Application of secoisolariciresinol in whitening
By applying ring-opening isolate larch resin phenol in Deer Fairy Grass extract, the problem of limited types of whitening effects of Deer Fairy Grass extract was solved, and significant tyrosinase inhibition and whitening activity were achieved.
Patent Information
- Application Number
- CN202411945711.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-04
AI Technical Summary
The development and research of deer fairy grass extracts are limited, and the development types of whitening effects of deer fairy grass extracts in the prior art are limited.
The whitening composition was prepared by ethanol extraction, petroleum ether and ethyl acetate extraction, chromatography purification and other steps.
The ring-opening isolate larch resin phenol can effectively inhibit tyrosinase activity and has significant whitening activity. The inhibition rate of tyrosinase at a concentration of 1 mg/ml is 55.78%, and the IC50 value is 0.7060 mg/ml.
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Figure CN119745705A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of traditional Chinese medicine extracts, and in particular to application of secoisolariciresinol in whitening. Background Art
[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
[0003] Deer fairy grass is the whole herb of Balanophora involucrata Hook.f. and Balanophora harlandii Hook.f., plants of the genus Balanophora in the family Banophoraceae. It is a dicotyledonous perennial parasitic fleshy herb, also known as Wenwang's pen and Jixinqi. Among them, Balanophora involucrata often parasitizes on the roots of plants of the genus Rhododendron, while Balanophora harlandii mainly parasitizes on the roots of plants such as Ironwood, Hainan pea vine, Indian blood vine, rock bean vine, dense flower bean, palm leaf fig, cold water flower, red mold water kudzu, water hemp, feather leaf white head tree, etc. It is distributed in Fujian, Hubei, Guangdong, Guangxi, Sichuan, Yunnan and other places, and is produced in Sichuan, Yunnan, Guizhou and Hunan.
[0004] The whole plant of Deer Herb is used as medicine, with a bitter, sweet and flat taste; cool in nature; and enters the heart, spleen, stomach and kidney meridians. It is beneficial to the kidney and nourishes yin; it has the effects of clearing away heat and stopping bleeding, strengthening yang and tonifying the kidney, regulating qi and strengthening the stomach, clearing away heat and detoxifying, stopping bleeding and promoting tissue regeneration. It is mainly used to treat kidney deficiency and low back pain; consumptive bleeding; hemorrhoid bleeding. It is used for impotence, spermatorrhea, edema, menorrhagia, leucorrhea, neurosis, chronic hepatitis, stomach pain, gastrointestinal bleeding, wind-heat macules, lung heat cough, and traumatic bleeding. It is mostly used in folk medicine, as Miao medicine, Zhuang medicine, Yi medicine, and is also used clinically. It plays an important role in the treatment of chronic hepatitis, traumatic bleeding, drunkenness, anti-tumor, sobering up, protecting the liver and lowering blood sugar.
[0005] Uneven skin tone, dullness and the formation of spots are generally caused by excessive melanin deposition. Tyrosine in melanocytes in the basal layer of the epidermis is oxidized to DOPA (DOPA) under the action of tyrosinase (TYR), and then further oxidized to dopaquinone, which is related to the formation of melanin. At present, there are few reports on the whitening active ingredients of Luxiancao. Therefore, in this study, the chemical components of Luxiancao were extracted and separated, and the isolated monomer components were studied for their in vitro anti-tyrosinase activity, in order to provide literature references for the development of the whitening active ingredients of Yi medicine Luxiancao.
[0006] The prior art states that a herbaceous deer stalk extract, preparation method and application thereof have the effect of inhibiting tyrosinase activity to achieve whitening, and the extracted ingredients include 3-methoxy-quercetin 1.92-10.38 mg / g, 3,4-dihydroxybenzoic acid 1.50-6.47 mg / g, apigenin 1.32-3.79 mg / g, kaempferol 1.97-8.59 mg / g, and herbaceous deer stalk sterone 25-500 μg / g. However, the prior art points out that there is synergistic whitening between the above multiple substances, and the types of extracts developed are limited. Summary of the invention
[0007] The purpose of the present invention is to provide an application of secoisolariciresinol in whitening to address the problem of limited development and research of the current chelidon grass extract, provide a new chelidon grass extract with whitening effect, and enhance the application value of the chelidon grass.
