TWI736818B - Extract from golden eye-grass (curculigo orchioides) and use in preparation of composition having activities of skin whitening and protecting dna - Google Patents

Abstract

The present invention relates to an extract from Golden eye-grass (Curculigo orchioides) and a use thereof. The Golden eye-grass extract has activities of skin whitening and protecting DNA, so that it can be added as an effective ingredient in a composition for skin whitening and protecting DNA.

Description

The use of Curculigo extract for the preparation of a composition with skin whitening and DNA protection activity

The present invention relates to the use of a plant extract, in particular to the use of a curculus extract to prepare a composition with skin whitening and DNA protection activities.

It is currently known that ultraviolet rays can cause wrinkles and pigment spots on the skin, and even cause cell aging, cancerization or death. Ultraviolet (ultraviolet; UV) or reactive oxygen species (reactive oxygen species; ROS) is one of the factors that cause a large number of free radicals. The activity of free radicals is high and unstable. Too many free radicals will be produced with the cell tissue in the body Oxidation reaction. After ultraviolet rays increase the activity of free radicals in the human body, under normal conditions, the human body has the ability to repair. However, excessive ultraviolet rays cause a large number of free radicals to be produced, causing tissue cells to lose their normal functions, and even damage DNA, reducing the cell's DNA repair ability, causing damage or mutations, and then causing cancer.

Curculigo (Golden eye-grass; Kali musli, Syah musli; Weevil-wort; Xian mao; Curculigo orchioides ), also known as ground brown roots, ground brown, single-grass roots, single-grass, single-leg curculus, codonopsis, curculigo Ginseng, Hainan ginseng, salsify or bud melon seeds, which are perennial herbs of the genus Curculio Amaryllidaceae. Distributed in Indonesia, Japan, Southeast Asia, Taiwan Island and China's Guangxi, Guangdong, Guizhou, Fujian, Yunnan, Zhejiang, Sichuan, Hunan, Jiangxi and other places. Curculigo is known to have liver protection, anti-cancer, immune regulation, anti-diabetic, wound healing and anti-asthmatic activities.

However, there is currently no document confirming whether Curculio vulgaris extract has skin whitening and DNA protection activities. In view of this, there is an urgent need to provide a new use of Curculio extract to expand its application.

Therefore, one aspect of the present invention is to provide a use of Curculigo orchioides extract for preparing a composition with skin whitening and DNA protection activities.

Another aspect of the present invention is to provide a curculio extract obtained through at least one distribution extraction step. The aforementioned Curculio extracts include any one or any combination of crude extracts, n-hexane extracts, ethyl acetate extracts, and n-butanol extracts.

Another aspect of the present invention is to provide a composition comprising a curculio extract obtained by the above-mentioned at least one partition extraction step.

According to the above aspect of the present invention, a curculus extract is proposed for the preparation of a composition with skin whitening and DNA protection activities. In this way, the above-mentioned Curculio vulgaris extract uses a lower alcohol solution to perform a rough extraction step on the roots or rhizomes of Curculigo to obtain the crude extract. The weight-volume ratio (g:mL) of the roots or rhizomes to the lower alcohol solution is 1:500 to 1:1000, and the lower alcohol solution is methanol solution or ethanol solution.

According to the above-mentioned embodiment of the present invention, the above-mentioned curculio extract uses n-hexane to perform the first distribution extraction step on the crude extract to obtain a n-hexane extraction layer and a water extraction layer, wherein the n-hexane extraction layer has a n-hexane extract .

According to the above-mentioned embodiment of the present invention, the above-mentioned curculio extract uses ethyl acetate to perform the second distributive extraction step on the water extraction layer to obtain an ethyl acetate extraction layer, wherein the ethyl acetate extraction layer has an ethyl acetate extract .

According to the above-mentioned embodiment of the present invention, the above-mentioned curculio extract uses n-butanol to perform the third distributive extraction step on the water extraction layer to obtain the n-butanol extraction layer, wherein the n-butanol extraction layer has the n-butanol extract .

According to another aspect of the present invention, which system using the application of curculin extract obtained in the extraction step as the whitening active ingredient at least one of the dispensing, an effective dose of whitening activity of this extract is Curculigo 25 μ g / mL to 50 μ g / mL.

According to another aspect of the present invention, the curculus extract obtained by the above-mentioned distributive extraction step at least once is used as an application to protect the active ingredients of DNA, and the effective dose of the curculus extract to protect the DNA activity is 100 μ g / mL to 500 μ g / mL.

