KR101566822B1 - Cosmetic containing herb extracts for improving atopic dermatitis - Google Patents

Abstract

The invention will be directed to a cosmetic composition for promoting improved atopic symptoms saengyakjae containing extract, more particularly, slender (Asiasari the bamboo sap Radix ), lobules ( Perillae Herba ), a device ( Perillae Semen ), horse riding ( Cimicifugae Rhizoma ), royal fashions ( Melandrii Herba ), Yoon Kyu ( Solani Nigri Herba , Arctii Semen , Potentillae Radix ), broiler ( Cinnamomi Cortex Spissus ), Hoelen rubra ), headache ( Phaseoli angularis Semen ), excess ( Ammomi Tsao - ko Frucuts ), ocherus ( Alpiniae Katsumadaii Semen ), buckwheat ( Fagopyrum esculentum ), honeybees ( Chenopodium album there is . centrorubrum ), the biceps ( Bistorta manshuriensis ), Lycoris squamigera), Island Thyme (Thymus quinquecostatus there is . japonica ), mugwort bat ( Senecio argunensis) and chamnamul (Pimpinella brachycarpa ) as an active ingredient, and can be used for improving atopic symptoms.

Bamboo sap * Herbal medicine * Atopy

Description

[0001] Cosmetic containing herb extracts for improving atopic dermatitis [0002]

The present invention relates to atopic symptoms improved promote skin cosmetic composition containing an extract saengyakjae, more particularly slender (Asiasari the bamboo sap Radix ), lobules ( Perillae Herba ), a device ( Perillae Semen ), horse riding ( Cimicifugae Rhizoma ), royal fashions ( Melandrii Herba ), Yoon Kyu ( Solani Nigri Herba , Arctii Semen , Potentillae Radix ), broiler ( Cinnamomi Cortex Spissus ), Hoelen rubra , Red Pox ( Phaseoli angularis Semen ), excess ( Ammomi Tsao - ko Frucuts ), ocherus ( Alpiniae Katsumadaii Semen ), buckwheat ( Fagopyrum esculentum ), honeybees ( Chenopodium album there is . centrorubrum ), the biceps ( Bistorta manshuriensis ), Lycoris squamigera), Island Thyme (Thymus quinquecostatus there is . japonica ), mugwort bat ( Senecio argunensis) and chamnamul (Pimpinella The present invention also relates to a skin cosmetic composition containing at least one herbal medicine extract selected from the group consisting of brachycarpa as an active ingredient.

Atopic dermatitis is a skin disorder characterized by a combination of symptoms such as strong cannibalism, dryness, severe keratinization, dandruff, erythema and swelling, skin cracking, severe dental eczema, It is an abnormal syndrome. It is a chronic disease that lasts for more than 10 years in many cases and causes great inconvenience to long-term life. In the past, infant and childhood began with the growth of the disease gradually improved, 7 to disappear before the age of 8 was the most common. However, since the 1970s, the rate of incidence has increased year by year due to pollution caused by rapid industrialization, environmental pollution, and the symptoms continue to adolescence and adulthood. Symptoms are associated with inflammation of the skin, accompanied by strong itching. Histamine overdose is a major cause of itching. If the skin is scratched by itching, erythema or eczematous lesions will appear. In severe cases, the skin will peel off or become fizzy (skin thickening).

Although there have been many research results and information on the cause of atopic dermatitis until now, definite theories have not yet been established about the precise pathogenesis and effective treatment methods due to the conflicting research data and complexity of the disease itself. Because of these problems, atopic dermatitis remedies, which have been developed to date, are more effective than the basic treatments such as steroids, oral antihistamines, hydroxyquinolines, tar preparations and hypoallergenic moisturizers, Products are dominant. However, any drug that has been developed so far does not fully meet the needs of patients. Until recently, topical steroids have been the main medicines for treating atopic dermatitis, but there is a desperate need for the development of safer nonsteroidal atopic dermatitis treatment without side effects due to resistance to long-term use and safety. Therefore, substances capable of simultaneously inhibiting the production of interleukin-2 and interleukin-4 and excessive proliferation of T cells, which are the final products of these cytokine responses, are more effective for treating atopic dermatitis than general immunosuppressive agents .

