JP2014210723A - Anti-aging composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-aging component that has excellent anti-aging effects and has an active ingredient in a high level of safety.SOLUTION: An anti-aging composition comprises lower alcohol-water extracts of Alpinia zerumbet leaves as an active ingredient.

Description

  The present invention relates to an anti-aging composition comprising a lower alcohol-water extract of ghetto leaves as an active ingredient.

  Being young is an aspiration common to all humankind, and various studies have been conducted to delay aging since a long time ago. Research on the mechanism of aging has been actively conducted as the basis of this research, and various theories have been presented. However, research on the mechanism of aging is still in the basic research stage, and it is difficult to say that an effective means for preventing aging, such as prolonging lifespan or delaying various symptoms characteristic of aging, has been established.

  Under such circumstances, several components that are thought to be effective in preventing aging have been reported. For example, the results of animal experiments have reported that vitamin E, vitamin C, etc. have the effect of delaying the aging phenomenon, but subsequent studies confirmed that these did not have a clear life-spanning effect. ing.

In addition, extensive research has been conducted on plant extracts, amino acids, polymer compounds, glycosides, etc., for example, an extract of Phyllanthus niruri (Patent Document 1), a specific degree of deacetylation. Chitosan (patent document 2), threonine or methionine (patent document 3), nicotinamide riboside (patent document 4) and the like have been reported to have an anti-aging action.

JP-A-8-176004 JP 2005-289839 JP2010-83848 JP2012-176862

“Okinawa no Chouju”, pp. 31-48 (1999) “J.Am.Coll.Nutr.”, 28,500s-516s (1999) "ACS Symposium Series 507", pp72-86 (1992)

  As described above, various studies and proposals have been made on active ingredients that give anti-aging effects. However, it is desired to provide active ingredients that are superior in anti-aging effects and have high safety. It is an object of the present invention to provide an anti-aging component.

  The present inventor previously reported that, as one of the reasons for longevity in Okinawa, local people have ingested healthy plants, which contributes to the longevity of local people (Non-Patent Document 1 and 2). Nakatani reported that the antioxidant compounds contained in Okinawan plants contributed to the health of the islanders (Non-patent Document 3). And it is known that many of these plants contain abundant polyphenols having an antioxidant effect.

Therefore, the present inventor paid attention to ghetto ( Alpinia zerumbet ), which is one of the plants that grows only in Okinawa in Japan. The extract obtained by extraction with a mixture of water has been found to have an extremely high polyphenol content and can extend the life of the organism, thereby completing the present invention.

  That is, this invention is an anti-aging composition which uses the lower alcohol-water extract of a ghetto leaf as an active ingredient.

  The anti-aging composition of the present invention is expected to suppress aging and prolong life. Therefore, the anti-aging composition of the present invention prevents the aging of a patient with an aging or age-related disease by ingesting it, and is applied to the skin etc. It is possible to prevent aging of the skin and to prevent generation of wrinkles, stains and the like.

It is drawing which shows the quantity of the polyphenol in each ghetto leaf extract. It is the figure which showed the radical scavenging action of the lower alcohol-water extract (ALP) of a ghetto leaf in relation to the concentration. Transgenic C.I. It is drawing which shows the condition where the expression of SOD-3 :: GFP was raised in Elegance CF1553. Transgenic C.I. It is drawing which shows the condition where the expression of HSP-16.2 :: GFP was raised in Elegance CL2070.

The ghetto ( Alpinia zerumbet ) used in the present invention is a perennial plant classified as a genus of the family Gingidae. Has been used as a traditional Chinese medicine.

In the present invention, the leaves and stems of this ghetto are used as the raw material for the extract.
As the leaves and stems, harvested ones may be used as they are, or those that have been dried to some extent may be used.

  In obtaining the ghetto leaf extract used in the present invention, the leaf portion or stem portion (hereinafter referred to as “ghetto raw material”), which is a raw material, is shredded or pulverized to an appropriate size, and then the lower alcohol- Extraction may be performed by dipping in a water mixture (hereinafter referred to as “alcohol mixture”).

  Examples of the lower alcohol used in the alcohol mixture in this extraction include ethanol, methanol, isopropanol, and the like, and ethanol is preferable from the viewpoint of safety and the like. Further, the mixing ratio of the lower alcohol and water in the alcohol mixture is preferably in the range of 20% to 80% lower alcohol aqueous solution. Furthermore, the amount of the alcohol mixture with respect to 1 part by weight of the ghetto raw material is preferably about 1 to 20 parts by weight.

  The temperature in the extraction is not particularly limited, but is preferably about 25 to 60 ° C. In addition, it is desirable to perform slow mechanical stirring for the extraction.