According to another aspect of the present invention, the above-mentioned Curculigo extract is used as a composition, wherein the composition is a cosmetic composition or a pharmaceutical composition.

In order to make the above and other objectives, features, advantages and embodiments of the present invention more comprehensible, the detailed description of the accompanying drawings is as follows: [FIG. 1A] is a diagram showing the effect of Curculio vulgaris extract according to an embodiment of the present invention Bar graph of mushroom tyrosinase activity in cells.

[Fig. 1B] is a bar graph showing the reduction of melanin content in cells by Curculio vulgaris extract according to an embodiment of the present invention.

[Figure 2A] is a nucleic acid electrophoresis diagram showing the protection of DNA from Curculio vulgaris extract under the treatment of _{hydrogen peroxide (H 2} O _{2) and UVB.}

[Figure 2B] is a bar graph showing the fluorescence intensity ratio of the supercoiled DNA and the open-circular DNA of each group in Figure 2A.

[Fig. 3A] is a bar graph showing the survival rate of cells co-cultured with Curculio vulgaris extract and HaCaT cells according to an embodiment of the present invention.

[Figure 3B] is a bar graph showing the survival rate of cells co-cultured with Curculio vulgaris extract and B16F10 cells according to an embodiment of the present invention.

[Figure 4] is a bar graph showing the in vitro nitric oxide (NO) free radical scavenging rate of Curculio vulgaris extract of an embodiment of the present invention.

The bearing, the present invention provides a method for the manufacture curculin extract having skin whitening composition of the active material and the protection of DNA, this curculin extract was based on the use of a lower alcohol solution Hypoxidaceae (Curculigo orchioides) of the root or rhizome (rhizomes) are obtained by the crude extraction step.

The lower alcohol solution referred to herein may be, for example, an alcohol solution with a carbon number of not more than 3, and may optionally be a methanol solution or an ethanol solution. In one embodiment, during the rough extraction step, the tuber or rhizome of Curculigo orchioides is crushed at no higher than room temperature (for example, 10°C to 40°C). Then, the crushed curculio tubers or rhizomes are added to a lower alcohol solution for extraction, and then filtered one or several times to remove residues using gauze, filter paper, centrifugation or other conventional methods to obtain filtrate. In this embodiment, the weight-to-volume ratio (g:mL) of the tuber to the lower alcohol solution is 1:500 to 1:1000.

Then, a dehydration step can be optionally performed, such as a vacuum concentration method, a freeze-drying method, and other conventional methods to remove the lower alcohol solution of the filtrate to obtain a crude extract. In an embodiment, the dehydration step may use a conventional method, such as a vacuum drying method, a vacuum freeze drying method, a reduced pressure concentration method, etc., and the present invention is not limited to the examples herein.

In one embodiment, n-Hexane and water are used to perform a partition extraction step on the crude extract prepared above to obtain a n-Hexane extraction layer and a water extraction layer. The obtained n-hexane extract layer is subjected to a dehydration step to remove excess water to obtain a n-hexane extract.

Then, using ethyl acetate (Ethyl acetate) to perform a partition extraction step on the water extraction layer prepared above to obtain an ethyl acetate extraction layer. The obtained ethyl acetate extract layer is subjected to a dehydration step to remove excess water to obtain an ethyl acetate extract.

After that, n-Butanol is used to perform a partition extraction step on the water extraction layer prepared above to obtain an n-Butanol extraction layer. The obtained n-butanol extract layer is subjected to a dehydration step to remove excess water to obtain an n-butanol extract.

The curculus extract may optionally include any one or any combination of crude extract, n-hexane extract, ethyl acetate extract, and n-butanol extract. Experiments have confirmed that the above Curculigo extracts have skin whitening and DNA protection activities, and can be used as effective ingredients in various compositions. In some embodiments, the aforementioned composition may be, for example, a cosmetic composition or a pharmaceutical composition. The cosmetic composition referred to herein is a mixture of Curculio vulgaris extract with a cosmetically or dermatologically acceptable carrier or medium, and presents a form suitable for topical application. The aforementioned "cosmetic or dermatologically acceptable" means that it is in contact with the skin or human skin appendages, and does not pose a risk of toxicity, intolerance, instability, allergic reactions and other risks suitable for users. Specifically, the cosmetic composition can be used in products for cleaning, protecting, maintaining, and beautifying the skin, such as facial cleansers, lotions, facial masks, cosmetics, and the like.