Korean Patent Laid-Open Publication No. 2008-0079396 discloses a composition for treating atopic skin diseases comprising natural vegetable and mineral materials including bamboo charcoal powder in association with improvement of atopic sensation containing bamboo ingredients, Korean Patent Publication No. 2004-0097026 There are atopic plants produced by mixing raw pine leaves, elm seeds, elm oil, raw pine needles, raw mulberry leaves, bamboo dew, raw pine, raw pine, raw pine, raw pine, licorice, raw green leaves, Herbal compositions for preventing and treating dermatitis are known. Korean Patent Laid-Open Publication No. 2004-0062230 discloses the improvement and prevention of atopic dermatitis containing extracts extracted from bamboo. In Korean Patent Publication No. 2004-0039664, a soap containing bamboo extract is known . Korean Patent Laid-Open Publication No. 2004-0039663 discloses that bamboo is heated at a high temperature to extract and extract bamboo extract (bamboo extract) in liquid form and is effective for the improvement and prevention of atopic dermatitis. However, there has not been developed a cosmetic composition for improving atopic symptoms containing a herbal medicine extract in bamboo sap, which is not a bamboo extract.

The object of the present invention is to provide a bamboo liquid which is capable of controlling an immune response abnormally activated in the skin, such as seesin, lobule, device, horse riding, royal flush, yonggu, The present invention further provides a pharmaceutical composition comprising at least one of herbal medicine extracts comprising at least one of herbal medicine extracts comprising at least one herbal medicine extract, , Contact dermatitis, and psoriasis, which is effective in improving the appearance of the skin.

The skin cosmetic composition according to the present invention is characterized in that the bamboo sap is added to the bamboo sap in the form of sesin, lobule, element, horse riding, fowl, fashion, , Mugwort bat, and mugwort, as an active ingredient.

According to the present invention, symptoms of atopic dermatitis can be improved.

The bamboo sap used in the present invention is produced as a by-product during periodic cutting to help growth during the bamboo cultivation process. The composition ratio of the content of the bamboo is varied depending on the environmental factors such as the area normally grown, It contains protein (0.08-0.7%), calcium (422mg / l), sugar (1.6-2.7g / l) and amino acid (7.6-30mg / l) .

Although the bamboo sap can be obtained from all bamboo trees, it is preferable from the standpoint of yield in terms of harvesting from the bamboo shoots, cotton bolls, and bamboo shoots, which are relatively useful weeds. As is commonly known, the bamboo roots are cut at the first node or just above the node of the second node (height: 10-20 cm), attached with a container, and then filtered with a filter cloth. The filtrate is generally sterilized and used in a cosmetic composition. It may be used as it is or may be used in powder form. Bamboo sap is preferably collected around May to June.

In the present invention, the bamboo sap solution may be used directly as an active ingredient of the cosmetic composition, or may be pretreated before discoloration and discoloration in storage before use and used as an active ingredient.

On the other hand, in the pretreatment method, bamboo sap immediately after harvesting is filtered with a filtration membrane having a pore size of 0.001 to 0.45 μm, preferably 0.2 μm, and anhydrous ethanol is added to the filtrate in an amount of 7.0 wt% By weight, preferably 7.0 to 50% by weight. If the size of the filtration membrane is less than 0.001 탆, the filtration takes a long time. If the size exceeds 0.45 탆, the filtration efficiency is ineffective.

That is, by filtering with a filtration membrane, protein having a large molecular weight which is likely to precipitate or denature during long-term preservation of bacteria, microorganisms and microorganisms present in bamboo sap is removed, and then anhydrous ethanol is added to inhibit the activity of the enzyme Prevent discoloration and detachment.

 The pre-treated bamboo sap is sealed immediately under a nitrogen atmosphere to prevent intrusion of external bacteria or microorganisms.

The content of the skin cosmetic composition of the bamboo sap may be appropriately selected according to the formulation, and may be selected within the range of 0.01 to 99.5% by weight, preferably 1.0 to 90.0% by weight based on the total weight of the composition.

The herbal medicine extracts used in the skin cosmetic composition of the present invention are herbal medicines which have been used for the purpose of symptom improvement and treatment of various diseases, as will be described in detail below.

Slender (Asiasari Radix) used in the present invention is used for purposes such as sentinel at the root of the perennial plant of jokdori pool (Asarum heterotropoides var. Mandshuricum) belonging to the little insect.- and (Aristolochiaceae) with, antipyretic relax analgesic, Jinhae action .

Perillae Herba is a perennial herb that belongs to the family Labiatae ( Perilla frutescens there is . acuta ), which is used to treat asthma, abdominal distension, cold and the like due to perspiration and detoxification efficacy.

