WO2009054606A1 - A composition comprising pentaphylloides fruticosa extract as an active ingredient - Google Patents
A composition comprising pentaphylloides fruticosa extract as an active ingredient Download PDFInfo
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- WO2009054606A1 WO2009054606A1 PCT/KR2008/004842 KR2008004842W WO2009054606A1 WO 2009054606 A1 WO2009054606 A1 WO 2009054606A1 KR 2008004842 W KR2008004842 W KR 2008004842W WO 2009054606 A1 WO2009054606 A1 WO 2009054606A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Abstract
The present invention relates to a composition containing Pentaphylloides fruticosa extract as an active ingredient. Pentaphylloides fruticosa extract of the present invention extracted by using water, alcohol or a mixture thereof as a solvent inhibits the activity of AP-1 (activator protein-1), a MMP-I transcription factor, inhibits type I collagenase, a skin matrix decomposing enzyme, but increases collagen, a major component of skin, so that it can be effectively used as a preparation for skin external application including cosmetics owing to its wrinkle and elasticity improving effects. In addition, Pentaphylloides fruticosa extract of the present invention has radical scavenging effect, so that it can also be effectively used as an antioxidative agent.
Description
[DESCRIPTION]
[invention Title]
A COMPOSITION COMPRISING PENTAPHYLLOIDES FRUTICOSA EXTRACT AS AN ACTIVE INGREDIENT
[Technical Field]
The present invention relates to a composition comprising Pentaphylloides fruticosa extract having anti- skin aging and anti-oxidative activities as an active ingredient.
[Background Art]
Skin aging is clinically divided into natural aging characterized by feeble and pale skin and delicate wrinkles and reduction of elasticity of skin, and photo aging characterized by rough wrinkles by sunlight, pigmentation and telangiectasia (Gilchrest B. A., J. Am. Acad. Dermatol. , 21: 610, 1989; Ma W., et al . , Clin. Exp. Dermatol. , 26: 592, 2001; Jenkins G., Mech. Ageing Develop., 123: 801, 2002). The changes or decrease of collagen which is a major component of skin has been known as a critical reason of clinical changes of skin such as wrinkles and reduction of elasticity. The changes or decrease of collagen is observed in both natural aging and photo aging (Lavker R. M., et al., J. Invest. Dermatol. ,
90: 325, 1988). Collagen deficiency observed during natural aging is allegedly resulted from the decrease of collagen generation itself or the increase of type I collagenase (Matrix Metalloproteinase-1 , MMP-I) decomposing collagen. UV irradiation induces type I collagenase biosynthesis in skin and the increased type I collagenase accelerates collagen decomposition in skin and further promotes extracellular matrix (ECM) decomposition (Millis A. J., et al., Exp. Cell. Res., 201: 373, 1992; Chung J. H., et al., J. Invest. Dermatol. , 117: 1218, 2001; Fisher G. J., et al . , J. Clin. Invest., 106: 663, 2000; Visse R. et al., Circ. Res., 92: 827, 2003; Vincenti M. P., Crit. Rev. Eukaryot Gene Expr. , 6: 391, 1996; Scharffetter-Kochanek K., et al., Exp. Gerontol. , 35: 307, 2000). So, recovering from collagen deficiency in human skin might be a way to recover from skin aging. Therefore, it is expected to prevent or recover from skin aging symptoms such as wrinkles, reduction of elasticity and skin drooping, by accelerating collagen biosynthesis or reducing MMP-I biosynthesis.
Every aerobic organism including human survives with energy metabolism using oxygen. When oxygen in vivo gets physical, chemical or biological stress, it turns into harmful reactive oxygen species (ROS) such as superoxide
anion radical (O2), H2O2, hydroxy radical (-0H), etc. These reactive oxygen species cause critical physiological disorder and if worse, they cause life-threatening disease. Organism has a self defense mechanism to eliminate reactive oxygen species, which is antioxidative mechanism. And lives have been evolved with the development of the antioxidative mechanism. However, generation of reactive oxygen species outmatching the defensive mechanism damage immune-related factors such as protein, DNA, enzyme and T- cell to cause various diseases. The reactive oxygen species attack unsaturated fatty acid, a component of cell membrane, to cause peroxidation. As a result, the accumulated lipid peroxide In vivo causes aging and other diseases (Sies H., et . al . , Philos Trans R Soc Lond B Biol ScI. 311:617, 1985; Neuzil J., et. al . , B±ochem J. 293: 601, 1993; Steinberg D, et. al . , N Engl J Med. 320: 915, 1989) .