The medical composition referred to here refers to the curculus extract as the active ingredient, mixed with a pharmaceutically acceptable carrier, and formulated for topical or oral administration using techniques known to those with ordinary knowledge in the field. Suitable dosage form of the medicine. The pharmaceutically acceptable carrier may contain one or more of the following preparations: solvents, emulsifiers, suspending agents, decomposers, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants Agents, absorption delay agents, liposomes, etc.

When applied to a whitening active composition, the composition may contain an effective dose of curculus extract at 25 μg/mL to 50 μg/mL.

When applied to a composition with DNA-protecting activity, the composition contains an effective dose of curculio extract at a dose of 100 μg/mL to 500 μg/mL to effectively protect DNA from damage.

It is supplemented that there is no particular limitation on the cause of the aforementioned DNA damage, and it can be damage caused by ultraviolet (ultraviolet; UV) or reactive oxygen species (ROS). The aforementioned ultraviolet rays may include, but are not limited to, ultraviolet A (UVA), ultraviolet B (UVB), ultraviolet C (UVC) and/or any combination of the foregoing. The reactive oxygen-containing species may include, but are not limited to superoxide (superoxide; O ₂ ^-), hydroxyl radical (hydroxy radical; ‧OH), peroxide radical (peroxide radical; ROO‧) or hydrogen peroxide (H ₂ O ₂ ) and so on.

The following uses several embodiments to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.

Example 1: Determination of the total phenol content and total flavonoid content of Curculio vulgaris extract

First, the rhizomes of Curculio vulgaris pulverized at no higher than room temperature (for example, 10°C to 40°C). Next, the crushed Curculio rhizome was added to the ethanol solution. Then, use gauze, filter paper, centrifugation or other conventional methods to filter once or several times to remove the residue, and then use a vacuum concentration method to perform a dehydration step to remove the ethanol solution to obtain a crude extract.

Next, the crude extract is partitioned and extracted using n-hexane and water to obtain a n-hexane extraction layer and a water extraction layer. The water extraction layer is then subjected to a partition extraction step with ethyl acetate to obtain an ethyl acetate extraction layer. Using n-butanol to perform a partitioning extraction step on the water extraction layer to obtain a n-butanol extraction layer.

The n-hexane extraction layer, the ethyl acetate extraction layer, and the n-butanol extraction layer obtained above are subjected to a dehydration step using a reduced pressure concentration method to remove excess water to obtain a n-hexane extract, an ethyl acetate extract, and N-Butanol extract. Any one or any combination of the crude extract, n-hexane extract, ethyl acetate extract, and n-butanol extract prepared here is the Curculigo extract of the present invention.

Next, the total phenolic contents (TPC) and total flavonoids content (TFC) of the curculio extract were determined. Take 1μL, 1000μg/ml crude extract, n-hexane extract, ethyl acetate extract and n-butanol extract respectively, add 250μL ten-fold diluted Folin-Ciocalteu's phenol reagent, and shake After mixing, let stand for 5 minutes, then add 200μL of 7.5% sodium carbonate (Na ₂ CO ₃ ) solution and 500μL of deionized water (ddH ₂ O), protect from light at room temperature for 1 hour, and use a spectrophotometer (e.g. Synergy ^TM 2 Multi-Mode Microplate Reader (BioTek, USA) measures the absorbance at a wavelength of 760nm. Make a calibration curve against the standard galic acid, and convert it into every gram of Curculio extract Contains gallic acid equivalents (gallic acid equivalents), to calculate the total phenolic content of Curculio vulgaris extract.

Separately take 1μL, 1000μg/ml crude extract, n-hexane extract, ethyl acetate extract and n-butanol extract, add 490μL deionized water and 3μL 5% sodium nitrite (NaNO ₂ ) to react for 6 minutes , Then add 30μL of 10% aluminum chloride (AlCl ₃ ) to react for 6 minutes, add 400μL of 4% sodium hydroxide (NaOH) and 40μL of deionized water, shake and mix uniformly, let stand for 15 minutes, measure with spectrophotometer Absorbance at 510nm wavelength. Make a calibration curve against Rutin standards. Calculate the total flavonoids of the flavonoids in the curculus extract by converting the rutin equivalents contained in each gram of the curculus extract.

