As a result of the proliferation of acne bacteria, anaerobic epidermis bacteria, which are formed on the microcotton thus formed, various low-molecular substances including free fatty acids are produced by lipolytic enzymes, which are known to cause inflammation.

To date, various therapies have been used to treat acne. For example, anti-androgens that inhibit sebum production, nonsteroidal anti-inflammatory agents used as anti-inflammatory agents, and resorcinol, sulfur and benzoylperoxide, which have antibacterial effects, and tetracycline, erythromycin, and macrocycline, which have antibiotic effects. And other methods of inhibiting activity such as bacillus bacilli. In recent years, a therapeutic method using vitamin A derivatives such as retinoic acid has also been widely used.

However, while these synthetic drugs have some effect on the treatment and prevention of acne, most of them have the problem that they can cause side effects such as skin redness, skin hypersensitivity reaction or photosensitive reaction.

Therefore, in recent years, in order to overcome the shortcomings of such synthetic drugs, extensive research has been conducted on natural products with high human stability and few side effects even after long-term use. However, the natural product therapeutics also vary depending on the type, but the separation and purification is not easy, it takes a long time for the separation of the active ingredient, there is a disadvantage in that the cost of raw material supply and processing is expensive.

In view of the above problems, the present inventors found that Seonhakchocho extract exhibited specific antimicrobial activity against Propionibacterium acnes while conducting research on acne prevention and treatment for herbal ingredients and plants. It became. In addition, by using the recently developed nano-technology to extract the pulverized Seonhakchoe to a fine level of nano-scale to extract the extraction efficiency and antimicrobial activity against propionibacterium acnes significantly improved the present invention was completed.

Accordingly, it is an object of the present invention to provide Seonchocho extract and its fractions as an acne prophylactic and therapeutic agent. In addition, to provide a pharmaceutical composition for preventing and treating acne containing the Seonhakcho extract and fractions thereof as an active ingredient.

The external preparation composition for preventing and treating acne provided by the present invention in order to achieve the technical problem as described above relates to an external preparation composition for preventing and treating acne containing an extract of Agrimonia pilosa Ledeb as an active ingredient, wherein the extract Silver pulverulent pulverized to make a powder having a diameter of 30 ~ 600 nanometers, solvent extraction of the powder with an aqueous methanol solution or an ethanol aqueous solution and then eluting the n-hexane fraction obtained by solvent fractionation with a mixture of n- hexane and water on a silica gel column. The basic characteristics of the present invention are obtained by using a mixed solvent of dichloromethane and ethyl acetate as the elution solvent.

At this time, in the external preparation composition for preventing and treating acne of the present invention as described above, the dichloromethane and ethyl acetate mixed solvent used as the elution solvent is particularly preferably a dichloromethane: ethyl acetate 2: 1 to 4: 1 mixed solvent. Do.
Referring to the present invention in more detail as follows.

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Seonhak vinegar used as a raw material in the present invention is a medicinal herb taken from a perennial herb of Rosaceae and dried. The scientific name is Agrimonia pilosa Ledeb. Its ingredients include agrimonin, agrimonolide, tannin, sterol, organic acid and saponin, and are effective in gastritis, gastric ulcer, enteritis and chronic diarrhea. It is widely known that it has significant effects on depression, physical weakness, nerve weakness, bleeding due to pulmonary tuberculosis or gastric ulcer, and hemorrhoidal bleeding.

In this regard, Korean Patent Laid-Open Publication No. 2005-0004355 discloses sunhak vinegar by conventional ethanol extraction method together with a description of a cosmetic composition made by mixing golden primrose, sunhakcho, omae, rhubarb and pine bark extract extracted by supercritical extraction method. In case of using the extract treated alone, it is known that the strain grows again even if the growth of the Propionibacterium acnes is stopped for a while.

However, the present invention improves the extraction efficiency by making Seonhakcho into nano-level particles through nanotechnology, and provides a purification technique using solvent fractionation and column chromatography, so that the active ingredient can be extracted with the most active composition. The structure is different from the above-described disclosed invention.

In addition, the present invention compared to the above-described disclosed invention, the cost is lower than the supercritical extraction method, but overcoming the disadvantages while maintaining the advantages of the solvent extraction method inferior extraction efficiency, which can be used alone as sunhakcho extract as an acne treatment Since the object and effect of the invention are different in that it provides a solution, the two inventions must be different from each other. That is, as the object of the present invention by inhibiting the activity of the Propionibacterium acnes ( propionibacterium acnes ) has not been reported until now to apply the sunhakcho extract alone for the purpose of preventing and treating acne.