Device ( Perillae Semen ) is a perennial herb that belongs to the Labiatae family, Perilla frutescens there is . acuta ), and it is used for the purpose of god dam, treatment of constipation.

Horseback riding ( Cimicifugae Rhizoma ) is a perennial plant belonging to the Ranunculaceae , stork ( Cimicifuga foetida ) or horseback riding ( Cimicifuga heracleifolia ). It has antimicrobial, detoxifying and sedative properties and is used for infectious diseases, headache treatment and so on.

Wangbul fashion (Melandrii Herba) is a biennial herbaceous jangguchae belonging to the Dianthus (Caryophyllaceae) (Melandrium firmum and Melandrium apricum . It is used for the treatment of amenorrhea, insufficiency of juice, gingko hemorrhage and the like because it has the efficacy such as hibernation , flowering , and Choi.

Solani Nigri Herba is an annual herbaceous plant belonging to Solanaceae ( Solanum nigrum ) as the outpost, anti-inflammation, cyanosis, detoxification, hibernation, and has the efficacy.

Arctium Semen is a fruit of a burdock ( Arctium lappa ), a biennial herb that belongs to the Compositae family, and has effects such as antibacterial activity, hypoglycemia, elimination of customs, elimination, and detoxification.

Potentillae Radix is a perennial herbaceous flower belonging to Rosaceae ( Potentilla chinensis ) or hairy flower ( Potentilla chinensis there is . concolor ), it has efficacy such as elimination of customs and detoxification, and is used for treating dysentery, rheumatoid arthritis, skin diseases and the like.

Broiler ( Cinnamomi Cortex Spissus ) is an evergreen tree belonging to the genus Lauraceae ( Cinnamomum loureirii , or Cinnamomum cassia , and has a sedative, antimicrobial, and dry texture.

Hoelen rubra is a reddish part of sclerotia of Poria cocos belonging to Polyporaceae and is used for diuretic, moist heat removal and so on.

Pediatric angularis Semen ) is a perennial herb that belongs to Leguminosae , Phaseolus calcaratus ) or red bean ( Phaseolus It is a seed of angularis . It is used to treat symptoms of various species, each jaundice, jaundice, hari, etc. due to its efficacy such as diuretic, humid, blood circulation, drainage and detoxification.

Excess ( Ammomi Tsao - ko Frucuts ) are perennial herbs belonging to the genus Zingiberaceae ( Amomum It is a fruit of tsao - ko , and it is used to treat symptom of malaria, flatulence, vomiting, hari, etc.

Alpiniae Katsumadaii Semen is a seed of perennial herb Alpinia katsumadai belonging to Zingiberaceae . It has the effects of dormancy, hunger, humidity and so on. ≪ / RTI >

Buckwheat ( Fagopyrum esculentum ) is an annual herb of Polygonaceae . Seeds are used for symptoms such as acute enteritis, chronic hari, and congestion, stems and leaves have hemostatic, capillary strengthening effects.

Chenopodium album there is . centrorubrum ) is an annual herb of Chenopodiaceae and is used for edible, medicinal or feed use. It has the efficacy of cheerfulness, humidification, insecticide, etc. and is used for branch office, construction, and tentative purpose.

Bass Tails manshuriensis ) is a perennial herb of Polygonaceae . Rhizomes are used to treat tetanus, fever, rubella , and intestinal mucus , with efficacy of cyanosis , gentleness and moisturizing .

Lycoris squamigera ) is a perennial herb belonging to Amaryllidaceae . It is used for symptoms such as throat and water because it has effects such as genomic, diuretic, detoxification, and lowermost.

Island Thyme (Thymus quinquecostatus there is . japonica ) is a perennial plant of Lepidoptera ( Labiatae ), which is used as a respiratory protective agent for bronchitis and as an analgesic for neuritis.

Senecio argunensis is a perennial herb of Compositae . It is used for the treatment of dysentery, acute conjunctivitis, sore throat, eczema, dermatitis and the like because the outpost has cyanosis and detoxification efficacy.

Pimpinella brachycarpa ) is a perennial herb of mountain and ( Umbelliferae ), edible or medicinal in outpost. Hypertension, prevention of stroke, neurosis, treatment of large bowel disease, hemostasis, and paralysis.

Although the herbal medicine extract used as an active ingredient in the skin cosmetic composition of the present invention has been proven to be effective in improving symptoms and treating various diseases as described above, I did.