The results showed that the total phenol content of the ethanol extract was equivalent to 18.8 mg/g gallic acid equivalent, and the total flavonoid content was equivalent to 64.3 mg/g rutin equivalent. The total phenol content of the n-hexane extract is equivalent to 3.1 mg/g gallic acid equivalent, and the total flavonoid content is equivalent to 107.2 mg/g rutin equivalent. The total phenol content of the ethyl acetate extract is equivalent to 32.8 mg/g gallic acid equivalent, and the total flavonoid content is equivalent to 232.6 mg/g rutin equivalent. The total phenol content of the n-butanol extract is equivalent to 25.5mg/g of gallic acid equivalent, and the total flavonoid content is equivalent to 151.5mg/g of rutin equivalent.

Example 2: Evaluation of Curculio extract inhibiting melanin production in cells

The melanogenesis of cells is mainly produced through the activation of tyrosinase by α-melanocyte-stimulating hormone (α-MSH). The reaction pathway is tyrosine catalyzed by tyrosinase to form dopa dihydroxyphenylalanine (DOPA), DOPA Once again catalyzed by tyrosinase, DOPAquinone is formed, which in turn leads to the synthesis of melanin. By inhibiting the activity of tyrosinase, the synthesis of melanin can be reduced. In this example, melanoma cancer cells were used as the analysis mode to evaluate the effects of Curculio vulgaris extract on inhibiting tyrosinase activity and melanin content in the cells.

1. Curculigo extract inhibits tyrosinase activity in cells

In this example, mushroom tyrosinase was used as an in vitro model to evaluate the effect of Curculio vulgaris extract on inhibiting the intracellular tyrosinase activity of melanoma cancer cells B16F10 (accession number: BCRC 60031). First, culture the B16F10 melanoma cells in a 96-well cell culture dish at 37ºC and 5% CO ₂ for at least 24 hours. Next, divide the cells into a control group (2μL of water per well), a-MSH treatment group (0.1μM of α-MSH per well), and each treatment group (0.1μM of α-MSH per well and 2μL of the difference in Example 1 Curculigo extract (25μg/mL to 50μg/mL)] or arbutin treatment group (0.1μM α-MSH and 2μL of 50μg/mL arbutin per well), treated for 72 hours. Then, add 18 μL of dimethyl sulfoxide (DMSO) to each well, mix well, and react at room temperature for 10 minutes, then add 155 μL of 2.5 mM _L- DOPA, and react at room temperature for 20 minutes. After that, use a commercially available multi-function microplate reader (such as Synergy ^TM 2 Multi-Detection Microplate Reader, BioTek Instruments, Inc., USA), according to the manufacturer (such as CellTiter 96 AQ, Promega, Madison.WI, USA) provided _{Measure the absorbance value (OD 492nm} ) of cells in each well at a wavelength of 492nm. Taking the OD _{492nm of the} control group as 100%, the tyrosinase activity (%) in the cells of each group was converted, and the result is shown in Figure 1A.

Please refer to FIG. 1A, which is a bar graph showing the activity of Curculigo vulgaris extract on intracellular tyrosinase activity according to an embodiment of the present invention. Among them, the X axis represents the control group (untreated B16F10 cells), the α-MSH group (the B16F10 cells with α-MSH), and each treatment group (with α-MSH and crude extract (concentrations respectively) from left to right. B16F10 cells with 25μg/mL and 50μg/mL); B16F10 cells with α-MSH and n-hexane extracts (concentrations of 25μg/mL and 50μg/mL respectively); α-MSH and ethyl acetate extracts (concentrations) 25μg/mL and 50μg/mL) B16F10 cells; B16F10 cells supplemented with α-MSH and n-butanol extract (concentrations of 25μg/mL and 50μg/mL, respectively) and the arbutin group (with α-MSH and arbutin) (Arbutin) (50μg/mL) B16F10 cells]. The Y axis is the activity (%) of tyrosinase in the cell.

The results in Figure 1A show that compared with the α-MSH group, after adding crude extract, n-hexane extract, ethyl acetate extract, and n-butanol extract, the tyrosinase activity in B16F10 cells decreased . Among them, the n-butanol extract has the best inhibitory activity on tyrosinase, followed by the n-hexane extract, followed by the ethyl acetate extract and the crude extract. Therefore, the above results confirm that Curculio vulgaris extract has the activity of inhibiting tyrosinase.

