A whitening root extract of Bletilla striata and its application

Abstract

The present invention relates to a whitening root extract of Bletilla striata and its application. The present invention obtains and separates and purifies three phenanthrene components from the white root for the first time, and 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol, 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene, and 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrene diol all have tyrosinase inhibitory activity. Among them, 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol has the strongest tyrosinase inhibitory activity, and in vivo experiments have proved that it has excellent whitening activity, which directly provides a material basis for the comprehensive development and utilization of non-medicinal parts of Bletilla striata.

Description

Technical Field

[0001] The invention relates to a Bletilla striata root extract with whitening activity and application thereof. Background Art

[0002] Tyrosinase is the most important rate-limiting enzyme for the synthesis of melanin in organisms. It is widely present in animals, plants, microorganisms and the human body. Its abnormal expression can lead to skin problems caused by pigmentation such as freckles and chloasma. However, due to safety and stability issues, only a small number of the tyrosinase inhibitors that have been discovered are used in skin whitening products. Natural tyrosinase inhibitors are used for skin whitening, with the advantages of rich resources, long application history, mild and long-lasting whitening effect, etc. The growing cosmetics market has an urgent need to develop natural, efficient and safe tyrosinase inhibitors from plants. Although natural tyrosinase inhibitors are highly favored, it is still very difficult to screen safe and effective tyrosinase inhibitors from huge natural resources due to their complex ingredients and low content.

[0003] Bletilla striata is derived from the orchid plant Bletilla striata Bletillastriata The dried tuber of (Thunb.) Reichb.f. has the effects of astringent hemostasis, detumescence and tissue regeneration, and is clinically used for hemoptysis, vomiting blood, traumatic bleeding, sore poisoning, skin cracking and other diseases, as well as tuberculosis hemoptysis, ulcer bleeding, etc. Since ancient times, Bletilla striata has been considered a holy product for whitening and skin care, and people often grind its dried tuber into powder for external application to achieve the effect of whitening. Modern chemical and pharmacological studies have shown that the main chemical components of Bletilla striata include bibenzyl, phenanthrene, flavonoids, polysaccharides, etc., which have antibacterial, antiviral, antioxidant and other pharmacological activities, but the research on its whitening activity is less. At present, it is mainly focused on Bletilla striata polysaccharides and Bletilla striata crude extracts, and whether the abundant small molecule components in Bletilla striata have whitening activity is still lacking in-depth research.

[0004] According to the description of Bletilla striata in Part I of the 2020 Chinese Pharmacopoeia, Bletilla striata is excavated in summer and autumn, the fibrous roots are removed, and the roots are washed. The fibrous roots account for about 1 / 3 of the root mass, but as a non-medicinal part, they are discarded during the harvesting and processing process, resulting in a great waste of resources. Current studies have shown that the fibrous roots and tuber parts of Bletilla striata have similar composition, and the total phenol content and tyrosinase inhibitory activity of the fibrous roots are higher, suggesting that the fibrous root part of Bletilla striata is very likely to have potential whitening medicinal value. However, the natural active ingredients with whitening effects in the fibrous roots of Bletilla striata have not yet been reported. Summary of the invention

[0005] In response to actual needs, the present invention provides a Bletilla striata root extract with whitening activity and application thereof.

[0006] The technical solution adopted by the present invention is:

[0007] A Bletilla striata root extract with whitening activity is prepared by the following method:

[0008] (1) Take the powder of the root of Bletilla striata, use methanol as solvent, and extract it three times by continuous reflux; combine the three extracts, evaporate the extract, and dry the residue to obtain a crude extract;

[0009] (2) The crude extract was separated by silica gel column chromatography, and the column was packed with 400 mesh silica gel. According to the mass ratio of sample: sample-mixed silica gel of 1:1-1.5, the sample was loaded by dry method and the column was packed by wet method. Then, the petroleum ether-ethyl acetate system was used for gradient elution with seven volume ratios of 16:5, 7:3, 13:7, 3:2, 1:1, 2:3, and 3:1. Each gradient fraction was collected with 1000 mL, and a total of 27 fractions were collected and named Fr.1-Fr.27. Fractions Fr.3-Fr.27 were detected by silica gel thin layer plate spotting, and the developing solvent was ethyl acetate-petroleum ether with a mass ratio of 1:1;