[0008] The technical solution of the present invention is as follows:
[0009] In one aspect, the present invention provides the use of secoisolariciresinol in preparing a whitening composition.
[0010] Preferably, the whitening composition includes whitening cosmetics, skin care products, medicines and the like.
[0011] Preferably, the structural formula of secoisolariciresinol is as follows Figure 1 As shown. White crystals, molecular formula is C 20 H 26 O6, ESI-MS m / z: 385.4 [M+Na] + , 1 H-NMR (400MHz, CD3OD) δH: 6.68 (2H, d, J = 7.9 Hz, H-5, 5'), 6.61 (2H, d, J = 1.9 Hz, H-2, 2'), 6.56 (2H, dd, J = 8.0, 1.9 Hz, H-6, 6'), 3.75 (6H, s, 3,3′-OCH3), 3.59(4H,m,H-9,9′), 2.68(2H,dd,J=13.8,6.9Hz,H-7a,7′a), 2.57(2H,dd,J=13.8,7.8Hz,H-7b,7′b), 1.92(2H,s,H-8,8′); 13 C-NMR (100 MHz, CD3OD)δ C:133.9(C-1,1′),113.4(C-2,2′),148.8(C-3,3′),145.5(C-4,4′),115.8(C-5,5 ′), 122.7(C-6,6′), 36.0(C-7,7′), 44.1(C-8,8′), 62.1(C-9,9′), 56.2(3-OCH3).
[0012] According to a preferred embodiment, the preparation and separation method of the secoisolariciresin phenol is as follows:
[0013] Step S1: drying the whole herb of Luxiancao, crushing and sieving it, heating and refluxing it with 85% ethanol, extracting it 5 times, combining the extracts and concentrating them under reduced pressure to obtain an ethanol extract;
[0014] Step S2: Take the ethanol extract of step S1, add water to suspend it, filter it through 400 mesh nylon cloth, and then extract it with 1:1 volume of petroleum ether and ethyl acetate for 5 times respectively, combine the parts and concentrate them under reduced pressure to obtain a petroleum ether part and an ethyl acetate part;
[0015] Step S3: Take the ethyl acetate fraction and mix it with 100-200 mesh silica gel in equal amounts of 1:1 and load it onto a silica gel column. Degrease it with 2 column volumes of petroleum ether, and then flush the column with petroleum ether: ethyl acetate in ratios of 30:1, 10:1, 5:1, and 1:1; and dichloromethane: methanol in ratios of 30:1, 20:1, 10:1, 5:1, and 1:1 to obtain 9 components.
[0016] Step S4: Take dichloromethane: methanol at a ratio of 20:1 and conduct MCI column chromatography with methanol-water (30:70-100:0) gradient elution, and combine to obtain three fractions according to the reverse phase elution order. Take methanol-water at a ratio of 60:40 and conduct normal silica gel chromatography (200-300 mesh), and conduct gradient elution with petroleum ether: ethyl acetate (15:1-5:1), and then separate by Sephadex LH-20 chromatography (methanol), and purify by semi-preparative liquid phase (acetonitrile-water 21:79; injection volume 35uL, volume flow rate 3mL / min; detection wavelength: 210nm, 230nm, 280nm) to obtain the compound secoisolariciresinol (tR=36.534min).
[0017] Another aspect of the present invention provides a whitening composition comprising secoisolariciresinol.
[0018] According to a preferred embodiment, the concentration of secoisolariciresinol is 0.1 mg / ml-1 mg / ml.
[0019] According to a preferred embodiment, the concentration of secoisolariciresinol is 0.5 mg / ml-0.7 mg / ml.