It was recently acknowledged that aging and adult diseases are caused by reactive oxygen species. Since then, studies to develop antioxidative agents which can regulate reactive oxygen species have been actively going on, for example anti-oxidative enzymes such as superoxide dismutase, peroxidase, catalase, glutathione peroxidase, and other natural low molecular antioxidants such as
tocopherol (vitamin E), ascorbic acid, carotenoid and glutathione, have been major targets of studies (Valko M., et. al., Int J Biochem Cell Biol. 39: 44, 2007; Terlecky SR., et. al., Biochlm Biophys Acta. 1763-1749, 2006). In addition, a synthetic antioxidative agent such as BHA ( 2 , 6-di-tert-butyl-4-hydroxyanisole ) has been developed and used in the field of medicine and food industry. However, after the report saying mass-administration of a synthetic antioxidative agent could cause cancer in animal tests (Rahimtula AD, et. al . , Br J Cancer. 45:935, 1982; Botterweck AA, et. al . , Food Chem Toxicol. 38: 599, 2000), consumers are tend to avoid taking a synthetic antioxidative agent, resulting in the decreasing demand of a synthetic antioxidative agent. Thus, it is required to develop a safer and more effective natural antioxidative agent (Masaki H., et. al . , Biol Pharm Bull. 18: 162, 1995) .
Pentaphylloides fruticosa is a shrub belonging to Rosaceae, which is distributed in Siberia, east of Russia, and far-east of Caucasus middle Asia. It grows on banks of river or lake or even on a rock wall of an alpine region of at least 2000 m above sea-level. Young bud is silvery gold and a mature tree is 20 ~ 150 cm in height and the leaf is thin and long in shape. It blossoms in June - August, the
flower is light yellow, and its separated bark of a stem is grey-brown. The aerial part of this plant contains vitamin C and P, carotin, phenolcarboxylic acids (caffeic acid, sinapic acid, ferulic acid, ellagic acid, cumaric acid) , catechins, flavonoids and triterpenoids . Leaves and flowers have been used as a medicine, which have antibacterial activity, phlegm discharging activity, hemostatic activity and astringent effect. The plant has been largely used to treat digestive disease, inflammatory disease in the liver, pneumonia, fever, etc. Besides, it has been used to treat diarrhea, mental illness and blood-related disease or as a supplement to treat heart disease, or as an orexigenic agent (folk remedy in Russia).
According to the study results of Pentaphylloides frutlcosa, it helps to recover from chronic hepatitis and lipid peroxidation in the liver (Kolpakov MA, et. al., Bull Exp Biol Med. 131: 470, 2001). Except these results, no other reports have been made on Pentaphylloides fruticosa. Examination on the components of the plant has not been made yet. There are no patents on Pentaphylloides fruticosa and studies on skin aging using the plant have not been reported yet.
Thus, the present inventors studied and completed this invention by confirming that Pentaphylloides fruticosa
has excellent inhibition effect on AP-I (activator proteinic reduces type I collagenase biosynthesis, increases collagen biosynthesis, has radical scavenging activity, but has no side effects, so that it can be effectively used as a skin-care agent for preventing and improving skin aging, as an antioxidative pharmaceutical composition or as a health improving functional food.
[Disclosure] [Technical Problem]
It is an object of the present invention to provide a pharmaceutical composition for anti-oxidation, preventing skin aging and improving wrinkles or a health improving functional food containing Pentaphylloides fruticosa extract as an active ingredient.
[Technical Solution]
To achieve the above object, the present invention provides an antioxidative agent containing Pentaphylloides fruticosa extract extracted by using water, alcohol or a mixture thereof as a solvent as an active ingredient.
The present invention also provides a preventive and therapeutic agent for a disease caused by the accumulation of reactive oxygen species containing Pentaphylloides fruticosa extract as an active ingredient.
The present invention further provides a composition for preventing skin aging and improving wrinkles containing Pentaphylloides fruticosa extract as an active ingredient.
The present invention also provides a health improving functional food for preventing skin aging and improving wrinkles containing Pentaphylloides fruticosa extract as an active ingredient.
In addition, the present invention provides a method for preventing and treating a disease caused by the accumulation of reactive oxygen species.
Hereinafter, the present invention is described in detail .
Pentaphylloides fruticosa extract of the present invention is preferably prepared by the method comprising the following steps, but not always limited thereto.
1) extracting dried Pentaphylloides fruticosa using an extraction solvent; 2) filtering the extract of step 1) after cooling; and
3) concentrating the filtered extract of step 2) under reduced pressure, followed by drying thereof.
In the above method, Pentaphylloides fruticosa of step 1) can be purchased or cultivated. Aerial part or
roots of Pentaphylloides fruticosa can be used, but aerial part is preferred.
In the above method, the extraction solvent is water, alcohol or a mixture thereof. Herein, the alcohol is preferably Ci - C4 lower alcohol and the lower alcohol is exemplified by ethanol or methanol. The preferable amount of the solvent is 5 - 15 times the amount of dried Pentaphylloides fruticosa, and more preferably 10 times the amount, but not always limited thereto. The temperature of the extraction is 80 - 120 °C and preferably 100°C, but not always limited thereto. The extraction time is preferably 2 - 24 hours and more preferably 3 hours, but not always limited thereto. The extraction is preferably- repeated 1 - 5 times and more preferably 3 times, but not always limited thereto.