[0010] (3) According to the thin layer chromatograms of different fractions, the fractions with spots in similar positions were merged, and Fr.3~Fr.7, Fr.8~Fr.11, Fr.12~Fr.20, Fr.21~Fr.24, Fr.25~Fr.26, and Fr.27~Fr.28 were merged, dried, and weighed to obtain 6 fractions, which were named Fr.1-1~Fr.1-6;

[0011] (4) The fraction Fr.1-4 was subjected to semi-preparative liquid phase separation using a high performance liquid chromatography system under the following conditions: chromatographic column H&E-C18 column (5 mm, 250 mm × 9.4 mm id), column temperature 30°C, detection wavelength 275 nm, flow rate 3 mL / min, elution gradient as shown in the following table, and two fractions were obtained, which were named Fr.1-4-1 and Fr.1-4-2;

[0012]

[0013] (5) The fraction Fr.1-4-2 was subjected to semi-preparative liquid phase separation using a high performance liquid chromatography system under the following conditions: chromatographic column H&E-C 18 Chromatographic column (5 μm, 250 mm × 9.4 mm id ), column temperature 30℃, detection wavelength 275 nm, flow rate 3mL / min, elution gradient as shown in the following table:

[0014]

[0015] The eluted product is collected and dried to obtain the Bletilla striata root extract with whitening activity.

[0016] Specifically, the main active ingredients of the extract include: 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol, 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene and 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrene diol.

[0017] The present invention obtains and separates and purifies three phenanthrene components from the fibrous roots of Bletilla striata for the first time, and 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol, 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene, and 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrene diol all have tyrosinase inhibitory activity. Among them, 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol has the strongest tyrosinase inhibitory activity, and in vivo experiments prove that it has excellent whitening activity.

[0018] The invention also relates to the application of the bletilla striata root extract in the preparation of a tyrosinase activity inhibitor.

[0019] Specifically, the present invention relates to the use of 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol in the preparation of tyrosinase activity inhibitors.

[0020] The invention also relates to the application of the Bletilla striata root extract in preparing whitening facial cream.

[0021] Specifically, the present invention relates to the use of 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol in the preparation of whitening cream.

[0022] The beneficial effects of the present invention are mainly reflected in that: 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol is separated from the fibrous roots of Bletilla striata, and it is confirmed for the first time that it has excellent tyrosinase inhibitory activity and obvious whitening activity in vivo; in addition, analogues 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene and 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrene diol are also separated, and both are confirmed for the first time to have tyrosinase inhibitory activity, which directly provides a material basis for the comprehensive development and utilization of non-medicinal parts of Bletilla striata. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The process flow chart of high-resolution bioactivity spectrum screening strategy;

[0024] Figure 2 The inhibition rates of tyrosinase monophenolase (a) and tyrosinase diphenolase (b) of different extracts from Bletilla striata fibrous roots;

[0025] Figure 3 This is the high-resolution biological activity spectrum of tyrosinase inhibition in Bletilla striata hairy roots;

[0026] Figure 4 It is the thin layer chromatogram of different fractions Fr.3-27 of silica gel column chromatography;

[0027] Figure 5 This is the HPLC chromatogram of Fr.1-4 (275 nm);

[0028] Figure 6 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol (a), 2, 7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene (b), 1-(4-hydroxybenzyl)-4, 8-dimethoxy-2, 7-phenanthrene-diol (c);

[0029] Figure 7 This is the hydrogen spectrum of 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol sample;

[0030] Figure 8 This is the carbon spectrum of 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol sample;

[0031] Fig. 9 This is the hydrogen spectrum of 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene sample;

[0032] Fig.10 This is the carbon spectrum of 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene sample;

[0033] Fig.11 This is the hydrogen spectrum of 1-(4-hydroxybenzyl)-4, 8-dimethoxy-2,7-phenanthrene diol sample;

