JP6055623B2 - Periodontal disease prevention and treatment composition and cosmetic preservative - Google Patents

Description

  The present invention relates to a composition for preventing and treating periodontal disease and a preservative for cosmetics.

  Chronic (adult) periodontitis is a chronic infectious disease caused by periodontal tissues mainly due to the growth of a group of bacteria called gram-negative anaerobic bacilli among the resident bacteria constituting the gingival crevicular flora. The morbidity rate increases with aging, and it is said that many of those in their 40s and beyond have localized or diffuse periodontitis. The entity of this disease is chronic purulent inflammation of the periodontal tissue. The prevention and treatment of this disease often depends on daily care by mechanical cleaning with a toothbrush and a surgical procedure, and further improvement in prevention and / or treatment means is desired. In recent years, the use of implants to supplement teeth lost due to periodontal disease, dental caries, etc. has become widespread, and the problem of peri-implantitis (peri-implant titis) has also arisen.

  Patent Document 1 includes at least two kinds of extract extracts and extract extracts of mastic (milky incense) among betel nuts (palm), licorice (licorice), nutmeg (meat rice), and yak mushrooms (benuses). A composition for preventing or treating periodontal disease is disclosed.

  Porphyromonas gingivalis (P.g) is a bacterium that is detected frequently and characteristically in periodontal disease patients. It has strong proteolytic activity and strong endotoxin that induces bone resorption. It is considered to be one of the causative bacteria of chronic periodontitis. Since the increase or decrease of the bacteria in the periodontal pocket coincides with the change in the pathology of chronic periodontitis, direct antibacterial activity against this bacteria or the inhibition of bone resorption that is one of the main symptoms of periodontal disease Certain substances are effective in preventing chronic periodontitis and suppressing progression. The composition of patent document 1 mainly exhibits an effect by suppressing periodontopathic bacteria.

  The present inventor searches for a component effective for the prevention and / or treatment of periodontal disease by an action mechanism different from that of the composition of Patent Document 1, and a plant called chicken blood rat has a strong osteoclast differentiation / It has been found that it has a life-prolonging inhibitory action, an antibacterial action against periodontal disease causative bacteria, and a reactive oxygen species removing action, and has applied for a patent (Patent Document 2).

  Bone tissue such as alveolar bone is a dynamic tissue that is formed by osteoblasts and is repeatedly broken and resorbed by osteoclasts. Osteoporosis occurs when the balance between bone resorption and bone formation is lost, but even in periodontal disease, the alveolar bone is destroyed by a similar mechanism and causes loss of teeth.

  Chicken blood rat has a strong osteoclast differentiation / life-prolonging action. Furthermore, chicken blood rat is also effective for the prevention or treatment of peri-implantitis (Peri-implantitis) (Patent Document 2).

On the other hand, the lotus (scientific name: Nelumbo nucifera) calls the lotus fruit of the water lily family the herb medicine name “Ishirenko”, and the seed excluding the pericarp is called the herb medicine name “Lotusme” or “Renko”. It has been used as a tonic.
In recent years, it has been proposed to use various herbal medicines to suppress bad breath by inhibiting methioninase derived from oral bacteria that is the key to the generation of methyl mercaptan, which causes bad breath, and lotus is used for this purpose. It is disclosed that it can do (patent document 3).
However, it has not been known that lotus has a bactericidal effect on oral bacteria such as periodontal pathogenic bacteria and cariogenic bacteria.

Japanese Patent No. 3389556 JP 2010-138100 A JP 2012-17338 A

Chicken blood rat has strong osteoclast differentiation / prolonging life-prolonging action and reactive oxygen species removing action, but chicken blood wisteria alone may not be satisfactory in terms of controlling periodontal disease bacteria. The inventor found that there is.
Therefore, the present inventor searches for a new plant-derived component effective in suppressing periodontopathic bacteria, and that lotus exhibits a remarkably excellent bactericidal effect on periodontopathic bacteria and cariogenic bacteria. I found it.
Furthermore, the present inventors have found that a combination of chicken blood and lotus can provide a high therapeutic effect even in patients with severe periodontal disease or transplanted implants.

An object of the present invention is to provide a composition for preventing and treating periodontal disease comprising a lotus extract as an active ingredient.
Another object of the present invention is to provide a composition for preventing / treating peri-implantitis (peri-implant titis) comprising a lotus extract and chicken blood as active ingredients.