[0020] According to a preferred embodiment, the whitening composition includes whitening cosmetics, skin care products or medicines.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1. The present invention provides an application of secoisolariciresinol in a whitening composition, and discloses a new secoisolariciresinol with whitening effect, which can inhibit the activity of tyrosinase and has whitening activity. When the concentration is 1 mg / ml, the inhibition rate of tyrosinase is 55.78%; IC 50 The value was 0.7060mg / ml, which was lower than that of the positive drug β-arbutin (IC 50 value is 0.3763mg / ml) is about twice as large. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the structural formula of secoisolariciresinol;
[0024] Figure 2 Secoisolariciresin phenol 1 H-NMR spectrum;
[0025] Figure 3 Secoisolariciresin phenol 13 C-NMR (DEPT) spectrum;
[0026] Figure 4 This is the concentration inhibition curve of the positive drug β-arbutin on tyrosinase;
[0027] Figure 5 This is the concentration inhibition curve of secoisolariciresinol on tyrosinase. DETAILED DESCRIPTION
[0028] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.
[0030] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
[0031] Example 1 Separation of Sequenyl Iso-larch Resinphenol from Herba Luxiancao Extract
[0032] Step S1: Take 70 kg of dried whole herb of Herba Luxiancao, crush and sieve (10 mesh), and use 3 times the amount of 85% ethanol to heat and reflux and extract 5 times, each time for 2 hours, combine and concentrate the extracts under reduced pressure to obtain 38 kg of extract.
[0033] Step S2: the ethanol extract was suspended in water, filtered through a 400-mesh nylon cloth, and extracted five times with 1:1 volume of petroleum ether and ethyl acetate, respectively. After combining the fractions, they were concentrated under reduced pressure to obtain a petroleum ether fraction (100 g) and an ethyl acetate fraction (3.4 kg).
[0034] Step S3: Take the ethyl acetate fraction (3.4 kg) and mix it with 100-200 mesh silica gel in an equal amount of 1:1, and load it onto a silica gel column. First, degrease it with 2 column volumes of petroleum ether, and then flush and segment the column with petroleum ether: ethyl acetate in a ratio of 30:1, 10:1, 5:1, 1:1; and dichloromethane: methanol in a ratio of 30:1, 20:1, 10:1, 5:1, 1:1 to obtain 9 components (Fr.1-Fr.9).
[0035] Step S4: Take the dichloromethane:methanol 20:1 segment (Fr.6, 30g) and subject it to MCI column chromatography with methanol-water (30:70-100:0) gradient elution, and combine to obtain three fractions Fr.6.1-Fr.6.3. Take methanol-water 60:40 segment Fr.6.2 and subject it to normal silica gel chromatography (200-300 mesh), with petroleum ether:ethyl acetate (15:1-5:1) gradient elution, and then separate it by Sephadex LH-20 chromatography (methanol), and purify it by semi-preparative liquid phase (acetonitrile-water 21:79; injection volume 35uL, volume flow rate 3mL / min; detection wavelength: 210, 230, 280nm) to obtain compound secoisolariciresinol (5.6mg, tR=36.534min).
[0036] The purified open-ring isolaric resin phenol was identified by nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum. Figure 2 and Figure 3 The spectrum shown. Figure 2 It can be seen that1 H-NMR (400MHz, CD3OD)δ H :6.68(2H,d,J=7.9Hz,H-5,5′), 6.61(2H,d,J=1.9Hz,H-2,2′), 6.56(2H,dd,J=8.0, 1.9Hz,H-6,6′), 3.75(6H,s,3,3′-OCH3) , 3.59(4H,m,H-9,9′), 2.68(2H,dd,J=13.8,6.9Hz,H-7a,7′a), 2.57(2H,dd,J=13.8,7.8Hz,H-7b,7′b), 1.92(2H,s,H-8,8′); 13 C-NMR (100 MHz, CD3OD)δ C :133.9(C-1,1′),113.4(C-2,2′),148.8(C-3,3′),145.5(C-4,4′),115.8(C-5,5 ′), 122.7(C-6,6′), 36.0(C-7,7′), 44.1(C-8,8′), 62.1(C-9,9′), 56.2(3-OCH3). from Figure 3 It can be seen that ESI-MS m / z: 385.4 [M+Na] + The structural formula of secoisolariciresinol is as follows Figure 1 shown.