[0034] Fig.12 This is the carbon spectrum of 1-(4-hydroxybenzyl)-4, 8-dimethoxy-2, 7-phenanthrene diol sample;

[0035] Fig.13 is the inhibition curve of β-arbutin on tyrosinase monophenolase;

[0036] Fig.14 is the inhibition curve of kojic acid on tyrosinase monophenolase;

[0037] Fig.15 The inhibition curves of the three compounds on tyrosinase monophenolase;

[0038] Fig.16 This is a typical graph of melanin signal intensity in the zebrafish head after sample treatment, and the dotted box is the analysis site. DETAILED DESCRIPTION

[0039] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0040] The raw materials and equipment used in this application, unless otherwise specified, are all commonly used raw materials and equipment in this field, and are all commercially available products. The methods used in this application, unless otherwise specified, are all conventional methods in this field.

[0041] Example 1: Tyrosinase inhibition activity test of different extracts from Bletilla striata root

[0042] 1. Sample preparation

[0043] Accurately weigh 3 portions of Bletilla striata root powder (5.0 g) into a conical flask, and accurately add 100 mL of 70% ethanol aqueous solution, methanol, and ethyl acetate, respectively, and ultrasonicate for 30 min at 50 °C. After filtering and recovering the solvent, 70% ethanol extract, methanol extract, and ethyl acetate extract of Bletilla striata root were prepared.

[0044] 2. Preliminary screening of whitening activity of different extracts from Bletilla striata root

[0045] (1) Screening of monophenolase inhibitory activity

[0046] The sample group, sample blank group, control group and control blank group were set up respectively. 10 μL of different Bletilla striata root extract solutions (5 mg / mL), 10 μL of sample solvent (DMSO) and 50 μL of tyrosinase solution (125 U / mL) were added to the 96-well plate in sequence, and incubated at 35 °C for 5 min with shaking. 50 μL of L-tyrosine solution (1 mM) was added to the sample group and the control group to start the reaction, and the reaction was monitored at 492 nm on an ELISA reader for 30 min, with the absorbance detected every 1 min. The absorbance at a wavelength of 492 nm A sample , A blank , A control and A blank’ The phenolase inhibition rate was calculated using the following formula.

[0047]

[0048] In the formula, A sample ——The absorbance of the sample set (including tyrosinase, substrate and inhibitor);

[0049] A blank——Absorbance of the sample blank group (containing tyrosinase and inhibitor);

[0050] A control ——The absorbance of the control group (containing tyrosinase, substrate and inhibitor solvent);

[0051] A blank’ ——Absorbance of the control blank group (containing tyrosinase and inhibitor solvent).

[0052] like Figure 2 a, Different extracts from Bletilla striata root showed a certain tyrosinase monophenolase inhibition rate. Since the methanol extract had a stronger tyrosinase monophenolase inhibition effect, methanol was selected as the extraction solvent in subsequent experiments.

[0053] (2) Screening of diphenolase inhibitory activity

[0054] The sample group, sample blank group, control group and control blank group were set up respectively. 10 μL of different Bletilla striata root extract solutions (5 mg / mL), 10 μL of sample solvent (DMSO) and 50 μL of L-solution (5 mM) were added to the 96-well plate in sequence, and incubated at 30 °C for 10 min. 50 μL of L-tyrosine solution (71.4 U / mL) was added to the sample group and the control group to start the reaction. The reaction was monitored at 492 nm on an ELISA reader for 5 min, and the absorbance was detected every 1 min. The absorbance at a wavelength of 492 nm A sample , A blank , A control and A blank’ The monophenolase inhibition rate was calculated using the above formula.

[0055] like Figure 2 b, Different extracts from Bletilla striata fibrous roots showed lower tyrosinase diphenolase inhibition rates.

[0056] Example 2: Screening of whitening active ingredients in Bletilla striata root based on high-resolution biological tyrosinase inhibition activity spectrum screening strategy

[0057] 1. Sample analysis

[0058] Accurately weigh 5.0 g of Bletilla striata root powder into a conical flask, accurately add 100 mL of methanol, and ultrasonicate for 30 min at 50 °C. Filter and recover the solvent to prepare the Bletilla striata root extract.