According to the present invention, the following periodontal disease prevention / treatment composition, peri-implant titis prevention / treatment composition, and cosmetic and oral cosmetic preservatives are provided.
1. A composition for preventing and treating periodontal disease comprising a lotus extract as an active ingredient.
2. Furthermore, the composition for periodontal disease prevention and treatment of 1 containing a chicken blood rat or its extract.
3. Furthermore, 1 containing a plant component selected from the group consisting of mastic, grapefruit, sykon (purple root), nutmeg, betel loaf, mugwort, prunus, buttonpi, oxon, daiou (daihuang), hornbill, yakso, bears, and propolis Or the composition for periodontal disease prevention and treatment according to 2.
4). The composition for periodontal disease prevention and treatment according to any one of 1 to 3, which is used in the form of a coating agent, a dentifrice, a mouth rinse (gargle), a rinse solution or a poultice.
5. 5. The composition for preventing and treating periodontal disease according to 4, wherein the coating agent is in a gel form.
6). The composition for periodontal disease prevention and treatment according to any one of 1 to 5, wherein the content of the lotus extract is 0.5% by weight or more.
7). The composition for periodontal disease prevention and treatment according to any one of 1 to 6, wherein the lotus extract is obtained by a supercritical extraction method using carbon dioxide as an extraction solvent.
8). A composition for preventing / treating peri-implantitis (peri-implant titis) comprising a lotus extract and chicken blood rat as active ingredients.
9. A preservative for cosmetics and oral cosmetics containing a lotus extract.

  ADVANTAGE OF THE INVENTION According to this invention, the composition for periodontal disease prevention and treatment which uses a lotus extract as an active ingredient can be provided.

It is a graph which shows transition of the therapeutic effect of subject A. It is a graph which shows transition of the therapeutic effect of subject B. It is a graph which shows transition of the therapeutic effect of subject C. It is a graph which shows transition of the therapeutic effect of subject D. It is a graph which shows transition of the therapeutic effect of subject E. It is a graph which shows transition of the therapeutic effect of test subject F. It is a graph which shows transition of the therapeutic effect of subject G. It is a graph which shows transition of the therapeutic effect of test subject H. It is a graph which shows transition of the therapeutic effect of test subject I. It is a graph which shows transition of the therapeutic effect of subject J. It is a photograph which shows the implant periodontitis affected part before the gel use of the subject A. It is the photograph which shows the implant periodontitis affected part of the test subject A about 3 months after gel use.

Hereinafter, the present invention will be described in detail.
The composition for periodontal disease prevention / treatment which uses the lotus extract of the present invention as an active ingredient contains the lotus extract as an active ingredient.

  The “lotus” used in the present invention is the scientific name Nelumbo nucifera, English name Lotus, and is a lotus perennial aquatic plant native to India.

The “lotus extract” used in the present invention is not particularly limited as long as it is extracted from a lotus plant, but is particularly preferably extracted from a leaf.
The extraction method is not particularly limited, but a supercritical extraction method is preferable. According to the supercritical extraction method, extraction at a low temperature is possible, the solvent does not remain in the extract, and the extract components are unlikely to deteriorate or denature. The extraction solvent is not particularly limited, and an appropriate solvent may be selected according to the subsequent processing of the extract, but it is preferable to use carbon dioxide.
The lotus extract extracted by the supercritical extraction method is usually a light amber, slightly viscous liquid.

The obtained lotus extract can be used after diluted or concentrated.
What is necessary is just to adjust suitably the lotus component density | concentration at the time of setting a lotus extract as a diluent according to the objective. For example, when the lotus extract is used as a preservative, particularly a preservative for cosmetics and oral cosmetics described later, the concentration of the lotus component is preferably 0.1% by weight or more. When used as an antibacterial agent, it is preferably 0.5% by weight or more, more preferably 1% by weight or more, and particularly preferably 3% by weight or more.
Further, the lotus extract may be further concentrated to form an extract, or may be freeze-dried to form a powder or granules.

  The lotus extract used in the present invention has a high bactericidal effect against all oral bacteria such as periodontopathic bacteria and cariogenic bacteria, as shown in the test examples described later. Therefore, the composition for periodontal disease prevention / treatment of the present invention containing a lotus extract as an active ingredient is effective for the prevention and treatment of periodontal disease.