The present invention is to effectively separate the active ingredient effective in the prevention and treatment of acne by the following method from the above-mentioned Sunhakcho ( Agrimonia pilosa Ledeb ) as described in more detail as follows.

First, Agrimonia pilosa Ledeb ( Agrimonia pilosa Ledeb ) is put into the hammer mill and the first crushing, and then into the ACM (Air classifying mill) mill to finely secondary crushing. Then, the second pulverized product is added to the ultrafine pulverizer to obtain nano-level sunhakcho powder.

Into the Seonhakcho powder obtained by the above method, an aqueous solution of alcohol such as methanol (MeOH) or ethanol (EtOH) is added and cooled, followed by extraction of the solvent. At this time, the amount of the alcohol solution used is 3 to 5 times better than that of Seonchocho powder.

The separated alcohol aqueous solution is concentrated under reduced pressure under the conditions of 50 to 60 mmHg and 30 to 40 ℃ to remove the alcohol in the extract, through which the alcohol component remaining more than necessary in the alcohol extract is removed to the next solvent Fractions can be made easily. In addition, for a part of the extract obtained here, the above-mentioned concentration under reduced pressure is repeated to obtain an extract in powder form.

The concentrated solution was taken in a fractional filter, and a mixed solution of n-hexane and water in a volume ratio of 5: 1 to 1: 5 was added to the concentrated solution at a volume ratio of 1: 2, and the solvent was fractionated twice to obtain an n-nucleic acid layer. Obtain a solvent fraction of the water layer. At this time, the activity of the n-hexane fraction is the most excellent. A portion of each solvent fraction is concentrated under reduced pressure to obtain a powder.

The n-hexane fraction may be purified by silica gel column chromatography while changing the composition of the eluent to selectively increase the amount of the effective ingredient effective for inhibiting the growth of acne bacteria.

It was confirmed that the Seonhakcho extract obtained in each step by performing the extraction and purification steps as described above has an effect of inhibiting the activity of Propionibacterium acnes .

On the other hand, the present invention includes an external preparation composition for preventing and treating acne containing Seonhakcho extract as an active ingredient, the herbal composition according to the present invention to prepare a face wash, soap, cream, ointment, etc. through a conventional formulation method. Can be. In addition, Seonhakcho extract of the present invention may be used as a preventive and therapeutic agent for acne by adding to basic cosmetics such as foam cleansing, lotion, essence and the like.

For example, in the case of preparing the face wash and soap, it is prepared by containing the Seonchocho extract in the usual face wash and soap base. At this time, in order to improve the acne treatment effect (Keratolytic or Exfoliating agents such as salicylic acid, alpha-hydroxy acid, beta-hydroxy acid or sulfur) ) Can be added. In addition, fragrances, pigments, fungicides, antioxidants, preservatives, moisturizers, etc. can be added and used, and thickeners, inorganic salts, synthetic polymer materials, etc. can also be used together for the purpose of physical property improvement.

In the preparation of creams, sunhakcho extract is contained in a cream base of general oil type (O / W), and fragrances, chelating agents, pigments, antioxidants, preservatives, etc. are used, while proteins, minerals, Synthetic or natural materials such as vitamins can be used.

Facial washes, soaps, creams, and the like can be easily prepared in accordance with conventional manufacturing methods, respectively, and can be prepared in any form, such as liquid, cream, paste and solid.

When using the extract of Sunhakcho according to the present invention, it is preferable to contain 0.01 to 10.0% by weight based on the total weight of the composition. If the content is less than 0.01% by weight, the effect is not sufficient to achieve the desired purpose. If the content is more than 10.0% by weight, the effect is not increased in proportion to the increase of the content. Because you can not.

However, in the case of using the purified product by silica column chromatography, since the content of the active ingredient is high, even if it contains only 0.01 to 1% by weight based on the total weight of the composition can exhibit the desired effect.

In addition, Seonhakchocho extract according to the present invention is a natural extract, so there is no concern of side effects compared to other synthetic medicines, and in fact, the toxicity test results of these extracts were found to have no effect on the living body.

Hereinafter, the present invention will be described in more detail based on Examples and Experimental Examples. However, these Examples and Experimental Examples are only intended to help the understanding of the present invention, the scope of the present invention is not limited by these Examples.