In this method, the concentration under reduced pressure of step 3) is preferably performed by using rotary vacuum evaporator, but not always limited thereto. The extract is preferably dried by freeze-drying, but not always limited thereto.
The present invention provides an antioxidative agent containing Pentaphylloides fruticosa extract as an active ingredient. The antioxidative agent herein is characteristically
designed to prevent and treat a disease caused by the accumulation of reactive oxygen species, and the disease herein is selected from the group consisting of liver disease, stroke, myocardial infraction, diabetic vascular disorder, hyperlipidemia, acute inflammation, rheumatism and cancer.
The present invention also provides a composition for the prevention of skin aging and the improvement of wrinkles containing Pentaphylloldes fruticosa extract as an active ingredient.
The prevention of skin aging and the improvement of wrinkles herein are characteristically achieved by the improvement of elasticity. The present inventors measured antioxidative activity of Pentaphylloides fruticosa extract by DPPH (1,1- diphenyl-2-picryl-hydrazyl) method (Leu SJ, et. al., Biol Pharm Bull. 29: 740, 2006). DPPH is a comparatively stable free radical and exhibits the maximum absorbance at 517 nm as a radical. But, when it is eliminated, it looses absorbance. Based on this, the antioxidative activity can be measured. As a result, Pentaphylloides fruticosa extract of the present invention demonstrated high DPPH radical scavenging activity. Therefore, Pentaphylloides fruticosa extract was confirmed to have antioxidative
effect. Active oxygen generated during metabolism is a cause of aging (Muller FL, et . al., Free Rad±c Biol Med. 43: 477, 2007). Pentaphyllo±des fruticosa extract of the present invention has antioxidative activity, so that it can be effectively used as a skin aging inhibitor.
To confirm whether Pentaphyllo±des fruticosa extract could inhibit AP-I (activator protein-1), which is a major transcription factor of MMP-I, the present inventors measured AP-I inhibition by reporter gene assay using a luciferase conjugated plasmid. As a result, Pentaphylloides fruticosa extract of the present invention was confirmed to inhibit AP-I dose-dependently, indicating that Pentaphylloides fruticosa extract of the present invention has skin aging preventive effect and anti- wrinkle and elasticity improving effect.
The present inventors further investigated if Pentaphylloides fruticosa extract could inhibit type I collagenase (MMP-I) biosynthesis mediated by tumor necrosis factor α(TNFα) or UV. Human fibroblasts were treated with TNFα or irradiated by UV A, and then treated with Pentaphylloides fruticosa extract of the present invention, followed by ELISA to quantify type I collagenase. As a result, Pentaphylloides fruticosa extract was confirmed to • inhibit type I collagenase biosynthesis dose-dependently. Therefore, it was confirmed
that Pentaphylloides fruticosa extract of the present invention has preventive and improving effect on the reduction of skin matrix material, loss of elasticity and wrinkles . The present inventors also investigated if Pentaphylloides fruticosa extract could accelerate collagen biosynthesis and inhibit collagen decomposition. Human fibroblasts were treated with TNFα and then treated with Pentaphylloides fruticosa extract of the present invention, followed by ELISA to quantify procollagen. As a result, Pentaphylloides fruticosa extract of the present invention was confirmed to accelerate collagen biosynthesis dose-dependently and to inhibit collagen decomposition mediated by TNFα. Therefore, it was confirmed that Pentaphylloides fruticosa extract of the present invention has improving effect on skin wrinkles and elasticity.
The composition of the present invention can include, in addition to Pentaphylloides fruticosa extract, one or more effective ingredients having the same or similar function to Pentaphylloides fruticosa extract.
Pentaphylloides fruticosa extract can be administered orally or parenterally and be used in general forms of pharmaceutical formulation.
Pentaphylloides fruticosa extract of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing Pentaphylloides fruticosa extract of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, lyophilized preparations and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can
contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.
The effective dosage of Pentaphylloides fruticosa extract of the present invention can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage is 0.1-100 mg/kg per day, preferably 30-80 rag/kg per day, and more preferably 50-60 mg/kg per day, and administration frequency is preferably 1-6 times a day.
Pentaphylloides fruticosa extract of the present invention can be administered alone or treated together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
The present invention also provides a composition for the prevention of skin aging and the improvement of wrinkles containing Pentaphylloides fruticosa extract as an active ingredient as a preparation for skin external application.
The preparation for skin external application containing Pentaphylloides fruticosa extract of the present invention can additionally include a supplement generally used in the field of skin science such as fatty
substance, organic solvent, resolvent, concentrate, gelling agent, softener, antioxidant, suspending agent, stabilizer, foaming agent, odorant, surfactant, water, ionic or non- ionic emulsifying agent, filler, sequestering agent, chelating agent, preserving agent, vitamin, blocker, moisturing agent, essential oil, dye, pigment, hydrophilic or hydrophobic activator, lipid vesicle or other components generally used in a preparation for skin external application. The amount of the above supplement can be determined as generally accepted in the field of skin science.
