TWI815236B - Composite extract of maxillaria tenuifolia, composition including the same and application thereof - Google Patents

Abstract

The present invention relates to a composite extract of Maxillaria tenuifolia, a composition including the same and an application thereof. The composite extract of Maxillaria tenuifoliaincludes a crude extract of M. tenuifoliafresh flowers and/or at least one fraction layer of column chromatogrphy after loading the crude extract. The crude extract of M. tenuifoliacan be selected from the group consisting of a hot water crude extract, an ethanol crude extract and and ethyl acetate (EtOAc) crude extract. The fraction layer includes 3,4-dihydroxy benzoic acid methyl ester and flavanthridin, and optionally with vanillic acid and mangiferin, and it has anti-diabetic, anti-oxidative, anti-allergic, anti-inflammatory and blue light blocking activites, thereby being as an effective ingredient for use in preparation of various compositions.

Description

Axillary orchid complex extract, compositions containing the same and applications thereof

The present invention relates to a plant extract and its application, in particular to a compound extract of Orchid axillaris with anti-diabetic, antioxidant, anti-allergic, anti-inflammatory and anti-blue light activities, a composition containing the same and its application.

Axillary lip orchid ( Maxillaria tenuifolia Lindl.), also known as coconut orchid, is a perennial herbaceous plant of the Orchidaceae family. It is mostly distributed in Central and South America. It likes a climate with sufficient sunshine, high temperature and humidity, and is easy to cultivate quantitatively. The leaves of the axillary orchid are linear, and there is a flat pseudobulb at the base of the plant. The flower stalk will be drawn out from the base of the bulb. The flowering period of axillary orchids is from late spring to early summer. Each pseudobulb can produce 2 or 3 flowers, with a flower diameter of about 3 to 5 cm. The flower color is bright red to dark red, and the flowers have a rich milk and coconut fragrance.

The extract obtained by extracting the flowers of A. axillaris with glycerin/water has anti-aging and anti-wrinkle activities and can be used in cosmetic compositions. However, there is currently little research on whether extracts obtained from A. axillaris flowers by other methods have other activities.

Therefore, one aspect of the present invention is to provide a compound extract of Orchid orchid, which includes a crude extract of fresh flowers of Orchid orchid and/or at least one partitioned layer obtained by column chromatography of the crude extract, wherein the crude extract The extract is selected from a group consisting of hot water crude extract, ethanol crude extract and ethyl acetate crude extract. The divided layers include 3,4-dihydroxy benzoic acid methyl ester (3,4-dihydroxy benzoic acid methyl ester). acid methyl ester) and flavanthridin.

Secondly, another aspect of the present invention is to provide a compound extract of Orchid axillaris, which is a crude extract of fresh flowers of Orchid axillaris and/or a plurality of divided layers obtained by column chromatography of the crude extract. The extract is selected from a group consisting of hot water crude extract, ethanol crude extract and ethyl acetate crude extract. These divided layers include 3,4-dihydroxybenzoic acid methyl ester, flavonoids, vanilla Vanillic acid and mangiferin.

Furthermore, another aspect of the present invention is to provide a composition for external use on skin, wherein the composition for external use on skin contains an in vitro effective dose of the above-mentioned A. axillaris complex extract as an active ingredient.

Furthermore, another aspect of the present invention is to provide an oral composition, wherein the oral composition uses an effective dose of the above-mentioned A. axillaris complex extract as an active ingredient.

In addition, another aspect of the present invention is to provide a use of the Orchid axillary complex extract for preparing an anti-diabetic composition, wherein the composition uses the above-mentioned Orchid axillary complex extract as an active ingredient.

In addition, yet another aspect of the present invention is to provide a use of a compound extract of Orchid axillaris for preparing an antioxidant composition, wherein the composition uses the Orchid axillary complex extract as an active ingredient as described above. .

In addition, another aspect of the present invention is to provide a use of the Orchid axillary complex extract for preparing an anti-allergic and anti-inflammatory composition, wherein the composition is made of the Orchid axillary complex extract as described above. as active ingredient.

In addition, a further aspect of the present invention is to provide a use of the Orchid axillary complex extract for preparing an anti-blue light composition, wherein the composition uses the above-mentioned Orchid axillary complex extract as an active ingredient. .

According to the above aspects of the present invention, a compound extract of Orchid axillaris is proposed. In one embodiment, the composite extract of Orchid axillaris is a crude extract of fresh flowers of Orchid axillaris and/or at least one fraction of the crude extract obtained by column chromatography. The crude extract may be selected from the group consisting of a hot water crude extract, an ethanol crude extract and an ethyl acetate crude extract. The divided layer may include methyl 3,4-dihydroxybenzoate and flavonoids, and the content of methyl 3,4-dihydroxybenzoate is 1 part by weight, and the content of flavonoids may be, for example, 4 to 5 parts by weight. share.

In the above embodiment, the aforementioned crude hot water extract contains decalactone (δ-decalactone) and kaur-16-ene.