2. Evaluation of Curculigo extract reducing the melanin content in cells

In this example, B16F10 cells were used as the cell model to evaluate the effect of Curculio vulgaris extract on inhibiting melanin content in cells. First, add B16F10 cells (1×10 ⁵ cells/mL, 1 mL per well) to a 24-well cell culture dish and place them in a 37°C, 5% CO ₂ incubator for at least 24 hours. The above-mentioned cells are divided into control group (1μL water per well), α-MSH treatment group (0.1μM α-MSH per well, 100μL per well), and each treatment group (0.1μM α-MSH and 1μL of Example 1) Curculigo extract (25μg/mL to 50μg/mL)] or arbutin treatment group (0.1μM α-MSH per well and 2μL of 50μg/mL arbutin)], _{cultured at 37°C, 5% CO 2} for 72 hours. After that, remove the culture medium, add 300 μL of 1 equivalent concentration (N) of sodium hydroxide (NaOH) to each well to dissolve the cells, and measure the cell lysate in each well at a wavelength of 405nm using the aforementioned commercially available multi-function microplate reader. The absorbance value (OD _405nm ). Taking the melanin content of the untreated B16F10 cells (control group) as 100%, the melanin content (%) in the cells of each group was converted, and the result is shown in Figure 1B.

Please refer to FIG. 1B, which is a bar graph showing the reduction of melanin content in cells by Curculio vulgaris extract according to an embodiment of the present invention. Among them, the X axis represents the control group (untreated B16F10 cells) and each treatment group (the B16F10 cells with α-MSH (α-MSH group) from left to right; α-MSH and crude extracts are added (concentrations respectively) B16F10 cells with 25μg/mL and 50μg/mL); B16F10 cells with α-MSH and n-hexane extracts (concentrations of 25μg/mL and 50μg/mL respectively); α-MSH and ethyl acetate extracts (concentrations) 25μg/mL and 50μg/mL) B16F10 cells; B16F10 cells supplemented with α-MSH and n-butanol extract (concentrations of 25μg/mL and 50μg/mL, respectively) and the arbutin group (with α-MSH and arbutin) (50μg/mL) of B16F10 cells]. The Y-axis is the intracellular melanin content (%).

The results in Figure 1B show that B16F10 cells with Curculigo extract (crude extract, n-hexane extract, ethyl acetate extract or n-butanol extract) have a higher intracellular melanin content than B16F10 cells added with arbutin The melanin content in the cells is low. Among them, the crude extract has the better effect on reducing the melanin content in the cells, followed by the n-hexane extract and the n-butanol extract, and then the ethyl acetate extract. Therefore, the above results confirm that Curculio vulgaris extract has the effect of reducing the melanin content in cells.

Example 3: Evaluation of the effect of Curculio vulgaris extract on DNA protection

Hydrogen peroxide (H ₂ O ₂ ) is metabolized and/or UVB irradiated to form hydroxyl free radicals, which attack DNA and damage the DNA structure. In this example, hydrogen peroxide and UVB irradiation were used to simulate the damage of free radicals to the supercoiled (SC) pUC119 plastid DNA, and the supercoiled pUC119 pUC119 plastid DNA was damaged to form an open circular type (open circular). ; OC) or linear (linear; LC) structure. The effect of curculus extract on DNA protection is evaluated by the ratio of supercoiled DNA and open-circular DNA.

In this example, 25 μg, 0.5 μg/μL of pUC119 plastid DNA (TaKaRa, Japan) was diluted with PBS, and the volume ratio of pUC119 plastid DNA to PBS was 1:9. Take 2μL of the diluted pUC119 plastid DNA and add 4μL of PBS, without adding (control group; oxidation and UVB injury group) or adding 2μL of the Curculio vulgaris extract of Example 1 with different concentrations (100μg/mL and 500μg/mL) , Do not apply (control group) or apply 2μL, 0.1mM hydrogen peroxide and 2μL, 0.5mM ferrous sulfate (FeSO ₄ ), do not irradiate (control group) or irradiation dose of 20mJ/cm ² UVB, and After waiting for 1 hour at 37°C. Use 0.8% agar gel for electrophoresis. The electrophoresis film was photographed using the image capture analysis system, and the result is shown in Figure 2A. Figure 2A After the fluorescence intensity of each color band is calculated by the image analysis software, the fluorescence intensity of the supercoiled DNA and the open circle DNA of the control group are respectively set as 1.0, and the supercoiled DNA or open circle DNA of each lane is converted The relative fluorescence ratio, the result is shown in Figure 2B. The higher the fluorescence ratio of supercoiled DNA or open-circle DNA, the better the ability of Curculio extract to protect DNA.