[0037] Example 2 Study on the effect of secoisolariciresinol on the activity of tyrosinase
[0038] 1. Tyrosinase Inhibition Activity Test
[0039] 1. Solution Preparation
[0040] (1) Preparation of 250U tyrosinase solution: Accurately pipette 25mL of PBS phosphate buffer (0.01mol / L, pH 6.5) into 25KU tyrosinase and mix well. Dispense into 25 tubes of 1000units / mL tyrosinase solution. Accurately take 0.25mL of PBS buffer and dilute to 1mL with 0.75mL of PBS buffer to obtain 250units / mL tyrosinase test solution. Place the test solution in a -20℃ refrigerator for later use.
[0041] (2) Preparation of L-tyrosine solution: Accurately weigh 2 mg of L-tyrosine powder into a 50 mL centrifuge tube, add 20 mL of PBS buffer, mix well to obtain a 0.1 mg / mL L-tyrosine solution, set aside for use, and prepare freshly for use each time.
[0042] (3) Preparation of sample solution: Accurately weigh 1 mg of secoisolariciresinol sample, add 30 uL DMSO to completely dissolve it, then accurately add 970 uL PBS and mix well to make the concentration 1 mg / mL. After diluting each sample with PBS, the concentrations are as follows: 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.062 5 mg / mL, 0.031 25 mg / mL; set aside.
[0043] 2. Determination of tyrosinase activity inhibition rate
[0044] The experiment was divided into a sample reaction group (6 concentrations of secoisolariciresinol + 250U tyrosinase reagent), a sample control group (6 concentrations of secoisolariciresinol + PBS buffer), a reagent reaction group (250U tyrosinase reagent + PBS buffer), and a reagent control group (PBS buffer).
[0045] For each sample, use a pipette to pipette sample solutions of various concentrations into a 96-well plate, 40uL per well. For the sample reaction group, add 40uL of prepared tyrosinase solution to each well; for the sample control group, add 40uL of PBS buffer to each well; for the reagent reaction group, add 40uL of prepared tyrosinase solution and an equal amount of PBS buffer to each well, and for the reagent control group, add 80uL of PBS buffer to each well.
[0046] After adding liquid, incubate at 37°C in a constant temperature box for 10 minutes, then add L-tyrosine solution with a spray gun, 40uL per well, and add to each group. After adding liquid, place the 96-well plate in a constant temperature box at 37°C and incubate for 20 minutes. After incubation, immediately put it into a multifunctional microplate reader to measure its absorbance at 492nm and record the data. Perform 3 parallel groups for each compound and each concentration experiment, use the average value of the 3 groups of data, and calculate the inhibition rate of the compound on tyrosinase according to the following formula:
[0047] Inhibition rate = [1 - (sample reaction - sample control) / (reagent reaction - reagent control)] × 100% III. Data processing and analysis
[0048] IC 50 The calculation results were calculated using IBM SPSS Statistics 26. The experiment of secoisolariciresinol was repeated three times to calculate the IC value of each compound for tyrosinase inhibition. 50 The mean and standard deviation were compared with the positive drug β-arbutin using a one-way ANOVA test. LSD and Duncan were used for post hoc comparison.
[0049] 4. Experimental Results and Analysis
[0050] 1. Tyrosinase inhibition test results
[0051] The results of the positive drug β-arbutin's inhibition of tyrosinase activity are as follows Figure 4 As shown in the figure, the inhibition rates from high to low are 71.62%, 69.18%, 60.42%, 48.81%, 31.74% and 14.29%, respectively. The inhibition rates of positive drugs on tyrosinase increase with the increase of test solution concentration. When the concentration is less than 1 mg / ml, the inhibition rate of positive drugs increases faster with the increase of concentration. When the concentration is greater than 1 mg / ml, the increase is slower, showing concentration-dependent inhibition. Its IC 50 The value is 0.3763mg / ml.