  When the non-toxic extract such as water-ethanol is used as the ghetto leaf extract obtained as described above, the extract may be used as it is, or it may be concentrated and used as a concentrated extract. Further, it may be further made into a dry powder or a paste by known drying means such as freeze-drying.

  The ghetto leaf extract thus obtained can be made into an anti-aging composition by combining it with a known carrier or compounding component.

  The carrier or compounding component used in the preparation of the anti-aging composition of the present invention varies depending on the form of the composition. For example, in the case of a form to be blended in food or drink or a form to be used as a medicine, it is well known. What is necessary is just to mix | blend with a foodstuff raw material or a foodstuff, or to combine with a known pharmaceutical carrier thru | or a mixing component, and to make a formulation. Among these, examples of forms as pharmaceuticals include internal preparations such as tablets, capsules, powders, internal liquids, fine granules, granules and the like. On the other hand, in the case of skin cosmetics, skin external preparations and the like for the purpose of tissue activation effect in cells such as skin tissue, it is blended with known external preparations or cosmetic ingredients, liniment, spray, lotion, An external form such as an aqueous solution, emulsion, ointment or powder may be used.

  In addition, although there is no restriction | limiting in particular, the compounding quantity of the ghetto leaf extract in the case of the form mix | blended with food-drinks or the form used as a pharmaceutical etc. does not have a restriction | limiting, but about 10-1000 mg as an adult daily dose, Preferably 50- About 200 mg is appropriate. Moreover, the compounding quantity of the ghetto leaf extract in the case of using as a dermatological cosmetics and skin external preparation is about 0.1-20 mass% normally of the whole composition, and about 1-5 mass% is especially preferable.

  The effect of the anti-aging composition of the present invention will be described in detail in Examples below. For example, in tests conducted to evaluate stress tolerance, in the ghetto leaf extract at concentrations of 2.5, 5 and 10 μg / mL. , C.I. Elegance showed increased stress tolerance in both the oxidation resistance test and the heat resistance test. Also, in the survival analysis, 100 μg / mL ghetto leaf extract was more C.I. than 100 μM resveratrol. It has been found that the survival time of elegance is significantly extended.

  Many researchers have found that polyphenols from blueberries, grape resveratrol, ginkgo biloba, epigallocatechin gallate from green tea, and phenolic antioxidants from spinach are C.I. Although it has been shown that it can have a beneficial effect on the aging of elegance, the present invention also shows that the polyphenols of the ghetto leaf extract are C.I. It is considered that the survival time of elegance is significantly increased.

  As a result of the examples described later, the polyphenol contained in the ghetto leaves is C.I. It shows that it has a beneficial effect on the survival of elegance and that it may have great potential in the search for natural compounds for diseases related to aging or aging.

  Next, the present invention will be described in more detail with reference to examples and formulation examples, but the present invention is not limited to these examples and the like.

Example 1
Preparation of ghetto leaf extract:
50 g of fresh ghetto ( Alpinia zerumbet ) leaves were treated in 500 ml of boiling water for 20 minutes and cooled. This treated water was filtered to obtain a filtrate as a crude extract, which was then vacuum dried at 40 ° C. The obtained crude extract was dissolved again in water (10 mg / mL) to obtain a hot water extract (ALH) of ghetto leaves. This was stored in the refrigerator until use (ALH).

  Similarly, 50 g of fresh ghetto leaves were extracted with 50% ethanol (500 ml) at 25 ° C. for 48 hours. The resulting extract was filtered and evaporated to dryness under reduced pressure at 40 ° C. This solid was dissolved in water to 10 mg / ml to obtain a lower alcohol-water extract (ALP) of ghetto leaves. This was stored at 4 ° C. until use.

  By changing the extraction solvent from 50% ethanol to hexane and ethyl acetate, a hexane extract fraction and an ethyl acetate extract fraction of ghetto leaves were obtained, respectively. These extracted fractions were also dried under reduced pressure at 40 ° C. to obtain a hexane extract (Alh) and an ethyl acetate extract (Ale) in the same manner as described above.

Example 2
Measurement of total phenol content of each extract obtained:
The total phenol content of each extract (excluding ALH) obtained in Example 1 was measured using a foreign reagent. For comparison, the total phenol content of the ghetto leaf itself was also measured. The result is shown in FIG.

  As is clear from FIG. 1, the total phenol content of ALP was 10 times that of the ghetto leaves themselves, and 7 times or more compared to the ethyl acetate extract.

Example 3
Antioxidant activity of ALP:
The in vitro antioxidant activity of ALP was examined by evaluating the scavenging activity of phenoprolamin (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt (ABTS) radical. The results are shown in FIG.