Please refer to FIG. 2A, which is a nucleic acid electrophoresis diagram showing the protection of DNA from Curculio vulgaris extract under the treatment of _{hydrogen peroxide (H 2} O _{2) and UVB.} Among them, the electrophoresis lanes (lane) from left to right are the control group (untreated pUC119 plastid DNA), the oxidation and UVB damage group (the pUC119 pUC119 plastid DNA was simultaneously administered with hydrogen peroxide, ferrous sulfate and UVB) and Each treatment group [simultaneously administered hydrogen peroxide, ferrous sulfate, UVB, and crude extracts (concentrations of 100μg/mL and 500μg/mL) pUC119 plastid DNA; simultaneously administered hydrogen peroxide, ferrous sulfate, UVB And n-hexane extracts (concentrations of 100μg/mL and 500μg/mL respectively) pUC119 plastid DNA; simultaneously administered with hydrogen peroxide, ferrous sulfate, UVB and ethyl acetate extracts (concentrations of 100μg/mL and 500μg, respectively) /mL) pUC119 plastid DNA; and simultaneously administered with hydrogen peroxide, ferrous sulfate, UVB and n-butanol extracts (concentrations of 100μg/mL and 500μg/mL, respectively) pUC119 pUC119 plastid DNA]. Taking the fluorescence intensity of the supercoiled DNA and the open-circle DNA of the untreated pUC119 plastid DNA (control group) as 1.0, calculate the relative fluorescence intensity ratio of the supercoiled DNA and open-circle DNA in each lane , And the result is shown in Figure 2B.

Please refer to FIG. 2B, which is a bar graph showing the fluorescence intensity ratio of the supercoiled DNA and the open-circular DNA of each group in FIG. 2A. Among them, the X axis from left to right represents untreated pUC119 plastid DNA (control group); pUC119 pUC119 plastid DNA (oxidation and UVB damage group) administered simultaneously with hydrogen peroxide, ferrous sulfate and UVB; simultaneously administered Hydrogen peroxide, ferrous sulfate, UVB and crude extracts (concentrations of 100μg/mL and 500μg/mL, respectively) pUC119 plastid DNA; simultaneously administered with hydrogen peroxide, ferrous sulfate, UVB and n-hexane extract (concentration 100μg/mL and 500μg/mL) pUC119 plastid DNA; simultaneously administered with hydrogen peroxide, ferrous sulfate, UVB and ethyl acetate extract (concentrations of 100μg/mL and 500μg/mL respectively) pUC119 pUC119 plastid DNA; pUC119 pUC119 plastid DNA simultaneously administered with hydrogen peroxide, ferrous sulfate, UVB and n-butanol extracts (concentrations of 100μg/mL and 500μg/mL, respectively). The Y-axis represents the fluorescence intensity ratio of supercoiled (SC) DNA to open circle (OC) DNA.

The results of Fig. 2A and Fig. 2B show that the n-hexane extract and ethyl acetate extract maintain a higher proportion of pUC119 plastid DNA in the supercoiled form, followed by the crude extract, and then the n-butanol extract. And adding n-hexane extract, ethyl acetate extract, 500μg/mL crude extract and 500μg/mL n-butanol extract of pUC119 plastid DNA, which maintains the amount of supercoiled DNA compared to oxidation and There are many UVB damage groups. Therefore, the above The results confirmed that Curculigo extract has the activity of protecting DNA from oxidation and UVB damage.

Example 4: Evaluation of the cytotoxicity of Curculio vulgaris extract

In this example, HaCaT cells or B16F10 cells were used for co-cultivation without addition or addition of curculus extracts to evaluate the cytotoxicity of curculus extracts.