According to another aspect of the present invention, a compound extract of Orchid axillary is proposed, which is a crude extract of fresh flowers of Orchid axillaris and/or a plurality of divided layers obtained by column chromatography of the crude extract. The crude extract may be selected from the group consisting of a hot water crude extract, an ethanol crude extract and an ethyl acetate crude extract. The aforementioned divided layer includes methyl 3,4-dihydroxybenzoate, flavonoids, vanillic acid and mangiferin, and the content of methyl 3,4-dihydroxybenzoate is 1 part by weight. The content of flavonoids can be, for example, 4 to 5 parts by weight, the content of vanillic acid may be, for example, 20 to 25 parts by weight, and the content of mangiferin may be, for example, 700 to 750 parts by weight.

In the above embodiment, the number of the aforementioned division layers may be, for example, three.

In the above embodiments, the aforementioned complex extract of Orchid axillaris has anti-diabetic, antioxidant, anti-allergic, anti-inflammatory and anti-blue light activities.

According to another aspect of the present invention, a composition for external use on skin is provided, wherein the composition for external use on skin uses an in vitro effective dose of Orchid axillary complex extract as an active ingredient, and the Orchid axillary complex extract is as above As described above, the effective dose in vitro may be, for example, 0.01 μg/μL to 0.5 μg/μL.

According to another aspect of the present invention, an oral composition is provided, wherein the oral composition uses an effective dose of a compound extract of Orchid axillary as an active ingredient, and the compound extract of Orchid axilla is as described above, And the effective dose may be, for example, 0.01 μg/μL to 0.5 μg/μL.

According to yet another aspect of the present invention, a use of the Orchid axillary complex extract for preparing an anti-diabetic composition is proposed, wherein the composition uses the above-mentioned Orchid axillary complex extract as an active ingredient.

According to yet another aspect of the present invention, a use of a C. axillaris complex extract for preparing an antioxidant composition is proposed, wherein the composition uses the above-mentioned C. axillaris complex extract as an active ingredient.

According to another aspect of the present invention, there is provided a use of the Orchid axillary complex extract for preparing an anti-allergic and anti-inflammatory composition, wherein the composition is made of the above-mentioned Orchid axillary complex extract. Active ingredients.

A further aspect of the present invention is to provide a use of the Orchid axillary complex extract for preparing an anti-blue light composition, wherein the composition uses the above-mentioned Orchid axillary complex extract as an active ingredient.

By applying the Orchid axillary complex extract of the present invention and the composition containing the same, the Orchid axillary complex extract may include 3,4-dihydroxybenzoic acid methyl ester and flavonoids, and selectively contains vanillic acid and mangiferin, It has anti-diabetic, antioxidant, anti-allergic, anti-inflammatory, anti-blue light and other activities, and can be used as an active ingredient and used to prepare various compositions.

It is to be understood that the foregoing general description and the following detailed description are exemplary only, and are intended to provide further explanation of the claimed invention.

If the definition or usage of a term in a cited document is inconsistent or contrary to the definition of the term herein, the definition of the term here shall apply and not the definition of the term in the cited document. Secondly, unless the context otherwise requires, singular terms may include the plural and plural terms may also include the singular.

As mentioned above, the present invention provides a compound extract of Orchidaceae, compositions containing the same and applications thereof, wherein the compound extract of Orchidaceae can include 3,4-dihydroxybenzoic acid methyl ester and flavonoids, and It selectively contains vanillic acid and mangiferin and has anti-diabetic, antioxidant, anti-allergic, anti-inflammatory, anti-blue light and other activities. It can be used as active ingredients and used to prepare various compositions.

In one embodiment, the "complex extract of Orchidaceae" referred to here may refer to a crude extract of fresh flowers of Orchidaceae and/or a plurality of divided layers obtained by column chromatography of the crude extract. In other embodiments, the compound extract of Orchid axillary includes an Orchid axillary extract and a Orchid axillary complex, wherein the Orchid axillary extract refers to the product obtained by removing the solvent from the crude extract of Orchid axillaris, and Orchid axillaris The compound refers to the product obtained by dividing the layers and removing the solvent.

"Fresh flowers" as used here means flowers that have been away from the plant for no more than 24 hours. It should be added that if the extraction material is not taken from the fresh flowers of A. axillaris, it is not expected to obtain an extract with active ingredients and effective dosage.