Example 1 Preparation of Seonhakcho powder

(a) 100 g of dried Seonhak vinegar was put into the hammer mill, and firstly pulverized into particles having a size of 200 to 300 mesh. Was added to an ACM (Air classifying mill) grinder, which was finely ground. And (c) the pulverized product of step (b) was put into a dry grinder capable of ultra-fine grinding using a pressure grinding rod and a ceramic ball to prepare 98 g of Seonhakcho powder having a diameter of 30 ~ 600 nanometers.

Example 2 Preparation of Seonchocho extract (I) using alcohol

80% (v / v) aqueous methanol solution was added to a volume of about 3 times with respect to Seonchocho powder obtained in Example 1, and the solvent was extracted after cooling for 20 hours. The methanol extract (I) thus obtained was concentrated under reduced pressure under the conditions of 50 to 60 mmHg and 30 to 40 ° C., and a part of the resultant concentrate was taken, and the above-mentioned concentration under reduced pressure was repeated to obtain a powdery extract.

Comparative Example 1

In order to compare the extraction efficiency according to the nano-materialization of the raw material, methanol extract (I-A) was prepared in the same manner as in Example 2 using Sunhakchoe of 1 ~ 3 cm size.

Experimental Example 1 Minimum Inhibitory Concentration (MIC) Measurement Experiment

MIC was measured to confirm the antimicrobial activity of acne bacteria of Sunhakcho extract (I) and Comparative extract (I-A) obtained in Example 2 and Comparative Example 1. First, in order to activate propionibacterium acnes, the test strain, the cells were preincubated for 48 hours in BHI liquid medium (Brain-Heart Infusion broth; 3.7%). A sample solution was prepared by adding Seonchocho extract (I) dissolved in 80% ethanol aqueous solution to a sterilized culture medium at regular concentration intervals, and then pre-cultivated propionibacterium acnes in 1.0 mL solution. Inoculated at a concentration of%. After incubation in an anaerobic incubator at 35 ° C. for 3 days, the growth of the bacteria was observed. As a result, MICs of sunhakcho extract (I) and comparative extract (I-A) for acne causing bacteria were 0.0025% and 1.0%, respectively.

Experimental Example 2 Antibacterial Activity Test (Paper Disc Test)

Paper Disc Test was performed as another method for confirming the antimicrobial activity against acne bacteria of Sunhakcho extract (I) and Comparative extract (I-A) obtained in Example 2 and Comparative Example 1. First, in order to activate the test strain Propionibacterium acnes was precultured in BHI medium for 48 hours. The culture medium thus prepared was plated in BHI solid medium (Brain-Heart Infusion broth 3.7%; agar 1.5%) and dried. Seonchocho extract (I) and Comparative extract (IA) obtained in Example 2 and Comparative Example 1 were dipped in a concentration of 20 μl on paper discs having a diameter of 8 mm at respective concentrations, and then placed on a solid medium prepared above. Incubated for 3 days. The antimicrobial activity was evaluated by observing the bacterial growth inhibition zone around the paper disc and measuring the size of the ring.

As a result, the bacterial growth inhibitory ring size of Sunhakcho extract (I) and comparative extract (I-A) were 61 mm (300 ug / mL) and 45 mm (500 ug / mL), respectively.

[Table 1] Comparative experiment of active material extraction efficacy according to the difference in miniaturization of raw materials

Example 3 Purification of Sunhakcho Extract by Solvent Fractionation

A portion of the Seonhakcho extract obtained in Example 2 was taken in a fractional filter, and a mixed solution of n-hexane and water (1: 1) was added at a volume ratio of 1: 2 to the concentrated solution, and the solvent was fractionated twice to obtain an n-hexane layer and a water layer. Fractions were concentrated under reduced pressure to give 320 mg of n-hexane extract and 530 mg of water extract.

Experimental Example 3 Antibacterial Activity Test of Each Solvent Fraction

In order to compare the antimicrobial activity of the solvent fraction obtained in Example 3 against acne bacteria (propionibacterium acnes), the antibacterial activity test was carried out in the same manner as in Experimental Example 2. At this time, each fraction was dissolved in methanol, and n-hexane extract (II) and water extracts were dipped 20 μl at each concentration, and the results are shown in Table 2 below.

[Table 2] Comparison of activities for each fraction

As can be seen in Table 2, the bacterial growth inhibition region of 19 mm was observed in the n-hexane extract (10 ug / ml) of the solvent fraction of Sunhakcho, and the bacterial growth inhibition region was observed in the water extract (500 ug / ml) It wasn't. Therefore, it was found that the antimicrobial component of Seonhakcho extract is a nonpolar substance.