  As is clear from FIG. 2, the radical activity increased depending on the concentration of ALP, indicating that ALP has antioxidant activity.

Example 4
Evaluation of the survival rate of nematodes against oxidative stress using ghetto leaf extract:
(1) Nematode species used and their maintenance Kaenolabditis. Caenohabdiyis elegans (hereinafter referred to as “C. elegans”) wild type, N2 (var. Bristol) and transgenic species CF1553 (muls84) and CL2070 (dvls70) together with Escherichia coli strain OP-50 Obtained from Loveditis Genetic Center (University of Minnesota). All species were routinely grown at 20 ° C. on Nematode Grouse Mediam (NGM) plates with E. coli OP-50 as a food source.

  Synchronization of nematode cultures is achieved by treating hermaphrodites with many eggs with bleach (50% sodium hypochlorite; 2.5M sodium hydroxide) and recovering L1 larvae hatched on NGM / OP50 plates Was achieved.

(2) Evaluation of resistance to oxidation and heat stress NGM plates were treated with different concentrations of ghetto leaf extract and quercetin. Plate treatment with ghetto leaf extract was performed by adding stock solution of ghetto leaf extract to the desired final concentration to NGM plates that were freshly autoclaved at 55 ° C. Age-synchronized nematode larvae (L1) were added to the treated NGM plate and maintained at 20 ° C. for 96 hours.

  Next, about 30 to 40 nematodes were placed in S-basal medium containing 0.5 mM hydrogen peroxide solution for 8 hours to give oxidative stress. After this, S-Basal medium containing both live and dead nematodes was transferred to a new NGM plate and the number of live and dead nematodes was counted. Survival was scored as the number responding to soft contact, similar to the original nematode on the plate. The results are shown in Table 1.

  As a result, ALH and ALP both increased the survival rate in nematodes exposed to oxidative stress, but the effects of ALH 125 μg / mL and ALP 10 μg / mL were almost equivalent, and the effect of ALP was higher. It became clear. Moreover, the survival rate of any of 62.5 to 250 μg / mL ALH and 2.5 to 10 μg / mL ALP was superior to quercetin 50 μg / mL.

Example 4
Evaluation of the survival rate of nematodes against heat stress using ghetto leaf extract:
Evaluation of heat resistance was carried out by determining whether a 5-day-old hermaphroditic wild type C.I. Performed by Elegance, N2 adults. The nematodes were transferred onto 3.5 cm NGM agar plates that had been fed as described above and then cultured at 35 ° C. Scores were made every 2 hours by counting dead nematodes that did not respond to soft contact with the platinum tip. Nematodes that died from drying on the sides of the plate were not counted. The results are shown in Table 2.

  From the results shown in Table 2, under the heat stress of 35 ° C., the wild type nematode N2 is viable in both ALH (62.5, 125, 250 μg / mL) and ALP (25, 50, 100 μg / mL). Improved. The value is the survival rate (%) in unit time, and Q50 means quercetin 50 μg / mL for comparison.

Example 5
The life-longening effect of elegance of ghetto leaf extract:
For survival assessment, synchronized nematodes (20-30 per plate) were alive. Both Koly OP-50 were seeded and maintained on 3.5 cm NGM plates, and given different concentrations (0, 25, 50 and 100 μg / mL) of ghetto leaf extract and resveratrol (100 μM) .

  On the first day of adulthood (day 4), 400 μL of 5-fluorodeoxyuridine (FUdR) was added to prevent growth. Nematodes are nourished with live E. coli OP-50 and every 4 days, 80 μL of live E. coli OP-50 is added to the NGM plate to prevent the nematode from starving. It was. Dead nematodes were counted every two days as described above. The results are shown in Table 3.

  From Table 3, the effect that the lifetime depends on the concentration was recognized. There was a significant increase in nematode estimated mean survival time at 50 and 100 μg / mL ALP. The mean survival for controls in this study also increased the mean survival by 8.27, 26.8 and 49.5% at 25, 50 and 100 μg / mL ALP.

  Treatment with resveratrol (100 μM) had an increase of 23.7% compared to the control, whereas the group treated with ALP100 had an increase of 20.8% and the line treated with resveratrol. It had the property of prolonging survival better than insects. In addition, nematodes treated with ALP50 also had the same property of extending survival as resveratrol.

  When no nematodes were alive any longer in the control group, approximately 40% of nematodes were still alive in the group treated with 100 μg / mL ALP. Similarly, the maximum survival time of nematodes was also significantly increased to 13.2, 32.6 and 60.9% at ALP concentrations of 25, 50 and 100 μg / mL, whereas resveratrol (100 μM) only increased by 33.5% compared to the control.