The "crude extract" referred to here is generally selected from the group consisting of hot water crude extract, ethanol crude extract and ethyl acetate crude extract. In other words, the "crude extract" referred to here refers to the crude extract obtained by soaking the fresh flowers of A. axillaris in hot water (for example, the temperature is 90°C to 100°C), ethanol or ethyl acetate and other crude extraction solvents. things. The weight-to-volume ratio, soaking time, soaking temperature, and number of soaking times of the fresh flowers of Orchid axillaris soaked in the crude extraction solvent can be based on conventional process conditions, and the present invention is not particularly limited. In one example, the weight-to-volume ratio (kg/L) of fresh flowers of A. axillaris soaked in the crude extraction solvent can be, for example, 1:1 to 1:3, but 1:1.8 to 1:2.0 is preferred. The time for soaking the fresh flowers of A. axillaris in hot water is not particularly limited, and can be, for example, 1 hour to 6 hours, but the present invention is not limited to this example. Fresh flowers of A. axillaris can be soaked in ethanol or ethyl acetate at room temperature or below 40°C. The soaking time is not particularly limited, and can be, for example, 12 hours to 72 hours, but the present invention is not limited to the examples here. The number of times that fresh flowers of A. axillaris can be soaked in ethanol or ethyl acetate can be, for example, 1 to 5 times, but 2 to 4 times is preferred. In some specific examples, the above-mentioned hot water crude extract can be further optionally subjected to a step of concentration under reduced pressure to remove the solvent to obtain an extract of Orchid axillaris, which contains at least one volatile component, such as δ-decalactone. and kaurene (kaur-16-ene).

In another embodiment, after the crude extract is subjected to column chromatography, single or multiple divided layers can be obtained. The "column chromatography" referred to here refers to passing the ethyl acetate extract of the fresh flowers of A. axillaris and dividing it with the eluent through a column, thereby obtaining at least one divided layer.

The column suitable for the above column chromatography method can be a commercially available silicone column, and the present invention is not particularly limited. The eluent suitable for the above column chromatography method is chloroform/methanol (CHCl ₃ /MeOH) with gradient concentration (20:1 to 2:1, volume ratio). In one embodiment, according to the order in which the products are extracted, the first to tenth divided layers can be obtained. In some examples, the above-mentioned A. axillaris extract includes one division layer, such as the third division layer, and may also be selectively combined with other division layers, such as the ninth division layer and/or the tenth division layer. The above eluent suitable for the third, ninth and tenth division layers contains solvents of different polarities, such as chloroform and methanol, and the volume ratio of chloroform/methanol can be, for example, 19:1 to 2:1.

In one embodiment, the above-mentioned divided layers may include a plurality of non-volatile components, such as 3,4-dihydroxy benzoic acid methyl ester, flavanthridin and other inseparable components. of ingredients. In another embodiment, in addition to methyl 3,4-dihydroxybenzoate and flavonoids, the divided layer may optionally include vanillic acid, mangiferin and other inseparable components. . The above-mentioned divided layers can be further selectively subjected to a vacuum concentration step to remove the solvent to obtain the axillary orchid complex.

In some examples, the "active ingredients" referred to herein refer to axillary complexes containing methyl dihydroxybenzoate, flavonoids and other inseparable components, and/or optionally containing decanolide and kauri The axillary orchid extract of ene is used to achieve anti-diabetic, antioxidant, anti-allergic, anti-inflammatory and anti-blue light activities. In other examples, the "active ingredient" referred to here may also refer to the axillary complex containing methyl dihydroxybenzoate, flavonoids, vanillic acid, mangiferin and other inseparable ingredients, and/or selectivity Axillaris extract contains decanolide and kaurene to further enhance anti-diabetic, antioxidant, anti-allergic, anti-inflammatory and anti-blue light activities.

The "anti-diabetic activity" referred to here refers to the activity of the above-mentioned A. axillaris extract to inhibit blood sugar, which can, for example, inhibit the activity of α-glucosidase.

The "antioxidant activity" referred to here may be, for example, the activity of the above-mentioned A. axillaris extract in inhibiting the generation of lipid hydroperoxides. The "anti-allergic and anti-inflammatory activity" referred to here can be exemplified by the ability of the above-mentioned A. axillaris extract to inhibit hexosaminidase activity and relieve atopic dermatitis.

The "anti-blue light activity" referred to here means that the above-mentioned A. axillaris extract can effectively increase the cell survival rate after blue light exposure.

What is explained here is that the present invention requires the selection of fresh flowers of a specific variety of orchid orchid. After specific extraction steps and processes, the crude extract of the fresh flower of orchid orchid and the compound extract of orchid orchid can exert anti-diabetic, anti-diabetic and anti-diabetic effects. Antioxidant, anti-allergic, anti-inflammatory, anti-blue light and other activities. If you choose other varieties of Orchid orchid or Orchid axillary crude extract or Orchid axillary complex extract not obtained from fresh flowers, you will not be able to have an effective dose of active ingredients, and it will be difficult to exert the above-mentioned anti-diabetic, antioxidant, and antioxidant effects. Allergy, anti-inflammatory, anti-blue light and other activities.

In the above embodiment, the compound extract of Orchid axillaris is a single divided layer or at least one divided layer obtained by column chromatography of the crude extract of fresh flowers of Orchid axillaris. This divided layer may include a plurality of non-volatile components. These non-volatile components may include 3,4-dihydroxybenzoic acid methyl ester and flavonoids, and the content of 3,4-dihydroxybenzoic acid methyl ester is 1 Parts by weight, the content of flavonoids may be, for example, 4 to 5 parts by weight.