Example 4 Primary Purification by Chromatography

As a result of Experiment 3, it was confirmed that n-hexane extract (II) exhibited the highest antimicrobial activity in the solvent fraction of Example 3, and silica gel column chromatography was performed on n-hexane extract (II). Thereby further purification. That is, 10 g of silica gel was filled in a glass column having an inner diameter of 15 mm and a length of 200 mm, and then subjected to chromatography by applying 250 mg of n-hexane extract (II) thereto. At this time, n-hexane, dichloromethane, ethyl acetate was used alone or mixed as the elution solvent. Each solvent was eluted and concentrated under reduced pressure to obtain each fraction.

Experimental Example 4 Antimicrobial Activity Test of the Chromatographic Extract Obtained in Example 4

In order to compare the antimicrobial activity of each solvent elution obtained in Example 4 against acne bacteria (propionibacterium acne tm) was carried out in the same manner as in Experiment 2, and dissolved in methanol for each concentration, each 20 μl was dropped. The results are shown in Table 3 below.

Table 3 Antimicrobial Activity Test of Primary Chromatography Extract

As can be seen from the results of Table 3, among the extracts obtained by the first chromatography, the bacterial growth inhibition region of CH ₂ Cl ₂ : EtOAc = 4: 1 extract (2.5 ug / mL) was the strongest as 18 mm. In other extracts, almost no bacterial growth inhibition zone was observed.

Example 5 Secondary Purification by Chromatography

As a result of Experiment 4, CH ₂ Cl ₂ : EtOAc = 4: 1 (III) among the various extracts of Example 4 showed the strongest antimicrobial activity. Photography was carried out. That is, 10 g of silica gel was charged into a glass column having an inner diameter of 15 mm and a length of 200 mm, and then subjected to chromatography by applying 75 mg of CH ₂ Cl ₂ : EtOAc = 4: 1 extract. At this time, a solvent in which n-hexane, dichloromethane and ethyl acetate was mixed was used as the eluting solvent, and the eluted solution was divided into fractions in order. The eluted fractions were concentrated under reduced pressure to give each fraction.

Experimental Example 5 Antimicrobial Activity Test of the Chromatographic Purified Product Obtained in Example 5

In order to test the antimicrobial activity against the acne bacteria (propionibacterium acnes) of the chromatographic fractions obtained in Example 5 was carried out in the same manner as in Experiment 2, each fraction was dissolved in methanol for each concentration 20 ㎕ each Dropped, the results are shown in Table 4 below.

Table 4 Antimicrobial Activity Test of Second Chromatography Extract

As a result, the growth inhibition of about 9.5 mm and 23 mm was observed only in the CH ₂ Cl ₂ : EtOAc = 19: 1 and CH ₂ Cl ₂ : EtOAc = 8: 3 (IV) fractions, respectively. Little growth inhibition area was observed.

[Examples 6 to 9 and Comparative Example 2] Preparation of Sunhakcho extract-containing ointment composition

The mixture of Seonchocho extract and Ointment base of the Seonchocho extract (I) obtained in Example 2 or the CH ₂ Cl ₂ : EtOAc = 8: 3 (IV) fraction obtained in Example 5 at a composition ratio as shown in Table 5 below The ointment composition was prepared by uniformly mixing the composition by stirring, heating, degassing and cooling in a homo mixer.

[Table 5] Unit: weight%

Experimental Example 6 Test of bactericidal power against acne bacteria of the ointment composition

In order to confirm the effect of preventing and treating acne for the ointment compositions of Examples 6 to 9 and Comparative Example 2, the following experiments were carried out. First, after acne bacteria (propionibacterium acnes) was inoculated in Petri dish and incubated in an anaerobic culture tank for 3 days and the colony number (N1) of acne bacteria was measured, 5% aqueous solution of the ointment composition of Examples and Comparative Examples After dropping on a petri dish and incubated in an anaerobic culture tank for 3 days, colony count (N2) was measured. Using colony numbers N1 and N2, the bactericidal rate [%, (N1-N2) / N1 ㅧ 100] against acne bacteria was determined.

The bactericidal effect is determined only when the sterilization rate for acne bacteria is greater than 90%, and the experimental results are shown in Table 6 below.

TABLE 6

[Examples 10 to 13 and Comparative Example 3] Preparation of Sunhakcho extract-containing liquid formulation

The mixture of Seonchocho extract of the Seonchocho extract (I) obtained in Example 2 or the CH2Cl2: EtOAc = 8: 3 (IV) fraction obtained in Example 5 and the liquid base in a composition ratio as shown in Table 7 in a homomixer The face wash composition was prepared by uniformly mixing the composition by stirring, heating, degassing and cooling.