[0059] Accurately weigh 20 mg of Bletilla striata root extract, reconstitute it with methanol, and prepare a 20 mg / mL solution. Perform sample analysis using high performance liquid chromatography. The analysis conditions are as follows:

[0060] The instrument used in this experiment is a high performance liquid chromatography system, including an online vacuum degasser, a high pressure binary gradient pump, a disk autosampler, and a column oven. Chromatographic column H&E-C18 column (5 mm, 250 mm × 4.6 mm id ), column temperature 30℃, detection wavelength 275 nm, flow rate 1 mL / min. The elution gradient is shown in Table 1.

[0061] Table 1 Gradient elution process

[0062]

[0063] 2. Construction of high-resolution tyrosinase inhibitory activity spectrum

[0064] High-resolution tyrosinase monophenolase inhibitory activity spectrum: After chromatographic separation, the sample microfractions were collected into 4 96-well plates. The concentration of the Bletilla striata fibrous root test sample was 20 mg / mL, and the injection volume was 20 μL. The fractions within 10-62 min were collected according to the retention time. The microfractions were collected into 336 microwells at a resolution of 6.5 point / min. Row H of each 96-well plate was left blank. The mobile phase of the microfractions in the 96-well plate was removed, 10 μL DMSO was added to each well to dissolve the dried fractions in the well, and then 90 μL PBS solution, 50 μL tyrosinase solution (125 U / mL) and 50 μL L-tyrosine solution (1 mM) were added in sequence. Incubate at 35°C for 5 min before starting the reaction. After adding L-tyrosine solution to start the reaction, monitor at a wavelength of 492 nm on the microplate reader for 30 min, and detect the absorbance value every 1 min. The inhibition rate of the fraction in each well on the tyrosinase monophenolase activity was calculated using the formula in step (2), and a curve showing the change of the monophenolase inhibition rate with the retention time was obtained.

[0065] High-resolution tyrosinase diphenolase inhibitory activity spectrum: After chromatographic separation, the sample microfractions were collected into four 96-well plates. The concentration of the Bletilla striata fibrous root test sample was 20 mg / mL, and the injection volume was 20 μL. The fractions within 10-62 min were collected according to the retention time. The microfractions were collected into 336 microwells at a resolution of 6.5 point / min. Row H of each 96-well plate was left blank. The mobile phase of the microfractions in the 96-well plate was removed, 10 μL DMSO was added to each well to dissolve the dried fractions in the well, and then 90 μL PBS solution, 50 μL L-dopa solution (5 mM) and 50 μL tyrosinase solution (71.4 U / mL) were added in sequence. Incubate at 30°C for 10 min before starting the reaction. After the tyrosinase solution was added to start the reaction, the absorbance value was detected every 1 min at a wavelength of 492 nm on the microplate reader for 6 min. The inhibition rate of the fraction in each well on the tyrosinase diphenolase activity was calculated using the formula in step (2), and a curve showing the change of the diphenolase inhibition rate with the retention time was obtained.

[0066] Combined with the HPLC chromatogram at 275 nm, a high-resolution biological activity spectrum of tyrosinase inhibition was obtained, such as Figure 3 shown.

[0067] In the high-resolution tyrosinase monophenolase inhibitory activity spectrum, the active ingredients corresponding to peaks 1 and 2 showed potential tyrosinase monophenolase inhibitory activity, among which the active ingredient corresponding to peak 1 was the strongest.

[0068] Example 3: Isolation of Potentially Active Compounds

[0069] 1. Sample preparation

[0070] 1 kg of Bletilla striata root powder was extracted with methanol as solvent for three times under continuous reflux (1:3 reflux material-liquid ratio, temperature 60℃-80℃), each time for 100 min; the three extracts were combined, evaporated to dryness, and the residue was thoroughly dried in a fume hood to obtain 43 g of dry extract.