In other embodiments, the extract of Orchid orchid can also be combined into a plurality of divided layers (for example, three) obtained by column chromatography of the crude extract of fresh flowers of Orchid orchid. These combined divided layers include a plurality of A non-volatile ingredient, including methyl 3,4-dihydroxybenzoate, flavonoids, vanillic acid and mangiferin. In some examples, the content of 3,4-dihydroxybenzoic acid methyl ester is 1 part by weight, the content of flavonoids can be, for example, 4 to 5 parts by weight, and the content of vanillic acid can be, for example, 20 to 25 parts by weight. parts by weight, and the content of mangiferin may be, for example, 700 to 750 parts by weight.

In some embodiments, the extract of Orchidaceae can be applied to external skin compositions, such as cosmetic compositions or pharmaceutical compositions. In this embodiment, the composition for external use on skin uses an in vitro effective dose of the above-mentioned Orchidaceae extract as an active ingredient, and the in vitro effective dose may be, for example, 0.01 μg/μL to 0.5 μg/μL.

In other embodiments, the Orchid axillary extract can be used in oral compositions, such as anti-diabetic compositions, anti-allergic and anti-inflammatory compositions, and anti-blue light compositions, wherein the oral composition is formulated with an effective A dose of the above-mentioned extract of Orchid axillary is used as an active ingredient, and the effective dose can be, for example, 0.01 μg/μL to 0.5 μg/μL.

It can be understood that the following specific extracts of Orchid axillaris, specific formulas, specific dosages, specific detection methods, viewpoints, examples and embodiments are only for illustration and are not intended to be limitations of the present invention. The principal features of this invention may be employed in various embodiments without departing from the spirit and scope of the invention. Therefore, those with ordinary knowledge in the technical field to which the present invention belongs can easily determine the necessary technical features of the present invention and make various changes and modifications to the present invention to suit different uses and conditions without departing from the spirit and scope of the present invention. Example 1 1.1 Extraction of raw materials

The extraction raw material used in this example is Maxillaria tenuifolia Lindl (Orchidaceae), whose scientific name is Maxillaria tenuifolia Lindl (Orchidaceae), which was provided and confirmed by Dr. Xiao Yuyun. The voucher specimen (NPTU140530) of A. axillaris is preserved in the Herbarium of National Pingtung University of Science and Technology (Pingtung, Taiwan). 1.2 Preparation of A. axillaris complex extract

Fresh flowers of A. axillaris (2.8 kg) were soaked in hot water (15 L) for 1 to 6 hours to obtain decanolide and kaurene (weight ratio 4:1 to 1:4). Fresh flowers (2.8 kg) of A. axillaris were soaked in ethyl acetate (EtOAc, 5 L × 3 times) at room temperature. The obtained crude extracts were combined and concentrated under reduced pressure to remove the solvent to obtain A. axillaris. Lip Orchid crude extract (dark brown concentrate, 47.1 g). Next, the C. axillaris crude extract is subjected to column chromatography using a silica gel column, and is eluted with gradient concentrations of chloroform/methanol (CHCl ₃ /MeOH). The first to first products can be obtained in the order in which the products are eluted. The tenth division level. The third divided layer was subjected to column chromatography using a silica gel column, and was eluted with chloroform/methanol (volume ratio 19:1) to obtain 3,4-dihydroxybenzoic acid methyl ester (formula I) ( 20 mg) and flavonoids (Formula II) (91 mg). The ninth divided layer was subjected to column chromatography using a silica gel column, and was eluted with chloroform/methanol (volume ratio 9:1) to obtain vanillic acid (formula III) (443 mg). The tenth divided layer was recrystallized using ethyl acetate/methanol (volume ratio 2:1), thereby obtaining mangiferin (formula IV) (14.6g). The third divided layer, the ninth divided layer and the tenth divided layer are combined and subjected to a concentration step under reduced pressure, thereby obtaining the axillary orchid complex.

The results of gas chromatography mass spectrometry analysis of compounds of Formula I to Formula IV are as follows. The compound of formula I is methyl 3,4-dihydroxybenzoate, white powder. UV λ max (MeOH) nm: 224, 275. IR ν (KBr) cm-1: 1685, 1604, 1521, 1438, 1301. 1H-NMR (400 MHz, Acetone-d6): δ 9.83 (1H, s, OH), 8.62 (1H, s, OH), 7.46 (1H, dd, J = 7.8, 2.0 Hz, H-6), 7.45 (1H, d, J = 2.0 Hz, H-2), 7.02 (1H, d, J = 7.8 Hz, H-5), 3.94 (3H, s, OCH3).

The compound of formula II is flavonoid, white powder. UV λ max (MeOH) nm: 205 (sh), 210 (sh), 230, 235 (sh). IR ν (KBr) cm-1: 3400, 2910, 1610, 1490, 1400, 1230, 1122, 1076. 1H-NMR (400 MHz, Acetone-d6): δ 8.23 (1H, d, 8 Hz, J = 8 . -4), 8.13 (1H, s, OH), 7.88 (1H, s, OH), 6.69 (2H, m, Ar-H), 6.46 (1H, s, Ar-H), 3.85 (3H, s , OCH3), 3.79 (3H, s, OCH3), 2.67 (4H, m, H-9, H-10).