The present invention also provides a composition for the prevention of skin aging and the improvement of wrinkles containing Pentaptiylloides frutlcosa extract of the present invention as an active ingredient as cosmetics.
The cosmetics containing Pentaphylloides fruticosa extract of the present invention as an active ingredient can be formulated as general emulsified cosmetics and solubilized cosmetics. The emulsified cosmetics are exemplified by lotion, cream and essence, and the solubilized cosmetics are exemplified by skin.
The proper formulation for the cosmetics can be exemplified by solution, gel, solid or dough anhydride, emulsion prepared by dispersing oil phase on water phase,
suspension, microemulsion, microcapsule, microgranule, ionic (liposome) or non-ionic vesicle, cream, skin, lotion powder, spray, and conceal stick. In addition, the cosmetics can be formulated as an aerosol composition containing an foam or compressed propellant.
The cosmetics of the present invention can include, in addition to Pentaphylloides fruticosa extract of the present invention, a supplement generally used in the field of cosmetics such as fatty substance, organic solvent, resolvent, concentrate, gelling agent, softener, antioxidant, suspending agent, stabilizer, foaming agent, odorant, surfactant, water, ionic or non-ionic emulsifying agent, filler, sequestering agent, chelating agent, preserving agent, vitamin, blocker, moisturing agent, essential oil, dye, pigment, hydrophilic or hydrophobic activator, lipid vesicle or other components generally used in cosmetics.
The present invention also provides a health improving functional food for the prevention of skin aging and the improvement of wrinkles containing Pentaphylloides fruticosa extract as an active ingredient.
The prevention of skin aging and the improvement of wrinkles herein are characteristically achieved by the improvement of elasticity.
Pentaphylloides frutlcosa extract of the present invention can be used as a food additive. In that case, Pentaphylloides frutlcosa extract can be added as it is or as mixed with other food components according to the conventional method. The mixing ratio of active ingredients can be regulated according to the purpose of use (prevention, health enhancement or treatment). In general, to produce health food or beverages, Pentaphylloides frutlcosa extract is added preferably by up to 15 weight part and more preferably by up to 10 weight part. However, if long term administration is required for health and hygiene or regulating health condition, the content can be lower than the above but higher content can be accepted as well since Pentaphylloides frutlcosa extract has been proved to be very safe.
The food herein is not limited. For example, Pentaphylloides frutlcosa extract can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included.
The composition for health beverages of the present invention can additionally include various flavors or
natural carbohydrates, etc, like other beverages. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and glucose alcohols such as xilytole, sorbitol and erythritol. Besides, natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent. The content of the natural carbohydrate is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 m# of the composition.
In addition to the ingredients mentioned above, Pentaphylloides fruticosa extract of the present invention can include in a variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. Pentaphylloides fruticosa extract of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 001-0.1 weight part per 100 weight part of
Pentaphylloides fruticosa extract of the present invention,
In addition, the present invention provides a method for the prevention and treatment of a disease caused by the excessive accumulation of active oxygen.
The method for the prevention and treatment above includes the step of administering an effective dose of the said Pentaphylloides fruticosa extract extracted by using water, ethanol or a mixture thereof as a solvent to a subject with a disease caused by the excessive accumulation of active oxygen.
In this method, the disease caused by the excessive accumulation of active oxygen is selected from the group consisting of liver disease, stroke, myocardial infraction, diabetic vascular disorder, hyperlipidemia, acute inflammation, rheumatism and cancer, but not always limited thereto.
Pentaphylloides fruticosa extract of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation. The formulation method for Pentaphylloides fruticosa extract of the present invention is described hereinbefore.
The effective dosage of Pentaphylloides fruticosa extract of the present invention can be determined according to weight, age, gender, health condition, diet,
administration frequency, administration method, excretion and severity of a disease. The dosage is 0.1-100 mg/kg per day, preferably 30-80 mg/kg per day, and more preferably 50-60 mg/kg per day, and administration frequency is preferably 1-6 times a day.
Pentaphylloides fruticosa extract of the present invention can be administered alone or treated together with surgical operation, hormone therapy, chemo-therapy and biological regulators.
[Advantageous Effect]
Pentaphylloides fruticosa extract of the present invention extracted by using water, alcohol or a mixture thereof as a solvent has radical scavenging activity, inhibits the activity of AP-I (activator protein-1), the MMP-I transcription factor, inhibits type I collagenase (MMP-I), the skin matrix decomposing enzyme, but increases collagen, one of major components of skin, so that it can be effectively used as a preparation for skin external application including cosmetics owing to its wrinkle and elasticity improvement effects. In addition, Pentaphylloides fruticosa extract of the present invention has no side effects, so that it can also be used as a pharmaceutical composition or health improving functional food.
