Preparation method of a natural plant extract and its application in preparing a product with whitening effect

Abstract

The present invention relates to the field of biological medicine technology, and specifically discloses a preparation method of a natural plant extract and its application in the preparation of a product with a whitening effect. The preparation method of the natural plant extract is specifically as follows: taking cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract, mixing them evenly to obtain a mixed extract, which is the natural plant extract. Since the natural plant extract of the present invention has tyrosinase inhibitory activity, it can be used as a tyrosinase inhibitor; further, it can also be used as an active ingredient to prepare a drug or skin care product with a whitening effect.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and specifically relates to a preparation method of a natural plant extract and its application in the preparation of products with whitening effects. Background Art

[0002] Melanin is a kind of dark or brown spots or patches that appear on exposed parts of the body such as the face, back of the hand, and neck, also known as freckles, liver spots or age spots. Factors such as ultraviolet radiation and aging can all lead to an increase in the production and deposition of melanin in the skin, and the excessive deposition of melanin is also considered to be the main cause of the formation of skin pigmentation such as age spots and freckles.

[0003] Tyrosinase, also known as polyphenol oxidase, catechol oxidase or tyrosinase, etc.; is a structurally complex multi-subunit copper-containing oxidoreductase that exists widely in microorganisms, animals, plants and the human body; it is also the rate-limiting enzyme that regulates melanin production; it can catalyze the production of melanin oxidized from tyrosine; therefore, developing a tyrosinase inhibitor can effectively reduce the production of melanin; further, its application in the preparation of whitening products has important application value. Summary of the Invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a natural plant extract with tyrosinase inhibitory activity.

[0005] The technical solution of the present invention is as follows:

[0006] A preparation method of a natural plant extract, specifically: taking cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract and mixing them evenly to obtain a mixed extract, that is, the natural plant extract.

[0007] The inventor surprisingly found in the research that the natural plant extract obtained by mixing the cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract prepared by the method of the present invention evenly has good tyrosinase inhibitory activity; and its tyrosinase inhibitory activity is significantly higher than that of the cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract prepared by the method of the present invention alone. This may be because the combination of the cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract can produce a synergistic effect in inhibiting tyrosinase.

[0008] Since the natural plant extract of the present invention has tyrosinase inhibitory activity, it can be used as a tyrosinase inhibitor, and further, it can also be used as an active ingredient to prepare drugs or skin care products with whitening effects.

[0009] Preferably, the weight ratio of cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract is 1-3:1-3:1-3.

[0010] Most preferably, the weight ratio of cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract is 1:1:1.

[0011] Preferably, the cactus extract is prepared by a method comprising the following steps:

[0012] Crush the cactus and mix it evenly with water to obtain a cactus slurry; then add chloroform to the cactus slurry for extraction to obtain a chloroform extract; concentrate and dry the chloroform extract to obtain the cactus extract.

[0013] Further preferably, the dosage ratio of cactus, water, and chloroform is 1 kg: 3-5 L: 3-5 L.

[0014] Most preferably, the dosage ratio of cactus, water, and chloroform is 1 kg: 4 L: 4 L.

[0015] Preferably, the ophiopogon japonicus extract is prepared by a method comprising the following steps:

[0016] Take ophiopogon japonicus, and then extract it with an organic solvent to obtain an organic solvent extract; concentrate and dry the organic solvent extract to obtain the ophiopogon japonicus extract.

[0017] Further preferably, the extraction is maceration extraction;

[0018] The organic solvent is ethyl acetate;

[0019] The time of the maceration extraction is 1-3 days;

[0020] Among them, the dosage ratio of ophiopogon japonicus and ethyl acetate is 1 kg: 5-10 L;

[0021] Most preferably, the dosage ratio of ophiopogon japonicus and ethyl acetate is 1 kg: 6 L.

[0022] Preferably, the sophora flavescens leaf extract is prepared by a method comprising the following steps:

[0023] Take sophora flavescens leaves, and then extract them with an organic solvent to obtain an organic solvent extract; concentrate and dry the organic solvent extract to obtain the sophora flavescens leaf extract.

[0024] Further preferably, the extraction is maceration extraction;

[0025] The organic solvent is chloroform;

[0026] The time of the maceration extraction is 1-3 days;

[0027] Among them, the dosage ratio of Sophora flavescens leaves to chloroform is 1 kg: 6-12 L;

[0028] Most preferably, the dosage ratio of Sophora flavescens leaves to chloroform is 1 kg: 8 L.

[0029] Further preferably, the preparation method of the natural plant extract further comprises the following steps:

[0030] Load the mixed extract onto a silica gel column, first elute with 2-4 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:34-36, and discard the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:34-36; then elute with 3-6 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:54-56, collect the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:54-56, and obtain the natural plant extract after concentration and drying.

[0031] Most preferably, first elute with 3 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:35, and discard the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:35; then elute with 5 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:55, collect the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:55, and obtain the natural plant extract after concentration and drying.