[Table 7] Unit: weight%

Experimental Example 7 Test of Sterilizing Power against Acne Bacteria in Liquid Composition

In order to confirm the effect of preventing and treating acne on the liquid composition of Examples 9 to 12 and Comparative Example 3, the experiment was performed in the same manner as in Experimental Example 6 and the results are shown in Table 8 below.

TABLE 8

[Examples 14 to 17 and Comparative Example 4] Preparation of Sunhakcho extract-containing face wash composition

The mixture of Seonchocho extract of the Seonchocho extract (I) obtained in Example 2 or the CH2Cl2: EtOAc = 8: 3 (IV) fraction obtained in Example 5 and the face wash base with a composition ratio as shown in Table 9 in the homomixer The face wash composition was prepared by uniformly mixing the composition by stirring, heating, degassing and cooling.

Table 9 Unit: Weight%

Experimental Example 8 Test of Bactericidal Activity against Acne Bacteria in Cleanser Composition

In order to confirm the effect of preventing and treating acne for the face wash composition of Examples 14 to 17 and Comparative Example 4, the experiment was performed in the same manner as in Experimental Example 7 and the results are shown in Table 10 below.

TABLE 10

[Examples 18 to 21 and Comparative Example 5] Preparation of Sunhakcho extract-containing soap composition

According to the composition as shown in Table 11 below, the Sunhakcho extract (I) or (IV) and the soap base were added to the mixer and mixed well, and then extruded, cut and molded in a soap manufacturing machine to prepare a solid soap composition.

[Table 11] Unit: weight%

(Soap base: Use what is listed in the Cosmetic Ingredients Standard.)

Experimental Example 9 Test of Bactericidal Activity against Acne Bacteria in Soap Compositions

In order to confirm the effect of preventing and treating acne on the soap composition prepared in Examples 18 to 21 and Comparative Example 5, the experiment was performed in the same manner as in Experiment 8 and the results are shown in Table 12 below.

TABLE 12

Examples 22 to 25 and Comparative Example 6 Preparation of Sunhakcho extract-containing cream composition

According to the composition as shown in Table 13 below, the Sunhakchoe extract (I) or (IV) is well emulsified in a mixer together with an oily ingredient, an aqueous ingredient, a surfactant, etc., which are cream base, and degassed, filtered, and cooled. And the cream composition of Comparative Example 6. To this, preservatives, flavors and pigments were added as additives and prepared in oil-in-water (O / W) emulsion form.

[Table 13] Unit: weight%

Experimental Example 10 Test of bactericidal power against acne bacteria

In order to confirm the effect of preventing and treating acne on the cream composition prepared in Examples 22 to 25 and Comparative Example 6 was the same experiment as in Experiment 9, the results are shown in Table 14 below.

TABLE 14

As shown in Examples 6 to 25, all of the external preparations with sunhakcho extract according to the present invention as an active ingredient showed that the bactericidal rate of acne bacteria is 90.0% or more, which is excellent in acne prevention and treatment effect.

The production method according to the present invention and Seonhakcho extract prepared using the same can be modified and applied in various forms within the scope of the technical idea of the present invention and is not limited to the above preferred embodiment. The present invention described above is capable of various substitutions, modifications, and changes without departing from the technical spirit of the present invention for those skilled in the art to which the present invention pertains. It is a matter of course that it is not limited, and should be determined including not only the claims described below but also the claims and equivalents.

Seolhakcho ( Agrimonia pilosa Ledeb ) extract according to the present invention has the effect of strongly inhibiting the growth of propionibacterium acnes known as the main causative bacteria to worsen acne symptoms.

In addition, the composition according to the present invention has no side effects on the human body, and finely pulverized sunhakchoe at the nanoparticle level to extract and purify in an optimal way to maximize the content of the active ingredient to effectively treat acne with a small dose Bar, it can be applied as an external preparation for acne prevention and treatment containing it as an active ingredient.

Claims (7)

An external preparation composition for preventing and treating acne containing Agrimonia pilosa Ledeb extract as an active ingredient. The solvent was extracted with n-hexane and then the solvent was fractionated with a mixture of water and n-hexane fractions were eluted on a silica gel column, using a dichloromethane: ethyl acetate 2: 1 ~ 4: 1 mixed solvent as the elution solvent External preparation composition for preventing and treating acne. The external preparation composition for preventing and treating acne according to claim 1, wherein the external preparation is a face wash, a soap, a cream, or an ointment. delete delete delete delete delete

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