Moreover, the composition for the prevention and treatment of peri-implantitis (peri-implant titis) of the present invention is characterized by comprising a lotus extract and chicken blood rat as active ingredients.
The lotus extract has a strong bactericidal action against various oral bacteria, and chicken blood rat has an action of suppressing alveolar bone resorption (Patent Document 2). When these were used together, a synergistic effect of strengthening the alveolar bone was obtained while suppressing periodontal pathogenic bacteria and cariogenic bacteria in general, and an excellent prevention and treatment effect for peri-implantitis was obtained. .

  By adding chicken blood wisteria or its extract to the lotus extract, the lotus extract has a high bactericidal effect on periodontal pathogenic bacteria and cariogenic bacteria, and strong osteoclast differentiation of chicken blood wisteria -Due to the synergistic effect due to the life-prolonging inhibitory action, not only periodontal diseases but also peri-implantitis (peri-implant titis) can be significantly prevented.

  “Chicken blood wisteria” used in the present invention is a known herbal medicine, and examples thereof include, but are not limited to, Leguminosae (S. hormanii Gagnep), S. sinensis Chung (S. sinensis Chung). et T, Chen), Millettia dielsiana Harms, M. nitida Benth. var. hirsutissima Z. Wei, M. gentiliana Level, beauty wisteria M. speciosa Champ., M. reticulata benth, Muchu sempervirens Hemsl., M. birdwoodiana Tuchcher, Brown castor bean (M. castanea Merr), Craspedolobium schochii Harms, Kadsura interior A. C. Smith, atypical southern crimson (K. b. ), And iron scatter (Schisandra propinqua (Wall.), Baill. Var. Sinensis Olib.).

  In the present invention, dried stems, vines, roots of the above-described various plants, vines, roots may be used as they are, or cut, crushed, etc., or dried stems, vines, The extract obtained by bringing the root into contact with a suitable solvent (for example, water or hot water) can also be used. Further, the shape of the extract is not particularly limited, and liquid, soft extract, powder, granule and the like can be used.

The extract of chicken blood rat used in the present invention can be produced, for example, as follows.
Extract the dried chicken stalk, vine, and roots by bringing them into contact with water or an organic solvent such as hot water or alcohol and heating as necessary. Or you may extract by the supercritical extraction method.
The concentration of the component of chicken blood in the extract of chicken blood may be adjusted as necessary. For example, 200 g of dried chicken blood is added to 1 L of water and heated to 20%. be able to. The obtained 20% extract can be further diluted and used as necessary.
The chicken blood wisteria extract may be further concentrated to form an extract, or may be freeze-dried to form a powder or granules.

The preservative for cosmetics and oral cosmetics of the present invention is characterized by containing a lotus extract.
The lotus extract used in the present invention has high antibacterial properties against oral bacteria and is useful as a preservative for cosmetics, particularly oral cosmetics.

  In addition, the composition for preventing and treating periodontal disease and the composition for preventing and treating periodontitis of the present invention (hereinafter referred to collectively as the composition of the present invention) includes the periodontal in addition to the above active ingredients. Medicinal ingredients effective for the prevention and treatment of diseases can also be added. Examples of such components include components having antibacterial activity against periodontal pathogenic bacteria, anti-inflammatory agents, and the like. Only 1 type may be used for said component and it may use it in combination of 2 or more type.

  As a component having antibacterial activity against periodontal pathogenic bacteria, a known antibacterial agent can be used. For example, mastic, grapefruit, sicon (purple root), nutmeg, betel loin, mugwort, grasshopper, buttonpi, hornon, daiou (daihuang) ), Plant components such as cabbage, yakumoso, kumaso, and propolis are preferred, and plant components such as mastic and grapefruit are more preferred.

The composition of the present invention can be used in various dosage forms, but it can be used as a coating agent or a poultice applied to an affected area such as the mouth, or a toothpaste, a mouthwash (gargle), a rinse It is preferable to use as.
The coating agent is preferably a gel.