The compound of formula III is vanillic acid, white powder. UV λ max (MeOH) nm: 203, 225, 289. IR ν (KBr) cm-1: 3400~2500, 1682, 1598, 1526, 1436, 1286. 1H-NMR (400 MHz, DMSO-d6): 12.51 (1H, br, COOH), 9.86 (1H, s, 4-OH), 7.43 (1H, dd, J = 8.8, 2.0 Hz, H-6), 7.42 (1H, d, J = 2.0 Hz, H - 2), 6.83 (1H, d, J = 8.8 Hz, H-5), 3.80 (3H, s, OCH3).

The compound of formula IV is mangiferin, a light yellow powder. UV λ max (MeOH) nm: 246, 260, 333, 374. IR ν (KBr) cm-1: 3500~2600, 2910, 1650, 1610, 1490, 1460, 1250, 1195, ¹ H-NMR (400 MHz , DMSO-d6): δ 13.83 (1H, s, 1-OH), 7.32 (1H, s, H-8), 6.79 (1H, s, H-5), 6.34 (1H, s, H-4) , 4.57 (1H, d, J = 9.6 Hz, H-1'), 4.04 (1H, dd, J = 9.6, 9.2 Hz, H-2'), 3.67 (1H, d, J = 11.2 Hz, H- 6'a), 3.39 (1H, dd, J = 11.2, 4.8 Hz, H-6'b), 3.18 (1H, t, J = 9.2 Hz, H-3'), 3.12 (2H, m, H- 4', H-5'). 13C-NMR (100 MHz, DMSO-d6): δ 179.1 (C-9), 164.0 (C-3), 162.0 (C-1), 156.4 (C-4a), 155.6 (C-6), 151.3 ( C-10a), 144.3 (C-7), 111.2 (C-8a), 107.8 (C-8), 107.6 (C-2), 102.5 (C-5), 101.4 (C-9a), 93.5 (C -4), 81.8 (C-5'), 79.2 (C-3'), 73.3 (C-1'), 70.9 (C-4'), 70.4 (C-2'), 61.7 (C-6' )). Example 2 2.1 Evaluation of the biosafety of A. axillaris complex

This example is based on the testing method for pesticide residues in food-Multiple Residue Analysis Method (4) announced by the Food and Drug Administration of the Ministry of Health and Welfare of Taiwan, and was commissioned by Lianqun Biotechnology Co., Ltd. (Kaohsiung, Taiwan) to test the compound of Example 1 of Orchidaceae. The results are shown in Table 1. The results in Table 1 show that the axillary orchid complex of Example 1 has no residual heavy metals and pesticides.

Table 1 Test items Content (ppm, mg/kg) Method detection limit (mg/kg) Lead (Pb) ND 4.0 Cadmium (Cd) ND 0.4 Arsenic(As) ND 3.0 Mercury (Hg) ND 0.05 251 pesticide residues ND 0.01 Note: ND means that the measured value is lower than the detection limit.

Secondly, the axillary orchid complex of Example 1 was co-cultured with fibroblasts, and an MTT test was performed to evaluate the cytotoxicity. The MTT test can be performed using conventional methods, which will not be described in detail here. The results are shown in Figure 1 .

Please refer to Figure 1, which is a bar chart of the cell viability of the Orchidaceae complex at different dilution concentrations according to one embodiment of the present invention. The horizontal axis represents the dilution factor of the Orchidaceae complex in Example 1, 0 It represents that the orchid orchid complex is not added, and 10x to 100x respectively represents the dilution of the orchid orchid complex stock solution (1000 ppm) in Example 1 from 100 times to 1000 times, with 1 ppm being regarded as 100%.

The results shown in Figure 1 show that after co-culture of the A. axillary complex in Example 1 with fibroblasts at different dilution concentrations, the cell survival rates were all above 85%, which means that the A. axillary complex does not have cytotoxicity. 2.2 Evaluation of α- glucosidase inhibitory activity of A. axillaris complex

This evaluation method is based on the colorimetric method disclosed by Kim et al (YM Kim et. al) in Nutrition ( Nutrition ), Issue 21, pages 756-761 in 2005, with a slight modification of α-glucosidase [EC 3.2.1.20, from Baker's yeast ( Saccharomyces cerevisiae )] inhibition test. Briefly, p -nitrophenyl α-D-glucopyranoside (pNPG) was dissolved in 0.1 M phosphate buffer solution to prepare a substrate solution and adjust its pH to pH 6.9 to simulate intestinal fluids. Briefly, α-glucosidase was dissolved in 0.1 M phosphate buffer solution (pH 6.9) to prepare a 1 U/mL stock solution. The stock solution was pre-incubated with 100 μL of the respective test solution (0.1% concentration of the A. axillary complex of Example 1) or a DMSO solution of the compounds of formulas I to IV) at 37°C for 10 minutes. Add 0.95 mM pNPG to the above reaction solution and perform an enzyme reaction at 37°C for 20 minutes. Afterwards, 1M sodium carbonate (Na ₂ CO ₃ ) solution was added to terminate the reaction. The activity of α-glucosidase was evaluated by detecting the product released from pNPG (i.e., p-nitrophenol) at 405 nm using a commercially available microplate reader (Biotek, Synengy 2). The results are shown in Figure 2A and Figure 2 As shown in 2B.