[0071] 2. Silica gel column chromatography and semi-preparative liquid phase separation

[0072] The extract was separated by silica gel column chromatography, 500 g of 400 mesh column chromatography silica gel was used to pack the column, 64 g of silica gel was mixed with sample (sample: mixed silica gel = 1:1-1:1.5), dry loading was adopted, wet loading was adopted, and then the petroleum ether-ethyl acetate system (by volume ratio) was used for gradient elution at seven ratios of 16:5, 7:3, 13:7, 3:2, 1:1, 2:3, 3:1, and 1000 mL was collected for each gradient fraction. A total of 27 fractions were collected and named Fr.1-27 respectively; Fr.3-27 was detected by silica gel thin layer plate spotting, and ethyl acetate-petroleum ether (1:1) was selected as the developing solvent.

[0073] like Figure 4 , according to the thin layer chromatograms of different fractions, the fractions with spots in similar positions were merged. Fr.3-7, Fr.8-11, Fr.12-20, Fr.21-24, Fr.25-26, Fr.27-28 were merged, dried and weighed. Six fractions were obtained and named Fr.1-1-6;

[0074] The six fractions Fr.1-1-6 were respectively subjected to HPLC analysis, and the elution conditions were the same as those in Example 2.1.

[0075] like Figure 5 , peaks 1 and 2 were mostly enriched in Fr.1-4.

[0076] The Fr.1-4 fraction (about 652.35 mg) was subjected to semi-preparative liquid phase separation. The instrument used in this experiment was a high performance liquid chromatography system. The chromatographic column was H&E-C18 column (5 mm, 250 mm × 9.4 mm id), the column temperature was 30°C, the detection wavelength was 275 nm, and the flow rate was 3 mL / min. The elution gradient is shown in Table 2. Two fractions were obtained, which were named Fr.1-4-1 (about 35 mg) and Fr.1-4-2 (about 60 mg).

[0077] Table 2: Gradient elution process

[0078] Time / min 0.1% formic acid water (%) Methanol (%) 0 70 30 30 60 50 35 0 100

[0079] Fr.1-4-2 was subjected to semi-preparative liquid phase separation. The instrument used in this experiment was a high performance liquid chromatography system. Chromatographic column H&E-C 18 Chromatographic column (5 μm, 250 mm × 9.4 mm id ), column temperature 30 ° C, detection wavelength 275 nm, flow rate 3mL / min. The elution gradient is shown in Table 3. Three samples were obtained, named compounds 1-3, and were identified as 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol, 2, 7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene, and 1-(4-hydroxybenzyl)-4, 8-dimethoxy-2, 7-phenanthrene diol. The structure is as follows Figure 6 As shown in (a)-(c).

[0080] Table 3: Gradient elution process

[0081] Time / min 0.1% formic acid water (%) Acetonitrile (%) 0 70 30 30 60 40 50 55 45

[0082] Example 4: Structural Identification

[0083] The components obtained in Example 3 were identified, and the identification data are as follows: 1 H-NMR and 13 C-NMR images Figure 7-14 As shown, Figure 7 This is the hydrogen spectrum of 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol sample; Figure 8 This is the carbon spectrum of 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol sample; Fig. 9 This is the hydrogen spectrum of 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene sample; Fig.10 This is the carbon spectrum of 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene sample; Fig.11 This is the hydrogen spectrum of 1-(4-hydroxyphenyl)-4,8-dimethoxy-2,7-phenanthrene diol sample; Fig.12 This is the carbon spectrum of 1-(4-hydroxyphenyl)-4, 8-dimethoxy-2, 7-phenanthrene diol sample.

[0084] 1-(4-Hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol: 1 H-NMR (600 MHz, CD3OD, δ): 7.97 (1H, d, J =8.4 Hz, H-5), 6.95 (2H, d, J =8.4 Hz, H-2 ， , 6 ， ), 6.65 (2H, d, J =9.0 Hz, H-3 ， , 5 ， ), 6.60 (1H, dd, J =8.4 Hz, 3.0 Hz, H-6), 6.59 (1H, d, J =3.0 Hz, H-8), 6.52 (1H, s, H-3), 3.94 (2H, s, CH2-α), 3.83 (3H, s, O-CH3), 2.57-2.48 (4H, m, -CH2- CH2); 13C-NMR (600MHz, CD3OD, δ): 118.5 (C-1), 157.2 (C-2), 99.1 (C-3), 155.6 (C-4), 130.3 (C-5), 114.6 (C-6), 156.0 (C-7), 113.5 (C- 8), 30.9 (C-9), 27.4 (C-10), 140.9 (C-1a), 117.4 (C-4a), 126.5 (C-5a), 140.4 (C-8a), 55.9 (-OCH3), 31.0 (CH2-α), 156.06 (C-4 ， ), 133.8 (C-1 ， ), 130.3 (C-3 ， , 5 ， ), 115.9 (C-2 ， , 6 ， )