[0032] The inventors further found in the research that the natural plant extract prepared from the mixed extract through the above elution conditions of the present invention has a significantly improved inhibitory effect on tyrosinase compared to the mixed extract obtained by simply mixing the cactus extract, Ophiopogon japonicus extract, and Sophora flavescens leaf extract evenly.

[0033] The present invention also provides an application of the natural plant extract prepared by the above preparation method in the preparation of a tyrosinase inhibitor.

[0034] The present invention also provides an application of the natural plant extract prepared by the above preparation method in the preparation of a product with whitening effect.

[0035] Beneficial effects: The present invention provides a natural plant extract with a brand-new composition; the natural plant extract is prepared by uniformly mixing the cactus extract, the ophiopogon japonicus extract, and the sophora flavescens leaf extract prepared by the method of the present invention, and it has good tyrosinase inhibitory activity. Since the natural plant extract of the present invention has tyrosinase inhibitory activity, it can be used as a tyrosinase inhibitor, and further, it can be used as an active ingredient to prepare drugs or skin care products with whitening effects. Detailed implementation manners

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] Example 1 Preparation of natural plant extract

[0038] Take the cactus extract, the ophiopogon japonicus extract, and the sophora flavescens leaf extract and mix them evenly according to a weight ratio of 1:1:1 to obtain a mixed extract, that is, the natural plant extract;

[0039] The cactus extract is prepared by a method comprising the following steps: crushing the cactus and mixing it evenly with water to obtain a cactus slurry; then adding chloroform to the cactus slurry for extraction to obtain a chloroform extract; concentrating and drying the chloroform extract to obtain the cactus extract; wherein, the dosage ratio of the cactus, water, and chloroform is 1 kg:4 L:4 L;

[0040] The ophiopogon japonicus extract is prepared by a method comprising the following steps: taking ophiopogon japonicus, and then extracting it with an organic solvent to obtain an organic solvent extract; concentrating and drying the organic solvent extract to obtain the ophiopogon japonicus extract; wherein, the extraction is maceration extraction; the organic solvent is ethyl acetate; the maceration extraction time is 2 days; the dosage ratio of ophiopogon japonicus to ethyl acetate is 1 kg:6 L;

[0041] The sophora flavescens leaf extract is prepared by a method comprising the following steps: taking sophora flavescens leaves, and then extracting it with an organic solvent to obtain an organic solvent extract; concentrating and drying the organic solvent extract to obtain the sophora flavescens leaf extract; wherein, the extraction is maceration extraction; the organic solvent is chloroform; the maceration extraction time is 2 days; the dosage ratio of sophora flavescens leaves to chloroform is 1 kg:8 L.

[0042] Example 2 Preparation of natural plant extract

[0043] (1) Mix the cactus extract, the ophiopogon japonicus extract, and the sophora flavescens leaf extract evenly according to a weight ratio of 1:1:1 to obtain a mixed extract;

[0044] (2) Load the mixed extract onto a silica gel column. First, elute with 3 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:35, and discard the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:35. Then, elute with 5 column volumes of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:55, collect the eluate eluted by the mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:55, and obtain the natural plant extract after concentration and drying;

[0045] The cactus extract described in step (1) is prepared by a method comprising the following steps: Crush the cactus and mix it evenly with water to obtain a cactus slurry; then add chloroform to the cactus slurry for extraction to obtain a chloroform extract; concentrate and dry the chloroform extract to obtain the cactus extract; wherein, the dosage ratio of the cactus, water, and chloroform is 1 kg:4 L:4 L;

[0046] The ophiopogon japonicus extract described in step (1) is prepared by a method comprising the following steps: Take ophiopogon japonicus, and then extract it with an organic solvent to obtain an organic solvent extract; concentrate and dry the organic solvent extract to obtain the ophiopogon japonicus extract; wherein, the extraction is maceration extraction; the organic solvent is ethyl acetate; the maceration extraction time is 2 days; the dosage ratio of ophiopogon japonicus to ethyl acetate is 1 kg:6 L;

[0047] The sophora flavescens leaf extract described in step (1) is prepared by a method comprising the following steps: Take sophora flavescens leaves, and then extract it with an organic solvent to obtain an organic solvent extract; concentrate and dry the organic solvent extract to obtain the sophora flavescens leaf extract; wherein, the extraction is maceration extraction; the organic solvent is chloroform; the maceration extraction time is 2 days; the dosage ratio of sophora flavescens leaves to chloroform is 1 kg:8 L;

[0048] The silica gel in the silica gel column in step (2) is silica gel with a mesh size of 200 - 300, and the weight of the silica gel is 40 times the weight of the mixed extract.

[0049] Preparation of the natural plant extract in Comparative Example 1

[0050] Step (1) of Comparative Example 1 is the same as that of Example 2; the difference lies in the silica gel column elution conditions in step (2); the specific steps of step (2) in Comparative Example 1 are as follows:

[0051] Load the mixed extract onto a silica gel column. First, elute with 3 column volumes of a mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:5, and discard the eluate eluted by the mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:5. Then, elute with 5 column volumes of a mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:55, collect the eluate eluted by the mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:55, and obtain the natural plant extract after concentration and drying.