Please refer to Figure 2A, which is a bar chart illustrating the percentage of the axillary orchid complex inhibiting α-glucosidase activity in vitro according to an embodiment of the present invention, in which acarbose (synthesis of α-glucosidase Inhibitor, the concentration is 1 mg/mL) as the positive control group. The results in Figure 2A show that the inhibition percentage of α-glucosidase activity of the axillary complex of Example 1 (concentration: 0.1%) (82.9%) is higher than that of acarbose (about 50%). The inhibition percentages of compounds of formula I to formula IV (concentration of 1.0 mg/mL) on α-glucosidase are 22.9%, 96.7%, 81.0% and 59.9% respectively, among which compounds of formula II to formula IV inhibit α-glucosidase The enzyme activity inhibition percentages are all higher than acarbose, indicating that the axillary orchid complex of Example 1 has anti-diabetic activity.

Please refer to FIG. 2B , which is a bar graph illustrating the percentage of inhibition of α-glucosidase activity in vitro by the axillary orchid complex according to another embodiment of the present invention. The results in Figure 2B show that the inhibition percentage of the activity of α-glucosidase by the Orchidium axillaris extract (concentration: 1.0 mg/mL) and the Orchidaceae complex (concentration: 0.1%) in Example 1 (Orchid axillary extract) substance (the inhibition percentage is 82.9%) are higher than acarbose (about 50%), which means that the axillary orchid complex of Example 1 has anti-diabetic activity. 2.3 Evaluate the antioxidant activity of A. axillaris complex

This evaluation method is based on the iron thiocyanate method disclosed by Chen et al (HM Chen et. al) in J. Agric. Food Chem. in 1995, with slight modifications to evaluate antioxidant activity. Briefly, 1.0 mL of 0.1 M sodium phosphate buffer solution (pH 7.0), 0.5 mL of distilled water, and 1.0 mL of 50 mM ethanol solution of linoleic acid (99.5%) were placed in a glass test tube (volume 5 mL). mix. Then, without changing the total volume, add the sample to be tested into the above buffer solution or ethanol. Then, seal the test tube and store it at 60°C in the dark. Afterwards, 20 μL of 30% ammonium thiocyanate and 20 μL of 20 mM ferrous chloride solution were added to each 80 μL of the reaction mixture. After waiting for 3 minutes, use a commercial spectrophotometer (Hitachi U-3900) to measure the absorbance of the reaction mixture at a wavelength of 500 nm. The results are shown in Figure 3.

The ferric thiocyanate method can be used to determine the antioxidant activity of the axillary orchid complex of Example 1 and the compounds of formulas I to IV, and water-soluble vitamin E is used as a reference compound. Unsaturated fatty acids (such as linoleic acid) are gradually peroxidized to hydroperoxides. In the presence of ferrous iron ions, lipid hydrogen peroxide can oxidize ferrous iron ions into ferric ions, and then the ferric ions coordinate with thiocyanate to form iron thiocyanate, which can be observed at a wavelength of 500 The absorbance of nm increases.

Please refer to Figure 3A, which is a bar graph illustrating the inhibition of linoleic acid peroxide in vitro by the axillary orchid complex according to an embodiment of the present invention, in which water-soluble vitamin E (trolox, concentration is 1 mg/mL) As a positive control group. As shown in the results in Figure 3A, the percentage of inhibition of linoleic acid peroxide production by the compound of Example 1 (concentration of 1.0 mg/mL) is higher than that of water-soluble vitamin E, which means that the compound of Example 1 has the ability to inhibit the formation of linoleic acid peroxide. Antioxidant activity.

Please refer to FIG. 3B , which is a bar graph illustrating the inhibition of linoleic acid peroxide in vitro by the axillary orchid complex according to another embodiment of the present invention. As shown in the results in Figure 3B, compounds of formula I and formula IV can almost completely inhibit lipid peroxidation of linoleic acid emulsion (concentration 1.0 mg/mL), while water-soluble vitamin E inhibits the percentage of peroxide generation in linoleic acid emulsion. is 76.3%. In comparison, the antioxidant activities of the compounds of formula II and formula III are relatively low (the percentages of inhibiting the formation of peroxide are 10.5% and 15.5% respectively). 2.4 Evaluation of anti-allergic activity of A. axillaris complex

This evaluation method first adds the test sample to RBL-2H3 cells (Rat basophilic leukemia-2H3). Next, allergens: 25 nM phorbol-12-myristate-13-acetate (PMA) and 1 μM calcimycin (A23187) were added for induction. 2 hours to induce allergic response in cells. Then, the activity of the allergy mediator β-hexosaminidase in the cells was detected, and the test was repeated three times. The results are shown in Figure 4 and Table 2.