[0085] 2, 7-Dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene: 1 H-NMR (600 MHz, CD3OD, δ): 9.42 (1H, d, J =9.6 Hz, H-5), 7.76 (1H, d, J =9.6 Hz, H-10), 7.49 (1H, d, J =9.0 Hz, H-9), 7.13 (1H, d, J =3.0 Hz, H-8), 7.09 (1H, dd, J =9.6 Hz, 3.0 Hz, H-6), 7.02 (1H, part of the A2B2 system, J =9.0 Hz, H-2 ， , 6 ， ), 6.91 (1H, s, H-3), 6.65 (1H, part of the A2B2 system, J =9.0 Hz, H-3 ， , 5 ， ), 4.34 (2H, s, CH2-α), 4.12 (3H, s, -OCH3); 13C-NMR (600MHz, CD3OD, δ): 114.8 (C-1), 153.9 (C-2), 100.2 (C-3), 158.9 (C-4), 130.5 (C-5), 117.2 (C-6), 155.3 (C -7), 112.0 (C-8), 128.2 (C-9), 124.6 (C-10), 134.1 (C-1a), 116.7 (C-4a), 125.8 (C-5a), 134.1 (C-8a), 134.2 (C-1 ， ), 156.0 (C-4 ， ), 130.1 (C-2 ， , 6 ， ), 115.9 (C-3 ， , 5 ， ), 30.6 (CH2-α), 56.0 (4-OCH3);

[0086] 1-(4-Hydroxybenzyl)-4, 8-dimethoxy-2, 7-phenanthrene diol: 1 H-NMR (600 MHz, CD3OD, δ): 9.23(1H, dd, J =9.6 Hz, 0.4 Hz, H-5), 7.92 (1H, d, J =9.6 Hz, H-9), 7.83 (1H, d, J =9.6Hz, H-10), 7.15 (1H, d, J =9.6 Hz, H-6), 7.03 (2H, m, H-2 , , 6 , ), 6.91 (1H, s, H-3), 6.66 (2H, m, H-3 , , 5 , ), 4.35 (2H, s, CH2-α), 4.11 (3H, s, 4-OCH3), 3.92(3H, s, 8-OCH3); 13C-NMR (600 MHz, CD3OD, δ): 114.8 (C-1), 154.1 (C-2), 100.1 (C-3), 159.1 (C-4), 125.8 (C-5), 117.8 (C-6), 146.7 (C-7), 142.2 (C-8), 121.4(C-9), 124.6 (C-10), 116.7 (C-4a), 126.7 (C-5a), 127.7 (C-8a), 134.1 (C-10a),134.1 (C-1 , ), 130.2 (C-2 , , 6 , ), 156.1 (C-4 , ), 115.9 (C-3 , , 5 , ), 61.5 (8-OCH3), 55.9 (4-OCH3), 30.6 (CH2-α).

[0087] Example 5: Verification of tyrosinase activity in vitro of active ingredients

[0088] A series of concentration gradients of β-arbutin, kojic acid, 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol, 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene, and 1-(4-hydroxyphenyl)-4, 8-dimethoxy-2, 7-phenanthrene-diol were prepared for in vitro activity verification, and the inhibition IC of the compounds on tyrosinase monophenolase was calculated using SPSS staistics18 software. 50 Value, the result is Figure 13-15 shown.