[0052] The results of the concentration inhibition of the compound on tyrosinase activity are as follows Figure 5 As shown in the figure, the inhibition rates are 55.78%; 39.96%; 22.53%; 13.02%; 1.58%; -5.13% from high to low. The inhibition rate of tyrosinase also increases with the increase of the test solution concentration. When the compound concentration is below 0.5 mg / ml, the inhibition rate increases rapidly with the concentration. When the concentration is greater than 0.5 mg / ml, the growth rate becomes slower, showing concentration-dependent inhibition. Its IC 50 The value is 0.7060mg / ml.
[0053] 2. Results Analysis
[0054] The results of the inhibition rate experiment showed that the inhibition rate of the compound to tyrosinase was 55.78% when the concentration was 1 mg / ml, and the inhibition rate of the positive drug β-arbutin was 69.18%. 50 The value was 0.7060mg / ml, which was lower than that of the positive drug β-arbutin (IC 50 The value is 0.3763mg / ml), which is about twice as large as that of the 1mg / ml compound. However, it also has strong tyrosinase inhibitory activity at 1mg / ml. The above results all indicate that the compound secoisolariciresinol has certain whitening activity.
[0055] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.
Claims
1. Application of secoisolariciresinol in preparing whitening composition.
2. The application according to claim 1, characterized in that: The whitening composition includes whitening cosmetics, skin care products or medicines.
3. The use according to claim 1, characterized in that: The secoisolariciresinol inhibits tyrosinase activity to achieve whitening.
4. The use according to claim 1, characterized in that: The structural formula of the seco-isolariciresin phenol is as follows:
5. The use according to claim 1, characterized in that: The seco-isolariciresin phenol is extracted from gypsum schrenkiana.
6. The use according to claim 1, characterized in that: The method for extracting secoisolariciresinol from Herba Luxiancao is as follows: Step S1: drying the whole herb of Luxiancao, crushing and sieving it, heating and refluxing it with 85% ethanol, extracting it 5 times, combining the extracts and concentrating them under reduced pressure to obtain an ethanol extract; Step S2: Take the ethanol extract of step S1, add water to suspend it, filter it through 400 mesh nylon cloth, and then extract it with 1:1 volume of petroleum ether and ethyl acetate for 5 times respectively, combine the parts and concentrate them under reduced pressure to obtain a petroleum ether part and an ethyl acetate part; Step S3: Take the ethyl acetate fraction and mix it with 100-200 mesh silica gel in equal amounts of 1:1 and load it onto a silica gel column. Degrease it with 2 column volumes of petroleum ether first, and then mix petroleum ether:ethyl acetate in the ratio of 30:1, 10:1, 5:1, and 1:1 in sequence. Dichloromethane:methanol was flushed and segmented at 30:1, 20:1, 10:1, 5:1, and 1:1 to obtain 9 components; Step S4: Take dichloromethane: methanol at a ratio of 20:1 and conduct gradient elution with methanol-water at a ratio of 30:70-100:0 on an MCI column chromatography, and combine to obtain three fractions in the reverse phase elution order, take methanol-water at a ratio of 60:40 and conduct gradient elution with petroleum ether: ethyl acetate at a ratio of 15:1-5:1, and then conduct separation by Sephadex LH-20 chromatography, and purify by semi-preparative liquid phase to obtain the compound secoisolariciresinol.
7. A whitening composition, characterized in that: Includes secoisolariciresinol.
8. A whitening composition according to claim 7, characterized in that: The concentration of the secoisolariciresin phenol is 0.1 mg / ml-1 mg / ml.
9. A whitening composition according to claim 8, characterized in that: The concentration of the secoisolariciresin phenol is 0.5 mg / ml-0.7 mg / ml.
10. A whitening composition according to claim 7, characterized in that: The whitening composition includes whitening cosmetics, skin care products or medicines.