  As described above, ALP having a high phenol content is C.I. It appears to have a substantive role in delaying aging of elegance, which is thought to have similar effects on other organisms.

Example 6
Fluorescence quantification and visualization test The GFP fluorescence of the population expressing GFP was assessed using an MTP-880 fluorometer (Corona Electric, Japan). Fourteen-day controls, 15 nematodes treated with 10 μg / mL ALP, were placed in Nunc 96-well microtiter plates (black, clear, flat-bottomed wells) in 100 μL phosphate buffer. Transferred and total GFP fluorescence was measured using a 490 nm excitation and 530 nm emission filter.

  The experiment was repeated twice in triplicate. Results were provided as the mean ± SE of 6 experiments. For fluorescent microscopy, a GFP photograph of the transgenic nematode was taken with an Olympus DP-70 microscope.

  As a result, ALP was transformed into transgenic C.I. It was found that Elegance CF1553 regulates to increase the expression of SOD-3 :: GFP.

  To investigate whether the increase in stress resistance was due to the regulation of genes that respond to ALP specific stress, the reactivity of the antioxidant enzyme SOD was examined to 10 μg / mL.

  The group treated with ALP showed significantly higher SOD-3 :: GFP brightness in photographs taken with a confocal laser microscope (not shown). Lightness quantification through a microplate reader showed that ALP was able to significantly regulate SOD-3 :: GFP expression at 14 μg / mL and above 143% (FIG. 3).

  Similarly, ALP is transformed into transgenic C.I. It was found that Elegance CL2070 regulates to increase the expression of heat shock protein HSP-16.2.

  The HSP-16.2 family of proteins is αβ-crystalline and homologous, and C.I. It was elegance and expressed in stress. C. Predicting life with elegance can serve as a stress-sensitive reporter. The higher level of HSP-16.2 :: GFP is C.I. Predict the longer average life expectancy of elegance.

  In this study, the effect of ALP on HSP-16.2 expression was investigated and C. Elegance has provided more clues to the protective effects of ALP. CL2070 nematodes containing the HSP-16.2 :: GFP reporter gene were heat shocked at 35 ° C. for 2 hours and recovered at 20 ° C. for 12 hours. The sample treated at 10 μg / mL showed higher HSP-16.2 :: GFP intensity (not shown) on a confocal laser microscope.

  The results of quantifying the intensity of fluorescence showed that ALP was adjusted to significantly increase the expression of HSP-16.2 :: GFP approximately 1.5 times higher in CL2070 (FIG. 4).

Product example 1
Beauty liquid:
ALP 100mg
Glycerin 4g
Ethanol 10g
Xanthan gum 300mg
Fragrance Fine amount Purified water Balance
Total 100g

Product example 2
Condylar grains:
Collagen peptide 500mg
ALP 30mg
Soy isoflavone 50mg
Reduced lactose balance Soybean oligosaccharide 36mg
Erythritol 36mg
Dextrin 25mg
Citric acid 24mg
Fragrance fine amount total 1200mg

Product example 3
Drink ：
ALP 50mg
Royal Jelly 10mg
Liquid sugar 10g
DL-sodium tartrate 10mg
Citric acid 100mg
Vitamin C 100mg
Vitamin E 200mg
Cyclodextrin 250mg
Potassium chloride 20mg
Magnesium sulfate 10mg
Purified water balance meter 100ml

  ALP (lower alcohol-water extract of ghetto leaves) contains a high content of polyphenols, which is expected to prolong the life of the organism.

  Therefore, daily intake of foods and medicines containing this is expected to prevent aging of patients with aging or diseases related to aging, and can be used as an anti-aging composition.

  In addition, when the above ALP is applied to the skin or the like, it can be expected to have a tissue activating effect on cells such as skin tissue, prevent skin aging, and prevent generation of wrinkles, spots and the like. .

Claims (6)

  An anti-aging composition comprising a lower alcohol-water extract of ghetto leaves as an active ingredient.   2. The anti-aging composition according to claim 1, wherein the lower alcohol-water extract is an extract with a 20 to 80% lower alcohol aqueous solution.   The anti-aging composition according to claim 1 or 2, wherein the lower alcohol is ethanol.   The anti-aging composition according to any one of claims 1 to 3, which is in the form of a pharmaceutical product.   The anti-aging composition of any one of Claims 1 thru | or 3 which is a form mix | blended with food-drinks.   The anti-aging composition according to any one of claims 1 to 3, which is in the form of a cosmetic for skin or an external preparation for skin.

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