[Description of Drawings]
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
Figure 1 is a graph illustrating the inhibition effect of Pentaphylloides frutlcosa methanol extract of the present invention on AP-I, the transcription factor. Figure 2 is a graph illustrating the inhibition effect of Pentaphylloides fruticosa methanol extract of the present invention on MMP-I biosynthesis mediated by TNFα.
Figure 3 is a graph illustrating the inhibition effect of Pentaphylloides fruticosa methanol extract of the present invention on MMP-I biosynthesis mediated by UV.
Figure 4 is a graph illustrating the accelerating effect of Pentaphylloides fruticosa methanol extract of the present invention on collagen biosynthesis. Figure 5 is a graph illustrating the inhibition effect of Pentaphylloides fruticosa methanol extract of the present invention on collagen decomposition mediated by TNFα.
[Mode for Invention]
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1 : Preparation of Pentaphyllo±des frutlcosa extract
<!-!> Preparation of Pentaphylloides fruticosa methanol extract
100 g of the dried Pentaphylloides frutlcosa aerial part (provided by NIOC, Novosivirsk Institute of Organic Chemistry, Russia) was pulverized, which was put in an extraction sack. The sack wad dipped in 10 times the volume of 100% methanol, followed by reflux-extraction at 100 °C for 3 hours. The extraction was repeated three times. The extract was cooled down to room temperature and filtered with a filter paper. The extract was concentrated under reduced pressure in a concentrator until the solution was completely evaporated to give crystalline. As a result, 7.3 g of extract was obtained from 100 g of the dried Pentaphylloides fruticosa aerial part.
<l-2> Preparation of Pentaphyllo±des frutlcosa water extract
Extraction was performed by the same manner as described in Example <1-1> except that water was used instead of methanol as an extraction solvent. As a result, 12.3 g of extract was obtained.
<l-3> Preparation of Pentaphylloides fruticosa ethanol extract Extraction was performed by the same manner as described in Example <1-1> except that 100% ethanol was used instead of methanol as an extraction solvent. As a result, 7.2 g of extract was obtained.
Experimental Example 1; Investigation of radical scavenging activity
The antioxidative activity of Pentaphylloides fruticosa extract was measured by DPPH ( 1, l-diphenyl-2- picryl-hydrazyl) method (Leu SJ, et. al., Biol Pharm Bull. 29: 740, 2006).
Pentaphylloides fruticosa extract obtained in Example 1 was serial-diluted (1/2, 10 steps) in 70% ethanol from the concentration of 100 μg/ml in a 96-well plate. DPPH ( 1, l-diphenyl-2-picryl-hydrazyl) prepared in 100 μM ethanol was added thereto to make total volume 200 /^/well.
Reaction was induced at 37 °C for 30 minutes, followed by- measuring OD52O with ELISA reader, which was calculated by the following mathematical formula 1. The control group was not treated with samples. Vitamin C and Trolox which have been known to have radical scavenging effect were used as positive controls. IC5O (inhibitory concentration) was determined by measuring the concentration that could be able to scavenge 50% of DPPH radical.
Mathematical Formula 1
DPPH radical scavenging activity (%) = (OD of control - OD of sample) /OD of control X 100
[Table l]
Radical scavenging activity of Pentaphylloides frutlcosa extract (IC50, unit: βg/mi)
As a result, as shown in Table 1, Pentaphylloides fruticosa extract of the present invention has high radical scavenging activity (see Table 1).
Experimental Example 2: Inhibition effect on AP-I (activator protein-1)
To investigate the inhibition effect of Pentaphylloides fruticosa extract of Example 1 on the major MMP-I transcription factor AP-I (activator protein-1), reporter gene assay was performed using a luciferase conjugated plasmid. HeLa cells (ATCC, CCL-2 ) were inoculated in a 24-well plate, followed by culture in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for 24 hours. When the transcription factor was activated, transfection was performed with AP-1-luciferase reporter plasmid DNA ( Stratagene ) designed to report the activation by using lipofectamin transfection reagent ( Invitrogen) . 24 hours after the transfection, Pentaphylloides fruticosa extract was added to the medium at different concentrations, which was stimulated by PMA (Phobol ester, Sigma). 24 hours later, the cells were lysed and the luciferase activity was measured by luminometer to investigate the inhibition effect. AP-I inhibition, that is the inhibition of transactivation of AP-I binding element, was measured. The measured level in the control group treated with PMA alone was regarded as 100 for comparison.
[Table 2] Inhibition effect of Pentaphylloides fruticosa
extract on AP-I
As a result, as shown in Table 2, Pentaphylloides fruticosa extract of the present invention inhibited AP-I dose-dependently (see Table 2).