In the present invention, the amount of the extract to be used is in vitro ( in vitro) is 0.001 to 10% by weight, based on the total weight of the composition on the basis of experiments, preferably 0.01 to 5% by weight. If the content of the extract is less than 0.001% by weight, the action as an active ingredient is difficult to expect. If the content of the extract is more than 10% by weight, the formulation stability may be deteriorated. From this viewpoint, it is more preferable to use it in an amount of 0.01 to 5% by weight.

The skin cosmetic composition according to the present invention can be used for the purpose of improving the symptom of the skin diseases such as atopic dermatitis, contact dermatitis or psoriasis, and further treating and preventing the object of the present invention. Do not.

When the present invention is provided as a functional cosmetic composition, it may be formulated as a formulation of a softening agent, a nutritional lotion, a massage cream, a nutritional cream, a pack, a gel, an essence or a skin adhesive type cosmetic composition or a lotion, ointment, And may be formulated into transdermal dosage forms such as patches or sprays. In the cosmetic composition of each formulation, components other than the above-mentioned essential components may be formulated by a person skilled in the art according to the formulation or purpose of use of other cosmetic compositions.

Hereinafter, the constitution and effect of the present invention will be described in more detail with reference to examples and test examples. However, the present invention is not limited to these examples.

<Reference Example 1> Production method of bamboo extract

100g of bamboo powder was obtained by pulverizing bamboo leaves and stem parts. It was immersed in 1000 g of purified water and eluted at 40 DEG C for 48 hours. Subsequently, the solids were separated by a centrifuge, and the separated supernatant was taken and filtered. 1.08 g of a bamboo extract was obtained in accordance with a conventional vacuum concentration method.

&Lt; Referential Example 2 > Preparation method of pretreated bamboo sap

The first or second nodal area was cut out from the root of bamboo, and the bamboo sap from the roots was collected using a connector attached to the cut-off area. The bamboo sap just after harvesting was passed through a filtration membrane having a pore size of 0.2 mu m, and then anhydrous ethanol was added to the filtrate at a concentration of 7.0 wt% to perform pre-treatment. 100 g of the thus-obtained bamboo sap solution was pulverized according to a conventional vacuum concentration method to obtain 1.05 g.

<Reference Example 4> Production method of bamboo extract

100g of bamboo powder was obtained by pulverizing bamboo leaves and stem parts. It was immersed in 1000 g of purified water and eluted at 40 DEG C for 48 hours. Subsequently, the solids were separated by a centrifuge, and the separated supernatant was taken and filtered. 1.08 g of a bamboo extract was obtained in accordance with a conventional vacuum concentration method.

&Lt; Referential Example 5 > Production method of herbal medicine extract

Dried and crushed 1 kg of each of the sesame, lobule, element, horse riding, royal fashion, yonggyu, woebangjae, Then, 95% ethanol of four times the weight (kg) was added, and the mixture was heated at 80 DEG C for 2 hours to extract an active ingredient. This was allowed to stand at room temperature for one day to precipitate impurities. The filtrate was filtered twice using a filter paper, and the filtrate was completely evaporated by using a reduced pressure evaporator and concentrated.

< Example  1 to 21 and Comparative Example 1 ~ 4> Of skin cosmetics (cream)  Produce

In order to use the composition of the present invention as a cosmetic, a composition containing the bamboo sap, bamboo extract, and herbal extract prepared in Referential Examples 1 to 5 was prepared in the composition shown in Table 1 below.

[Table 1]

Ingredients (% by weight) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 Bamboo extract - - - - - - - 10 Bamboo sap 10 10 10 10 10 10 10 - Sesin extract 0.1 - - - - - - - Leaf extract - 0.1 - - - - - - Element extract - - 0.1 - - - - 0.1 Horse riding extract - - - 0.1 - - - - Kingfisher fashionable extract - - - - 0.1 - - - Yonggu extract - - - - - 0.1 - - Extract from wisteria - - - - - - 0.1 - Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Stearic acid 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 Stearyl alcohol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Glycerol stearate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Arachidyl behenyl alcohol and arachidyl glucoside 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Cetylaryl Alcohol and Cetearyl Glucoside 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Caprylic / capric triglyceride 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 Cyclomedicone 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 Triethanolamine 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

[Table 2]