In this implementation, first, HaCaT cells or B16F10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY), 10% fetal bovine serum (Hazelton Product, Denver, PA). , USA) and 1% penicillin-streptomycin (penicillin-streptomycin) in 5% CO ₂ , 37 ℃ incubator. Then inoculate HaCaT cells or B16F10 cells in a 96-well cell culture dish at a cell concentration of 1×10 ⁴ _{cells/well, and culture them in a 37°C, 5% CO 2} incubator for at least 24 hours. Then, in each well of the 96-well cell culture plate, without adding (control group) or adding different concentrations (25μg/mL to 100μg/mL) of the fresh culture solution of Curculio vulgaris extract of Example 1 for a total of 24 Hour. Then, use bromide 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide; MTT] method to evaluate the cell viability of HaCaT cells or B16F10 cells.

First, add 10 μL of 5 mg/mL MTT to each well of a 96-well cell culture plate _{. After incubating for 4 hours at 37°C and 5% CO 2} , remove the supernatant, and then add 100 μL of DMSO dissolved MTT. After shaking for 5 minutes, measure the absorbance (OD _570nm ) at a wavelength of 570nm to calculate the cell viability. Taking the cell survival rate of HaCaT cells or B16F10 cells (control group) without addition of Curculigo extract as 100%, calculate the survival rate of the cells under different concentrations of Curculia extract. The results are shown in Figure 3A (HaCaT cells) and Figure 3B (B16F10 cells). For example, the higher the cell survival rate, the lower the toxicity of Curculio curculio extract to cells.

Please refer to FIG. 3A, which is a bar graph showing the survival rate of the cells co-cultured with Curculio vulgaris extract and HaCaT cells according to an embodiment of the present invention. Among them, the X axis from left to right represents crude extract (concentrations of 25μg/mL, 50μg/mL, 100μg/mL) and n-hexane extract (concentrations of 25μg/mL, 50μg/mL, 100μg/mL) , Ethyl acetate extract (concentrations of 25μg/mL, 50μg/mL, 100μg/mL) and n-butanol extract (concentrations of 25μg/mL, 50μg/mL, 100μg/mL). The Y axis represents the cell survival rate (%) of HaCaT cells.

The results in Fig. 3A show that after 24 hours of co-cultivation of HaCaT cells and Curculio extract, the cell survival rate was maintained at more than 80%, indicating that Curculia extract is not cytotoxic to HaCaT cells.

Please refer to FIG. 3B, which is a bar graph showing the survival rate of cells co-cultured with Curculio vulgaris extract and B16F10 cells according to an embodiment of the present invention. Among them, the X axis from left to right represents the crude extract (control group and concentration 25μg/mL, 50μg/mL, 100μg/mL crude extract), n-hexane extract (control group and n-hexane extract with concentration of 25μg/mL, 50μg/mL, 100μg/mL), ethyl acetate Ester extract (control group and ethyl acetate extract with concentration of 25μg/mL, 50μg/mL, 100μg/mL) and n-butanol extract (control group and concentration of 25μg/mL, 50μg/mL, 100μg, respectively /mL of n-butanol extract). The Y axis represents the cell survival rate (%) of B16F10 cells.

The results in Fig. 3B show that after 24 hours of co-cultivation of B16F10 cells and Curculio extract, the cell survival rate is maintained above 75%, indicating that Curculia extract is not cytotoxic to B16F10 cells.

Example 5: Scavenging ability of Curculio vulgaris extract on nitric oxide (NO) free radicals

Nitric oxide (NO) is freely distributed in the body and plays an important role in many physiological functions, defenses and information transmission. However, due to the high activity and instability of free radicals, too many free radicals will produce oxidative reactions with the cells and tissues in the body, which will cause the cells and tissues to lose their normal functions and even damage DNA.

In this example, take 2 μL and different concentrations (25 μg/mL to 500 μg/mL) of the crude extract, n-hexane extract, ethyl acetate extract, and n-butanol extract of Example 1, and perform monoxide oxidation. Determination of nitrogen radical scavenging ability. Do not add (control group) or add the aforementioned Curculio extracts of different concentrations to a 96-well microporous plate, then add 10 mM sodium nitroprusside (sodium nitroprusside), react at room temperature for 2 hours, then add Cres' reagent (Griess reagent) [Containing 0.1% naphthylenediamine dihydrochloride (NED) compound, 5% phosphoric acid (H ₃ PO ₄ ) and 1% sulfanilamide] After reacting at room temperature for 10 minutes, _{The absorbance (OD 546nm} ) was measured at a wavelength of 546nm. _{Take the OD 546nm} without addition of Curculigo extract (control group) as 100%, and calculate the nitric oxide free radical scavenging rate (%). The lower the measured absorbance value, the better the antioxidant capacity of Curculigo, and the higher the scavenging rate of nitric oxide free radicals.