Experimental Example 3; Inhibition effect on TNFα mediated type I collagenase (MMP-I) biosynthesis
Human fibroblasts directly separated from neonatal foreskin were inoculated in a 48-well plate at the concentration of 104 cells/well, followed by culture. 24 hours later, the concentration of TNFα (tumor necrosis factor α) was adjusted to 20 ng/ml and the medium was replaced with fresh ones containing different concentrations of Pentaphylloides fruticosa extract of Example 1. On the 2nd day of culture, the supernatant was harvested, followed by ELISA (AP biotech RPN2610) to
quantify type I collagenase. The measured level in the control group treated with TNFα alone was regarded as 100 for comparison.
[Table 3]
Inhibition effect of Pentaphylloides frutlcosa extract on TNFα mediated type I collagenase biosynthesis
As a result, as shown in Table 3, Pentaphylloides fruticosa extract of the present invention reduced TNFα mediated type I collagenase biosynthesis dose-dependently (see Table 3 ) .
Experimental Example 4; Inhibition effect on UV mediated type I collagenase (MMP-I) biosynthesis
Human fibroblasts directly separated from neonatal foreskin were inoculated in a 48-well plate at the
concentration of 104 cells/well, followed by culture. 24 hours later, the cells were irradiated with UV A by 15 J/cm2. The medium was replaced with fresh ones containing different concentrations of Pentaphylloides fruticosa extract of Example 1. On the 2nd day of culture, the supernatant was harvested, followed by ELISA (AP biotech RPN2610) to quantify type I collagenase. The measured level in the control group treated with UV A alone was regarded as 100 for comparison.
[Table 4]
Inhibition effect of Pentaphylloides fruticosa extract on UV mediated type I collagenase biosynthesis
As a result, as shown in Table 4, Pentaphylloides fruticosa extract of the present invention reduced UV A mediated type I collagenase biosynthesis dose-dependently
(see Table 4)
Experimental Example 5: Acceleration of collagen biosynthesis in skin cells
Human fibroblasts directly separated from neonatal foreskin were inoculated In a 48-well plate at the concentration of 104 cells/well, followed by culture. 24 hours later, the medium was replaced with fresh ones containing different concentrations of Pentaphylloides fruticosa extract of Example 1. On the 2nd day of culture, the supernatant was harvested, followed by ELISA (Takara MKlOl) to quantify procollagen. The measured level in the non-treated control group was regarded as 100 for comparison.
[Table 5]
Accelerating effect of Pentaphylloides fruticosa extract on collagen biosynthesis
As a result, as shown in Table 5, Pentaphylloides fruticosa extract of the present invention accelerated collagen biosynthesis dose-dependently (see Table 5).
Experimental Example 6j Inhibition of collagen decomposition
Human fibroblasts directly separated from neonatal foreskin were inoculated in a 48-well plate at the concentration of 104 cells/well, followed by culture. 24 hours later, the concentration of TNFα (tumor necrosis factor α) was adjusted to 20 ng/ml and the medium was replaced with fresh ones containing different concentrations of Pentaphylloides fruticosa extract of Example 1. On the 2nd day of culture, the supernatant was harvested, followed by ELISA (Takara MKlOl) to quantify procollagen remaining as not-decomposed. The measured level in the control group treated with TNFα alone was regarded as 100 for comparison. The level of the non- treated group was also counted for comparison.
[Table 6]
Inhibition effect of Pentaphylloides fruticosa
extract on collagen decomposition
As a result, as shown in Table 6, Pentaphylloides fruticosa extract of the present invention inhibited TNFα mediated collagen decomposition dose-dependently (see Table 6).
Experimental Example 7; Acute toxicity test of Pentaphylloides fruticosa extract Acute toxicity test was performed in mice via oral administration as follows to confirm the stability of Pentaphylloides fruticosa extract. 6-week old SPF mice were used in the test for acute toxicity. Pentaphylloides fruticosa extract of the present invention was dissolved in 0.9% NaCl and orally administered once to 10 mice per group fasted for 17 hours at the concentrations of 2 g/kg
and 5 g/kg. Death, clinical symptoms, and weight changes in mice were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with eyes during autopsy.
The results showed that the extract did not cause any- specific clinical symptoms, weight change, or death in mice. No change was observed in hematological tests, biochemical tests of blood, and autopsy. Pentaphylloldes fruticosa extract of the present invention is evaluated to be a safe substance since it does not cause any toxic change in mice up to the level of 5 g/kg and its estimated LD50 value is much greater than 5 g/kg in mice.
The Manufacturing Examples of the composition for the present invention are described hereinafter.
Manufacturing Example 1; Preparation of pharmaceutical formulations
<!-!> Preparation of powders
Extract of Example <1-1> 2 g
Lactose 1 g
Powders were prepared by mixing all the above components, which were filled in airtight packs according
to the conventional method for preparing powders .
<l-2> preparation of tablets
Extract of Example <1-1> 100 mg Corn starch 100 mg
Lactose 100 mg
Magnesium stearate 2 mg
Tablets were prepared by mixing all the above components by the conventional method for preparing tablets.
<l-3> Preparation of capsules
Extract of Example <1-1> 100 mg
Corn starch 100 mg
Lactose 100 mg Magnesium stearate 2 mg
Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules.