  In addition to the above components, the composition of the present invention includes a solvent, a fragrance, a refreshing agent, a sweetener, a colorant, a surfactant, a lubricant, a thickener, a pH adjuster, an antiseptic, depending on the application and dosage form. Additives usually used for the oral cavity such as agents, excipients, anti-inflammatory agents and medicinal components can be further included. Only 1 type may be used for the said additive and it may use it in combination of 2 or more type.

  As the solvent, it is preferable to use a biocompatible material such as ethanol or water.

  As fragrances, menthol, peppermint oil, spearmint oil, orange oil, lemon oil, eucalyptus oil, mint oil, acacia oil, fennel oil, citrus oil, columnus oil, camphor oil, Nikkei oil, cinnamon oil, coconut leaf oil, Terpenes such as rose oil, sandalwood oil, clove oil, herb oil, banana oil, apple oil, methyl salicylate, carvone, anethole, limonene; and blended fragrances.

Sweeteners include saccharin, saccharin sodium, xylitol, stevioside, stevia extract, rebaudioside, paramethoxycinnamic aldehyde, neohesberyl dihydroxychalcone, perilartin, thaumatin, glycyrrhin, glycyrrhizin monoglucoside, hernandultin, trehalose, Examples include aspartame and sorbit.
Moreover, you may mix | blend plum fruit extract, lactic acid, sodium lactate, etc. as a seasoning.

  Examples of the colorant include legal dyes such as blue No. 1 and yellow No. 4, titanium dioxide, and carmel.

  Examples of the surfactant include an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant. Specifically, alkyl sulfate, alkyl benzene sulfonate, sucrose fatty acid ester, lactose fatty acid ester, lauroyl sarcosine salt, N-acyl glutamate, α-olefin sulfonate, 2-alkyl-N-carboxy-N— Hydroxyethyl imidazolium betaine, N-acyl taurine salt, alkylol amide, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil or fatty acid ester thereof, polyglycerin fatty acid ester, sorbitan fatty acid ester, fatty acid ester, polyethylene glycol fatty acid ester And propylene glycol fatty acid esters.

  Lubricants include sugar alcohols such as sorbitol, maltitol, xylitol, and lactitol; polyhydric alcohols such as glycerin, 1,3-butylene glycol, 1,2-pentanediol, polyethylene glycol, polypropylene glycol, and dipropylene glycol. Can be mentioned.

  Examples of thickeners include carboxyvinyl polymer, sodium carboxymethylcellulose (cellulose gum), methylcellulose, hydroxyethylcellulose, carrageenan, alkali metal alginate such as sodium alginate, julan gum, xanthan gum, guar gum, tragacanth gum, karaya gum, vegetal gum, gum arabic, etc. Examples of gums include polyvinyl alcohol, polyvinyl pyrrolidone, silica gel, aluminum silica gel and the like.

  Examples of pH adjusters include organic acids such as citric acid, malic acid, phosphoric acid and acetic acid and salts thereof; sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, calcium carbonate, calcium hydrogen carbonate, ammonium carbonate, hydrogen carbonate Ammonium, potassium potassium carbonate, lithium carbonate, urea, amino acid oligomer, sodium chloride, calcium chloride, calcium nitrate, calcium sulfate, calcium glycerophosphate, calcium hydroxide, etc., calcium lactate, calcium acetate, calcium malonate, citric acid Organic acid calcium salts such as calcium acid, calcium gluconate, calcium glycerate, calcium tartrate, calcium phytate and the like.

  Examples of the preservative include paraoxybenzoates such as methylparaben, ethylparaben, butylparaben, isopropylparaben, and propylparaben, benzoates such as sodium benzoate, alkyldiaminoethylglycine hydrochloride, phenoxyethanol, and sorbic acid.

  Anti-inflammatory agents include, for example, glycyrrhizic acid and its salts, glycyrrhetinic acid and its salts, tranexamic acid, lysozyme chloride, buckwheat extract, soft sugar extract, dl-α tocopherol acetate, dl-α tocopherol nicotinate, ipsiloaminocaproic acid, Examples include azulene sulfonate, dihydrocholesterol, epidihydrocholesterin and the like.