Please refer to FIG. 4, which is a bar graph showing the scavenging rate of nitric oxide free radicals in vitro of Curculio vulgaris extract according to an embodiment of the present invention. In Figure 4, the X axis represents crude extracts from left to right (control group and crude extracts with concentrations of 25μg/mL, 50μg/mL, 100μg/mL, 500μg/mL), n-hexane extract (control group) Group and concentration of 25μg/mL, 50μg/mL, 100μg/mL, 500μg/mL n-hexane extract), ethyl acetate extract (control group and concentration were 25μg/mL, 50μg/mL, 100μg/mL, respectively) , 500μg/mL ethyl acetate extract) and n-butanol extract (control group and n-butanol extract with concentrations of 25μg/mL, 50μg/mL, 100μg/mL, 500μg/mL) and rutin ( The concentration is 50μg/mL and 100μg/mL). The Y axis represents the scavenging rate (%) of nitric oxide free radicals.

The results in Figure 4 show that as the concentration of Curculigo extract increases, the scavenging ability of nitric oxide radicals also increases. Among them, the crude extract has better scavenging ability for nitric oxide radicals, followed by ethyl acetate extract and n-butanol extract, followed by n-hexane extract, which confirms that Curculigo extract has the ability to remove The effect of nitric oxide free radicals.

In summary, it is demonstrated by the above several examples that Curculio curculata extract has skin whitening and DNA protection activities, so it can be applied to the preparation of skin whitening and DNA protection activities such as cosmetic or pharmaceutical compositions. .

It should be added that although the present invention takes a specific process, a specific analytical method or a specific instrument as an example to illustrate the use of the curculio extract of the present invention to prepare a composition with skin whitening and DNA protection activities, the present invention Anyone with ordinary knowledge in the technical field knows that the present invention is not limited to this. Without departing from the spirit and scope of the present invention, the curculus extract of the present invention is used to prepare skin whitening and DNA-protecting compositions. The purpose can also be carried out by other processes, other analytical methods or other instruments.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can make various modifications without departing from the spirit and scope of the present invention. Modifications and modifications, therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent application scope.

Claims (3)

A kind of Curculia vulgaris extract is used to prepare a medicinal composition that promotes skin whitening, improves the elimination of nitric oxide free radicals and protects DNA activity, wherein the Curculia vulgaris extract is a n-butanol extract, and the extraction method includes: A rough extraction step is performed on a tuber or stem (rhizomes) of Curculigo orchioides with a lower alcohol solution at 10°C to no higher than 40°C, and a weight-volume ratio of the tuber or the rhizome to the lower alcohol solution (g:mL) is 1:500 to 1:1000 to obtain a crude extract, and the lower alcohol solution is methanol solution or ethanol solution; the crude extract is subjected to a first partition extraction step using n-hexane and water To obtain a n-hexane extraction layer and a first water extraction layer, wherein the n-hexane extraction layer has a n-hexane extract, and the total phenol content of the n-hexane extract is equivalent to 3.1 mg/g gallic acid equivalent The total amount of flavonoids in the n-hexane extract is equivalent to 107.2 mg/g rutin equivalent; the first water extraction layer is subjected to a second partition extraction step with ethyl acetate to obtain an ethyl acetate extraction layer and a The second water extraction layer, wherein the ethyl acetate extraction layer has an ethyl acetate extract, the total phenol content of the ethyl acetate extract is equivalent to 32.8 mg/g gallic acid equivalent, and the ethyl acetate extract The total amount of flavonoids is equivalent to 232.6mg/g rutin equivalent; and the second water extraction layer is subjected to a third partition extraction step using n-butanol to obtain a n-butanol extraction layer, wherein the n-butanol extraction layer With the n-butanol extract, the total phenol content of the n-butanol extract is equivalent to 25.5mg/g gallic acid equivalent, and the total flavonoid content of the n-butanol extract is equivalent to 151.5mg/g rutin equivalent. For the purposes described in item 1 of the scope of patent application, the cents An effective dose of the extract of cylindrica for promoting skin whitening is 25μg/mL to 50μg/mL. The use described in item 1 of the scope of patent application, wherein an effective dose of the curculus extract for protecting the DNA activity is 100 μg/mL to 500 μg/mL.

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