<l-4> Preparation of pills
Extract of Example <1-1> 1 g
Lactose 1.5 g
Glycerin 1 g
Xylitol 0.5 g Pills were prepared by mixing all the above
components according to the conventional method for preparing pills. Each pill contained 4 g of the mixture.
<l-5> Preparation of granules Extract of Example <1-1> 150 fflg
Soybean extract 50 mg
Glucose 200 mg
Starch 600 mg
All the above components were mixed, to which 100 mg of 30% ethanol was added. The mixture was dried at 60°C and the prepared granules were filled in packs.
Manufacturing Example 2 : Preparation of foods
Foods containing Pentaphylloides fruticosa extract of the present invention were prepared as follows.
<2-l> Preparation of flour food
0.5 ~ 5.0 weight% of the extract of Example <l-2> was added to the flour. Health enhancing foods such as bread, cake, cookies, crackers and noodles were prepared with the flour mixture according to the conventional method.
<2-2> Preparation of dairy products
5 - 10 weight% of the extract of Example <l-2> was added to milk. Health enhancing dairy products such as
butter and ice cream were prepared with the milk mixture according to the conventional method.
<2-3> Preparation of Sun-Sik Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders.
Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders.
The extract of Example <l-2> was concentrated under reduced pressure, spray-dried and pulverized to obtain 60- mesh dry powders.
Sun-Sik was prepared by mixing the dry powders of the grains, seeds and the extract of Example <l-2> according to the below ratio.
Grains (brown rice: 30 weight%, Yulmu: 15 weight%, barley: 20 weight%),
Seeds (wild sesame: 7 weight%, black soybean: 8 weight%, black sesame: 7 weight% ) ,
Dry powders of the extract of Example <l-2> (3 weight%) ,
Ganoderma lucldum (0.5 weight%), Rehmannia glutinosa (0.5 weight%)
Manufacturing Example 3: Preparation of beverages
<3-l> Preparation of health beverages
Extract of Example <l-2> 1000 rag
Citric acid 1000 mg Oligosaccharide 100 g
Maesil (Prunus mume) Extract 2 g
Taurine 1 g
Purified water up to 900 ml
The above constituents were mixed according to the conventional method for preparing health beverages. The mixture was heated at 85 °C for 1 hour with stirring and then filtered. The filtrate was loaded in 2 liter sterilized containers, which were sealed and sterilized again, stored in a refrigerator until they would be used for the preparation of a composition for health beverages.
The constituents appropriate for favorite beverages were mixed according to the preferred mixing ratio but the composition ratio can be adjusted according to regional and national preferences, etc.
<3-2> Preparation of vegetable juice
Health enhancing vegetable juice was prepared by- adding 5 g of the extract of Example <l-2> of the present invention to 1,000 m^ of tomato or carrot juice according to the conventional method.
<3-3> Preparation of fruit juice
Health enhancing vegetable juice was prepared by- adding 1 g of the extract of Example <l-2> of the present invention to 1,000 ml of apple or grape juice according to the conventional method.
Manufacturing Example 4 : Preparation of cosmetics
Functional cosmetics containing Pentaphylloides frutlcosa extract of the present invention as an active ingredient can be prepared. The present inventors prepared functional cosmetics, which are exemplified by such emulsified cosmetics as lotion, cream and essence; and such solubilized cosmetics as skin, etc.
<4-l> Preparation of emulsified cosmetics
Emulsified cosmetics were prepared according to the composition shown in Table 7. The method for the preparation is as follows. 1) heating the mixture comprising the raw materials
1-9 at 65-70°C;
2) adding the raw material 10 to the mixture of step i);
3) dissolving the mixture comprising the raw materials 11-13 by heating at 65-70°C;
4) slowly adding the mixture of step 2) during performing step 3), followed by emulsification at 8,000 rpm for 2-3 minutes;
5) dissolving the raw material 14 in water and then adding the solution to the mixture of step 4), followed by emulsification for 2 minutes;
6) weighing the raw materials 15-17, which were added to the mixture of step 5), followed by emulsification for 30 seconds; and
7) degassing the mixture of step 6) finished with the emulsification process and then cooling thereof at 25-35 °C to give emulsified cosmetics.
[Table 7]
<4-2> Preparation of solubilized cosmetics
Solubilized cosmetics were prepared according to the composition shown in Table 8. The method for the preparation is as follows.
1) adding the raw materials 2-6 to the raw material 1 (purified water), which were dissolved by using Agi-mixer;
2) adding the raw materials 8-11 to the raw material 7 (alcohol) and completely dissolved; and
3) slowly adding the mixture of step 2) to the mixture of step 1), followed by solubilization.
[Table 8]
Compositions of solubilized formulations 1, 2 and 3
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .
Claims
[CLAIMS]
[Claim l]
An antioxidative agent containing Pentaphylloides frutlcosa extract extracted by using water, alcohol or a mixture thereof as a solvent as an active ingredient.