[0089] As shown in the figure, the IC values ​​of β-arbutin and kojic acid are 50 The value is the positive control. The active compound 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol corresponding to peak 1 has the strongest inhibitory activity against tyrosinase monophenolase, IC 50 The value was 11.89 μM, which was much better than the positive control β-arbutin (IC 50 = 328.02 μM) and kojic acid (IC 50 =30.33 μM). 2,7-Dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene and 1-(4-hydroxyphenyl)-4,8-dimethoxy-2,7-phenanthrene diol also showed strong inhibitory activity against tyrosinase monophenolase IC 50 The values ​​were 184.60 μM and 54.54 μM, respectively, which were better than the positive control β-arbutin.

[0090] Example 6: In vivo whitening efficacy verification of active ingredients

[0091] Wild-type AB strain zebrafish were randomly selected in a 6-well plate, with 30 in each well (experimental group). The samples were dissolved in water (concentrations are shown in Table 4), the positive control arbutin was 3000 µg / mL, and a normal control group was set up at the same time, with a volume of 3 mL per well. After treatment at 28℃ for 48 h, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. The data were analyzed and collected using Image-Pro Plus advanced image processing software. The melanin signal intensity of the zebrafish head was analyzed, and the whitening effect of the sample was evaluated based on the statistical analysis results of this indicator. The statistical processing results are expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant. Under the experimental conditions of this experiment, 1-(4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol has a whitening effect, see for details. Fig.16 .

[0092] Table 4: Sample whitening efficacy evaluation test results (n = 10)

[0093]

[0094] Compared with the normal control group, ***p<0.001

[0095] The above description is only a specific embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent exchange made by using the present invention, or directly or indirectly applied in other related technical fields, is also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a Bletilla striata root extract having whitening activity, comprising the following steps: (1) Take the powder of the root of Bletilla striata, use methanol as solvent, and extract it three times by continuous reflux; combine the three extracts, evaporate the extract, and dry the residue to obtain a crude extract; (2) The crude extract was separated by silica gel column chromatography, and the column was packed with 400 mesh silica gel. According to the mass ratio of sample to sample silica gel of 1:1-1.5, the sample was loaded by dry method and the column was packed by wet method. Then, the petroleum ether-ethyl acetate system was used for gradient elution with seven volume ratios of 16:5, 7:3, 13:7, 3:2, 1:1, 2:3, and 3:

1. Each gradient fraction was collected with 1000 mL, and a total of 27 fractions were collected and named Fr.1-Fr.

27. Fractions Fr.3-Fr.27 were detected by silica gel thin layer plate spotting, and the developing solvent was ethyl acetate-petroleum ether with a mass ratio of 1:1; (3) According to the thin layer chromatograms of different fractions, the fractions with spots in similar positions were merged, and Fr.3~Fr.7, Fr.8~Fr.11, Fr.12~Fr.20, Fr.21~Fr.24, Fr.25~Fr.26, and Fr.27~Fr.28 were merged, dried, and weighed to obtain 6 fractions, which were named Fr.1-1~Fr.1-6; (4) The fractions Fr.1-4 were separated by semi-preparative liquid chromatography using a high performance liquid chromatography system under the following conditions: chromatographic column H&E-C 18 The chromatographic column was 5 mm, 250 mm × 9.4 mm, the column temperature was 30 °C, the detection wavelength was 275 nm, the flow rate was 3 mL / min, and the elution gradient was as shown in the following table. Two fractions were obtained and named Fr.1-4-1 and Fr.1-4-2; (5) The fraction Fr.1-4-2 was subjected to semi-preparative liquid phase separation using a high performance liquid chromatography system under the following conditions: chromatographic column H&E-C 18 Chromatographic column, 5 μm, 250 mm × 9.4 mm, column temperature 30 ° C, detection wavelength 275 nm, flow rate 3 mL / min, elution gradient is shown in the following table: The eluted product is collected and dried to obtain the Bletilla striata root extract with whitening activity.

2. The method according to claim 1, characterized in that The main active ingredients of the extract are: 1-(4-hydroxybenzyl)-4-methoxy-9, 10-dihydrophenanthrene-2, 7-diol, 2,7-dihydroxy-1-(4-hydroxybenzyl)-4-methoxyphenanthrene and 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrene diol.

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