Medicinal ingredients include allantoin, tocopherol acetate, isopropylmethylphenol, dextrose, chlorophyll, copper chlorophyll sodium, flavonoids, mutanase, lysozyme, amylase, protease, lytic enzyme, superoxide dismutase, epsilon aminocaproic acid, aluminum allantoin, aluminum chlorohydroxy Allantoin, dihydrocholestanol, bisabolol, glycerophosphate, water-soluble inorganic phosphate compound, sodium fluoride, sodium monofluorophosphate, tin fluoride, potassium fluoride, sodium fluorosilicate, aluminum fluoride, silver fluoride , Fluorides such as hexylamine hydrofluorate, decanolamine hydrofluoride, octadecenylamine hydrofluoride, DOO acid, zinc citrate, zinc chloride, copper gluconate, chlorhexidine gluconate, copper chloride, polyphosphates, pyrophosphates, vitamins A, vitamin C, vitamin E, vitamin B _6, vitamins such as pantothenic acid salt, Examples include amino acids such as glycine, lysine, and histidine, polyphenol compounds such as sodium chloride, sodium bicarbonate, aluminum lactate, potassium nitrate, sarcosinate, and catechins, and crude drugs.

[Test Example 1]
The following test verifies that the lotus extract has bactericidal action against periodontopathic bacteria and cariogenic bacteria.

(1) Manufacture of lotus extract 1 kg of dried lotus leaf is cut and ground, soaked in hot water for 2 hours, added with yeast for food, fermented for 3 days, heated and sterilized, and filtered through a cloth filter. The filtrate was obtained. From this filtrate, 5 mL of a thin amber and slightly viscous lotus extract was obtained by a supercritical extraction method using carbon dioxide as an extraction solvent.

(2) Manufacture of chicken blood wisteria, bear cake, grapefruit, and propolis extracts Like the lotus extract, chicken blood wisteria, bear bear, grapefruit, and propolis extracts were obtained by the supercritical extraction method.

(3) Test strains and culture medium Oral bacteria Streptococcus mutans Ingritt strain (abbreviated as S.m in Table 1, the same applies hereinafter), Streptococcus gordonii DL-1 strain (S. g), Lactobacillus casei ATCC 393 strain (LCC) C), Actinomyces viscosus ATCC15987 strain (A.v), Porphyromonas gingivalis ATCC33277 strain (P.g), Fusobacterium nucleum ATCC25586 strain (F.n) and Prevotella strain 26. aureus ATCC 10231 strain (Sa) and E cherichia coli HB101 strain (E.c) tried today.

The above bacterial culture was transformed into brain heart infusion medium (BHI; Becton, Dickinson and company, sparks, MD), yeast extract (5 mg / mL), hemin (5 μL / mL), and vitamin K ₁ (1 μL / mL). using the added medium (BHI · Y · H · VK 1 liquid medium), and intraoral bacteria under anaerobic conditions _{(N 2: 80%, H} 2: 10%, CO 2: 10%), at 37 ° C. After culturing for 48 hours, it was used for the test.

(4) Minimum Measurement of the Inhibitory Concentration (MIC) (1) and (2) the resulting test試抽exudates stock solution, 2-fold serial dilutions up to ^{2 -14} in sterile phosphate buffered saline (PBS) Then, 100 μL of the test strain adjusted in (3) above was added to 1 mL of each diluted solution, and then inoculated into ₁ mL of BHI • Y • H • VK ₁ liquid medium and cultured for 48 hours. The extract concentration at which growth inhibition was observed was defined as the minimum growth inhibition concentration (MIC). Table 1 shows the results expressed in terms of dilution ratio.

It can be seen that the lotus extract can obtain a bactericidal effect with a dilution solution of 8192 times or more against periodontal pathogenic bacteria, cariogenic bacteria and general bacteria.
On the other hand, it can be seen that some chicken blood wisteria, bear bear, grapefruit and propolis show an excellent bactericidal effect depending on bacteria, but some show a bactericidal effect only up to about 4 to 16 times dilution.
For Fusobacterium nucleatum, which is a periodontopathic bacterium, the lotus extract exhibits a significantly higher bactericidal effect than other extracts.