In addition, the axillary orchid complex of Example 1 (concentration 1.0%) was directly applied to the skin surface of the back or back of the hand of the patient with atopic dermatitis, left for 2 minutes or used once a day for 2 weeks, and the skin was observed with the naked eye. The results of changes in redness and swelling on the surface are shown in Figures 5A to 5F.

Please refer to Table 2 and Figure 4, which illustrates the results (Table 1) and bar chart (Figure 4) of inhibiting the activity of β-hexosaminase by the axillary orchid complex according to one embodiment of the present invention, in which the commercially available Cordyceps militaris extract (concentration 5% by weight and volume) was sold as a positive control group, and the DMSO group (i.e., control group) was used as the activity level of β-hexosaminase as 100%, and PBS (blank control group) was used As a blank control group.

Table 2 Group 1 2 3 Average value of β-hexosaminase activity Standard deviation (SD) PBS (i.e. blank control group) 12.763 12.681 12.956 12.8 0.141184 DMSO (i.e. control group) 103.916 97.877 98.207 100 3.395367 5% axillary orchid extract 62.228 62.626 59.046 61.3 1.962139 5% Axillary Orchid Complex 28.451 27.172 25.977 27.2 1.237238 Commercially available Cordyceps militaris 45.976 50.255 46.569 47.6 2.318336

The results in Table 2 and Figure 4 show that the activity percentage of β-hexosaminase treated with the axillary orchid complex of Example 1 (concentration is 5%) is 27.2%, which is much lower than that of the Cordyceps militaris extract (47.6% ), representing Example 1, the axillary orchid complex has anti-allergic activity.

Please refer to Figures 5A to 5F, which are photos showing the application of axillary orchid complexes to patients with atopic dermatitis according to several embodiments of the present invention, which respectively show the back (Figure 5A, Figure 5C, Figure 5E) Or the skin on the back of the hand (Figure 5B, Figure 5D, Figure 5F) before use (Figure 5A, Figure 5B), 2 minutes after use (Figure 5C, Figure 5D), and after use once a day for 2 weeks (Figure 5E, Figure 5E 5F) photo.

The results from Figure 5A to Figure 5F show that after the axillary orchid complex of Example 1 (concentration is 1%, equivalent to 0.01 μg/μL) is applied to the skin of atopic dermatitis patients, relief can begin 2 minutes after use. Symptoms of redness and swelling; after being used once a day for 2 weeks, the symptoms of redness and swelling can be effectively alleviated, indicating that the axillary lip orchid complex in Example 1 has anti-allergic activity. 2.5 Evaluate the anti-blue light activity of A. axillaris complex

This evaluation method first adds axillary complex (concentration 50 μg/mL) to adult retinal pigment epithelial cell line-19 (ARPE-19 cells), and commercially available lutein (concentration 50 μg/mL) was used as the positive control group, and DMSO was used as the blank control group, and cultured for 0, 24, 48, and 72 hours. During the culture period, take out the cell culture plate every 24 hours and illuminate the cells with blue light or a dark room (equivalent to no light) for 1 hour. After the last irradiation, use the Cell Counting Kit-8 (CCK-8 kit) and calculate the relative cell survival rate according to the method provided by the manufacturer. The results are shown in Figure 6.

Please refer to Figure 6, which is a bar graph showing the cell survival rate of the axillary orchid complex according to several embodiments of the present invention, in which the cell survival rate of the blank control group under 0-hour illumination is taken as 100%.

The results shown in Figure 6 show that ARPE-19 cells were co-cultured with axillary orchid complex before irradiation with blue light. After 72 hours of culture, the cell survival rate was indeed improved and was better than commercially available lutein. Representative Example 1 Lip Orchid Complex has anti-blue light activity. 2.6 Numerical analysis

The numerical values described below are expressed as mean ± standard error (SEM). Numerical analysis of multiple comparisons was performed using t-test, 95% confidence interval, one-way ANOVA and Tukey’s test. Numerical analysis was performed using commercially available software Prism 7.0 (GraphPad Software Inc., USA). P＜0.05 was used to judge that there was a statistically significant difference.

To sum up, the present invention uses specific varieties of fresh flowers of Orchid axillaris, specific extraction methods, specific active ingredients and specific ratios, specific analysis modes or specific evaluation methods only to illustrate the Orchid axillary complex, Compositions containing the same and their applications. However, those with ordinary knowledge in the technical field to which the present invention belongs will understand that other extraction methods, other inseparable components, other proportions of active ingredients, other analysis modes or other methods may be used without departing from the spirit and scope of the present invention. Other evaluation methods can also be used for axillary orchid complexes, compositions containing the same and their applications, and are not limited to the above.

According to the above embodiments, the orchid orchid complex of the present invention and the composition containing the same have the advantage that the orchid orchid complex is a crude extract of the fresh flowers of orchid and/or the crude extract is processed through the column layer At least one divided layer obtained by the analytical method has anti-diabetic, antioxidant, anti-allergic, anti-inflammatory, anti-blue light and other activities. In the future, it can be used as an active ingredient and used to prepare various compositions, such as external skin compositions (such as cosmetic compositions) , pharmaceutical compositions), oral compositions (such as anti-diabetic compositions, anti-allergic and anti-inflammatory compositions, anti-blue light compositions), etc.