[Claim 2]
The antioxidative agent according to claim 1, wherein the alcohol is Ci - C4 lower alcohol.
[Claim 3]
The antioxidative agent according to claim 2, wherein the lower alcohol is ethanol or methanol.
[Claim 4]
The antioxidative agent according to claim 1, wherein the antioxidative agent has radical scavenging activity.
[Claim 5] The antioxidative agent according to claim 1, wherein the antioxidative agent inhibits the activity of the transcription factor AP-I (activator protein-1).
[Claim 6] A preventive and therapeutic agent for a disease
caused by the excessive accumulation of active oxygen containing Pentaphylloides fruticosa extract extracted by using water, alcohol or a mixture thereof as an active ingredient.
[Claim 7]
The preventive and therapeutic agent according to claim 6, wherein the disease caused by the excessive accumulation of active oxygen is selected from the group consisting of liver disease, stroke, myocardial infraction, diabetic vascular disorder, hyperlipidemia, acute inflammation, rheumatism and cancer.
[Claim 8] A composition for the prevention of skin aging and the improvement of wrinkles containing Pentaphylloides fruticosa extract extracted by using water, alcohol or a mixture thereof as a solvent as an active ingredient.
[Claim 9]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the alcohol is Ci - C4 lower alcohol.
[Claim lθ]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the lower alcohol is ethanol or methanol.
[Claim ll]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition has radical scavenging activity.
[Claim 12]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition inhibits the activity of the transcription factor AP-I (activator protein-1).
[Claim 13]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition inhibits type I collagenase (matrix metalloproteinase-1, MMP-I) biosynthesis.
[Claim 14]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition promotes collagen biosynthesis.
[Claim 15]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition inhibits collagen decomposition.
[Claim 16]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition is formulated as a preparation for skin external application.
[Claim 17]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition is provided as cosmetics.
[Claim 18]
The composition for the prevention of skin aging and the improvement of wrinkles according to claim 8, wherein the composition improves skin elasticity.
[Claim 19]
A health improving functional food for the prevention of skin aging and the improvement of wrinkles containing
Pentaphylloides fruticosa extract extracted by using water, ethanol or a mixture thereof as a solvent as an active ingredient .
[Claim 20]
A method for the prevention and treatment of a disease caused by the excessive accumulation of active oxygen containing the step of administering an effective dose of Pentaphylloides fruticosa extract extracted by using water, ethanol or a mixture thereof as a solvent to a subject with a disease caused by the excessive accumulation of active oxygen.
[Claim 21] The method for the prevention and treatment of a disease caused by excessive accumulation of active oxygen according to claim 20, wherein the disease caused by the excessive accumulation of active oxygen is selected from the group consisting of liver disease, stroke, myocardial infraction, diabetic vascular disorder, hyperlipidemia, acute inflammation, rheumatism and cancer
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2007-0107086 | 2007-10-24 | ||
| KR1020070107086A KR20090041532A (en) | 2007-10-24 | 2007-10-24 | A composition comprising pentaphylloides fruticosa extract as an active ingredient |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2009054606A1 true WO2009054606A1 (en) | 2009-04-30 |
Family
ID=40579690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2008/004842 WO2009054606A1 (en) | 2007-10-24 | 2008-08-20 | A composition comprising pentaphylloides fruticosa extract as an active ingredient |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20090041532A (en) |
| WO (1) | WO2009054606A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040175439A1 (en) * | 2001-03-02 | 2004-09-09 | Benoit Cyr | Plant extracts and compositions comprising extracellular protease inhibitors |
| US20060228426A1 (en) * | 2002-08-30 | 2006-10-12 | Benoit Cyr | Plant extracts for treatment of angiogenesis and metastasis |
| US20070122492A1 (en) * | 2004-11-18 | 2007-05-31 | Stephen Behr | Plant extracts and dermatological uses thereof |
-
2007
- 2007-10-24 KR KR1020070107086A patent/KR20090041532A/en not_active Application Discontinuation
-
2008
- 2008-08-20 WO PCT/KR2008/004842 patent/WO2009054606A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040175439A1 (en) * | 2001-03-02 | 2004-09-09 | Benoit Cyr | Plant extracts and compositions comprising extracellular protease inhibitors |
| US20060228426A1 (en) * | 2002-08-30 | 2006-10-12 | Benoit Cyr | Plant extracts for treatment of angiogenesis and metastasis |
| US20070122492A1 (en) * | 2004-11-18 | 2007-05-31 | Stephen Behr | Plant extracts and dermatological uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| KOLPAKOV MA ET AL.: "Hepatoprotective properties of aqueous extract from Pentaphylloides fruticosa during chronic toxic hepatitis, 1", BULL EXP BIOL MED., vol. 131, no. 5, May 2001 (2001-05-01), pages 470 - 472 * |
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| Publication number | Publication date |
|---|---|
| KR20090041532A (en) | 2009-04-29 |
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