[Test Example 2]
A gel preparation having the following formulation was produced using a composition obtained by adding the extract of chicken blood obtained in (2) to the lotus extract obtained in Test Example 1 (1), and periodontal disease and peri-implantitis (Peri-implant titis) for one or both patients (10), apply gel to the affected area with fingers and massage 1 to 3 times a day after brushing for 1 to 3 months The results of examining the improvement status of one or both of periodontal disease and peri-implantitis with dentist appearance observation (findings / evaluation) and patient's sense are shown in FIG.
In all 10 patients selected as subjects, improvement of periodontal disease or peri-implantitis was observed even if the periodontal treatment was performed by a dentist for a certain period of time or the patient himself was brushed. There were cases that could not be improved or improved beyond the current level.

(Gel formulation)
Lotus extract 3% by weight
30% by weight of chicken blood rat extract
Glycerin 30-50% by weight
Citric acid (20% aqueous solution) 0.1% by weight
Cellulose gum 5-10% by weight
Stevia 0.01-0.05% by weight
Purified water remaining
Total 100% by weight

The evaluation criteria for each finding item in FIGS. 1 to 10 are as follows.
Pus:
[5] Extends to nipple and attached gingiva [3] Found in nipple and gingival margin [1] "0" found in part of nipple Not at all

Redness:
[5] Extends to nipple and attached gingiva [3] Found in nipple and gingival margin [1] Found in part of nipple [0] Not found at all

bleeding:
[5] bleeding naturally [3] recognized by acupressure [1] slightly detected by acupressure [0] not detected by acupressure

Drainage:
[5] Naturally draining [3] Recognized by finger pressure [1] Slightly recognized by finger pressure [0] Not recognized by finger pressure at all

Gingival color:
[5] Dark purple [3] Dark red [1] Bright red [0] Light red

upset:
[5] Lingually and vertically [3] Strongly lingually recognized [1] Slightly lingually recognized [0] Range of physiological sway For patient A, an implant is implanted Therefore, there was no upset.

Patient feeling:
[5] No change [3] A little better [1] Tightened [0] Very good

  It has been recognized as a dentist's finding and evaluation that periodontal disease symptoms have been greatly improved in any patient who has not been able to improve the therapeutic effect of conventional periodontal disease. It can also be seen from the patient's own sense that the dentist's findings cannot be evaluated that the symptoms are greatly improved.

Patient A is a case with not only periodontal disease but also peri-implantitis. 11a and 11b show photographs showing the condition of the affected part of the peri-implantitis at the start of the test and the condition of the affected part after about three months from the start of the test.
In FIG. 11a, it can be seen that the affected part that was swollen red and congested so that the implant part could not be seen was cured until the implant part was completely visible as shown in FIG. 11b.

  A composition containing a lotus extract and chicken blood wisteria has a synergistic effect of suppressing periodontal pathogenic bacteria and cariogenic bacteria while strengthening the alveolar bone, preventing periodontal disease higher than before. -Expected to have therapeutic effects and preventive and therapeutic effects on peri-implantitis.

The composition of the present invention is useful for periodontal disease prevention / treatment.
The composition of the present invention, particularly a composition in which chicken blood is blended with a lotus extract is useful for the prevention and treatment of peri-implantitis (peri-implant titis).
The composition of the present invention is also useful as a preservative for cosmetics, particularly oral cosmetics.

Claims (6)

A method for producing a composition for preventing and treating periodontal disease comprising a supercritical extract of a lotus leaf and a supercritical extract of chicken blood as an active ingredient ,
A method in which the extraction solvent used for the supercritical extraction is carbon dioxide . In addition, a claim containing a plant component selected from the group consisting of mastic, grapefruit, shikon (purple root), nutmeg, betel loaf, mugwort, green grass, button pi, dragon, daiou (daihuang), archaea, yakuso, kumagi, and propolis Item 2. A method for producing a composition for preventing and treating periodontal disease according to Item 1. The manufacturing method of the composition for periodontal disease prevention and treatment of Claim 1 or 2 used with the form of a coating agent, a dentifrice, a mouth rinse (gargle), a rinse liquid, or a poultice. The method for producing a composition for preventing and treating periodontal disease according to claim 3, wherein the coating agent is in a gel form. The method for producing a composition for preventing and treating periodontal disease according to claim 1, wherein the content of the lotus leaf extract is 0.5% by weight or more. A method for producing a composition for preventing / treating peri-implantitis (peri-implant titis) comprising a supercritical extract of a lotus leaf and a supercritical extract of chicken blood as an active ingredient ,
A method in which the extraction solvent used for the supercritical extraction is carbon dioxide .