Although the invention has been disclosed above in several specific embodiments, other embodiments are also possible. Therefore, the spirit and scope of the appended claims of the present invention should not be limited to the embodiments contained herein.

without

In order to make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the detailed description of the accompanying drawings is as follows: [Fig. 1] is a bar chart illustrating the cell survival rate of axillary orchid complex extracts at different dilution concentrations according to an embodiment of the present invention. [Fig. 2A] is a bar graph illustrating the percentage of inhibition of α-glucosidase activity in vitro by a compound extract of Orchid axillaris according to one embodiment of the present invention. [Fig. 2B] is a bar graph illustrating the percentage of inhibition of α-glucosidase activity in vitro by a C. axillaris complex extract according to another embodiment of the present invention. [Fig. 3A] is a bar graph illustrating the inhibition of linoleic acid peroxide in vitro by the complex extract of Orchid axillaris according to one embodiment of the present invention. [Fig. 3B] is a bar graph illustrating the inhibition of linoleic acid peroxide in vitro by the complex extract of Orchidaceae according to another embodiment of the present invention. [Fig. 4] is a bar graph illustrating the inhibition of β-hexosaminase activity by the complex extract of Orchid axillaris according to one embodiment of the present invention. [Fig. 5A] to [Fig. 5F] are photos showing the application of axillary orchid complex extract to patients with atopic dermatitis according to several embodiments of the present invention. [Fig. 6] is a bar graph showing the cell survival rate of the A. axillaris complex extract according to one embodiment of the present invention.

Domestic storage information (please note in order of storage institution, date and number) without Overseas storage information (please note in order of storage country, institution, date, and number) without

Claims (13)

A compound extract of Orchid orchid, comprising at least one partitioned layer obtained by silica gel column chromatography of an ethyl acetate crude extract of fresh flowers of Orchid orchid, wherein the divided layer includes 3,4-dihydroxybenzoic acid methyl ester (3,4-dihydroxy benzoic acid methyl ester) and flavanthridin, and the content of the 3,4-dihydroxy benzoic acid methyl ester is 1 part by weight, and the content of the flavonoid is 4 parts by weight to 5 parts by weight. The compound extract of Orchid axillary as described in claim 1, further includes a hot water crude extract of the fresh flowers of Orchid axillaris, and the hot water crude extract contains decalactone ( δ -decalactone) and kaurene ( kaur-16-ene). A compound extract of Orchid axillary, which is a plurality of divided layers obtained by silica gel column chromatography of the ethyl acetate crude extract of fresh flowers of Orchid orchid, wherein the divided layers include 3,4-dihydroxybenzene Methyl formate, flavonoids, vanillic acid and mangiferin, and one of the contents of 3,4-dihydroxybenzoic acid methyl ester is 1 part by weight, and one of the flavonoids is 4 parts by weight. to 5 parts by weight, the vanillic acid content is 20 to 25 parts by weight, and the content of mangiferin is 700 to 750 parts by weight. The axillary orchid complex extract as described in claim 3, further including It contains a hot water crude extract of fresh flowers of the axillary orchid, and the hot water crude extract contains decalactone (δ-decalactone) and kaur-16-ene. The A. axillaris complex extract as described in claim 3, wherein the number of one of the divided layers is three. The compound extract of Orchid axillary as described in claim 3, wherein the compound extract of Orchid axillaris has anti-diabetic, antioxidant, anti-allergic, anti-inflammatory and anti-blue light activities. A skin external use composition, wherein the skin external use composition uses an in vitro effective dose of axillary orchid complex extract as an active ingredient, and the axillary orchid complex extract is any one of claim 1 to claim 6 As described above, the effective dose in vitro is 0.01 μg/μL to 0.5 μg/μL. The external composition according to claim 7, wherein the skin external composition is a cosmetic composition or a pharmaceutical composition. An oral composition, wherein the oral composition uses an effective dose of Orchid axillary complex extract as an active ingredient, and the Orchid axillary complex extract is as described in any one of claim 1 to claim 6, And the effective dose is 0.01 μg/μL to 0.5 μg/μL. A kind of axillary orchid complex extract is used for preparing anti-diabetic medicine The use of a composition, wherein the composition uses the axillary orchid complex extract as described in any one of claims 1 to 6 as an active ingredient. A use of the Orchid axillary complex extract for preparing an antioxidant composition, wherein the composition uses the Orchid axillary complex extract as described in any one of claim 1 to claim 6 as an active ingredient. A use of the Orchid axillary complex extract for preparing an anti-allergic and anti-inflammatory composition, wherein the composition uses the Orchid axillary complex extract as described in any one of claims 1 to 6 as a component. Active ingredients. A use of the Orchid axillary complex extract for preparing an anti-blue light composition, wherein the composition uses the Orchid axillary complex extract as described in any one of claim 1 to claim 6 as an active ingredient.