Ingredients (% by weight) Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Comparative Example 2 Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 Bamboo extract - - - - - - - 10 Bamboo sap 10 10 10 10 10 10 10 - Gwangnyeongchi extract 0.1 - - - - - - - Broiler extract - 0.1 - - - - - - Root Extract - - 0.1 - - - - - Red Pepper Extract - - - 0.1 - - - 0.1 Excess extract - - - - 0.1 - - - Herbal Extract - - - - - 0.1 - - Buckwheat extract - - - - - - 0.1 0.1 Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Stearic acid 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 Stearyl alcohol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Glycerol stearate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Arachidyl behenyl alcohol and arachidyl glucoside 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Cetylaryl Alcohol and Cetearyl Glucoside 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Caprylic / capric triglyceride 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 Cyclomedicone 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 Triethanolamine 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

[Table 3]

Ingredients (% by weight) Example 15 Example 16 Example 17 Example 18 Example 19 Example 20 Comparative Example 3 Comparative Example 4 Purified water To 100 To 100 To 100 To 100 To 100 To 100 To 100 To 100 Bamboo extract - - - - - - 10 10 Bamboo sap 10 10 10 10 10 10 - - Sweet pea extract 0.1 - - - - - 0.1 - Bombyx mori extract - 0.1 - - - - - 0.1 Extract - - 0.1 - - - - - Island Thyme Extract - - - 0.1 - - - - Mugwort bat extract - - - - 0.1 - - - Truffle extract - - - - - 0.1 - - Vegetable hydrogenated oil 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Stearic acid 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 Stearyl alcohol 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Glycerol stearate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Polyglyceryl-10pentastearate and behenyl alcohol and sodium stearoyl lactylate 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Arachidyl behenyl alcohol and arachidyl glucoside 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Cetylaryl alcohol
& Cetearyl glucoside 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 PEG-100 Stearate & Glycerololate & Propylene Glycol 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Caprylic / capric triglyceride 11.00 11.00 11.00 11.00 11.00 11.00 11.00 11.00 Cyclomedicone 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 Triethanolamine 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

<Test Example 1> Test for improvement of atopic symptoms in human body

In order to confirm the effect of improving the atopic symptoms by the composition of the present invention, the cream prepared in Comparative Examples 1 to 4 and Examples 1 to 20 was applied to panels to evaluate the degree of improvement of atopic dermatitis. For each of the 48 panelists who thought that there was a symptom of dermatitis showing similar symptoms, the composition of each of the following Tables 1 to 3 was tested twice daily at 24 to 26 ° C and 75% And applied to the face for 12 weeks. At the end of the test, subjects were asked to fill out a questionnaire and conducted a subjective efficacy evaluation. In the questionnaire, symptoms of atopic dermatitis were classified into three categories according to the symptoms of "fever", "dryness", "keratinization", "dandruff", "erythema", "swelling", "skin crack" . When the symptom was present, the degree of improvement was evaluated for each symptom, and the improvement was evaluated by an individual to evaluate the improvement effect of atopic symptoms. The results are shown in Tables 4-6.

[Table 4]

Application group (number of respondents) (persons) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Significant improvement 2 2 2 2 2 2 2 - Improving - - - - - - - One No change - - - - - - - One worse - - - - - - - -

[Table 5]

Application group (number of respondents) (persons) Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Comparative Example 2 Significant improvement 2 2 2 2 2 2 2 - Improving - - - - - - - - No change - - - - - - - 2 worse - - - - - - - -

[Table 6]

Application group (number of respondents) (persons) Example 15 Example 16 Example 17 Example 18 Example 19 Example 20 Comparative Example 3 Comparative Example 4 Significant improvement 2 2 2 2 2 2 - - Improving - - - - - - One One No change - - - - - - One One worse - - - - - - - -

It can be seen from the above Tables 4 to 6 that the composition containing the herbal medicine extract in the bamboo sap of the present invention further improves the symptoms of the atopic dermatitis as compared with the case where the composition containing the herbal medicine extract is further applied to the bamboo extract .

Claims (3)

a) bamboo sap; And b) One species selected from the group consisting of sezin, lobules, element, horse riding, royal fashions, yonggye, woogyeo, stomach, broiler, red ginseng, red head, Herb medicine extract; As an active ingredient, a cosmetic composition for enhancing skin atopic symptoms, The bamboo sap solution is contained in an amount of 5 to 15% by weight based on the total weight of the composition, and the herbal medicine extract is contained in an amount of 0.1 to 1% by weight based on the total weight of the composition. Composition. The method according to claim 1, (a) filtering the bamboo sap from bamboo with a filtration membrane having a pore size of 0.001 to 0.45 mu m; And (b) adding anhydrous ethanol in an amount of 7.0 to 50% by weight to the filtrate filtered in the step (a); Wherein the pretreatment step comprises the steps of: delete