[0052] Preparation of the natural plant extract in Comparative Example 2

[0053] Step (1) of Comparative Example 2 is the same as that of Example 2; the difference lies in the silica gel column elution conditions in step (2); the specific steps of step (2) in Comparative Example 2 are as follows:

[0054] Load the mixed extract onto a silica gel column. First, elute with 3 column volumes of a mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:5, and discard the eluate eluted by the mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:5. Then, elute with 5 column volumes of a mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:20, collect the eluate eluted by the mixed organic solvent composed of chloroform and methanol at a volume ratio of 100:20, and obtain the natural plant extract after concentration and drying.

[0055] Experimental Example 1 Tyrosinase inhibition activity

[0056] (1) Mix 130 μL of phosphate buffer with a pH of 6.8, 10 μL of tyrosinase phosphate buffer solution with a concentration of 33.3 U / mL, and 10 μL of DMSO solution of the test extract with a concentration of 1 mg / mL evenly. Then add L-DOPA solution and continue to mix evenly until the final concentration of L-DOPA solution is 0.5 mM. Measure the OD value at 490 nm after 15 min, and record it as the OD value of the extract.

[0057] (2) Mix 130 μL of phosphate buffer with a pH of 6.8, 10 μL of tyrosinase phosphate buffer solution with a concentration of 33.3 U / mL, and 10 μL of DMSO solution evenly. Then add L-DOPA solution and continue to mix evenly until the final concentration of L-DOPA solution is 0.5 mM. Measure the OD value at 490 nm after 15 min, and record it as the control OD value.

[0058] (3) Calculate the tyrosinase inhibition rate; the calculation formula for the tyrosinase inhibition rate is: Tyrosinase inhibition rate (%) = [(control OD value - extract OD value) / control OD value] × 100%.

[0059] The extract to be tested is the natural plant extract prepared in Examples 1 and 2 and Comparative Examples 1 and 2; as well as the cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract prepared according to the method described in Example 1; the test results are shown in Table 1.

[0060] Table 1. Test results of tyrosinase inhibitory activity of the natural plant extract of the present invention

[0061] Tyrosinase inhibition rate Natural plant extract prepared in Example 1 71.6％ Natural plant extract prepared in Example 2 95.9％ Opuntia extract prepared in Example 1 36.4％ Ophiopogon japonicus extract prepared in Example 1 41.2％ Sophora flavescens leaf extract prepared in Example 1 45.8％ Natural plant extract prepared in Comparative Example 1 77.9％ Natural plant extract prepared in Comparative Example 2 55.2％

[0062] It can be seen from the experimental results in Table 1 that for the natural plant extract prepared in Example 1, its tyrosinase inhibition rate reached 71.6%, showing good tyrosinase inhibitory effect; at the same time, its tyrosinase inhibition rate was much higher than that of the cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract prepared according to the method described in Example 1; this indicates that: the natural plant extract obtained by uniformly mixing the cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract prepared by the method of the present invention has a much higher tyrosinase inhibitory activity than the individual cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract prepared by the method of the present invention. The combination of the cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract prepared according to the method of the present invention can produce a synergistic effect in inhibiting tyrosinase.

[0063] It can be seen from the experimental results in Table 1 that for the natural plant extract prepared in Example 2, its tyrosinase inhibition rate reached 95.9%, showing a further significant increase compared with Example 1, and having very excellent tyrosinase inhibitory activity. This indicates that: the natural plant extract prepared by further subjecting the mixed extract obtained by uniformly mixing the cactus extract, ophiopogon japonicus extract, and sophora flavescens leaf extract to the silica gel column elution conditions of the present invention has a further significant increase in the inhibitory effect on tyrosinase compared with the mixed extract; making the prepared natural plant extract have more excellent tyrosinase inhibitory activity.

[0064] As can be seen from the experimental results in Table 1, for the natural plant extract prepared in Comparative Example 1, although its tyrosinase inhibition rate is higher than that in Example 1, the increase is not significant, and the increase is much smaller than that of the natural plant extract prepared in Example 2. For the natural plant extract prepared in Comparative Example 2, its tyrosinase inhibition rate not only does not increase compared with Example 1, but instead decreases significantly. This shows that: the silica gel column elution conditions of the present invention are very crucial. If the silica gel column elution conditions are not properly selected, a large amount of active ingredients with tyrosinase activity cannot be enriched; thus resulting in significant differences in the tyrosinase inhibition rates of the prepared natural plant extracts. The above experimental results indicate that after mixing the cactus extract, the ophiopogon japonicus extract, and the sophora flavescens leaf extract evenly to obtain a mixed extract, only the natural plant extract prepared under the silica gel column elution conditions of the present invention can further significantly improve the tyrosinase inhibition activity; while the natural plant extracts prepared under other silica gel column elution conditions cannot further significantly improve the tyrosinase inhibition activity.

[0065] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Preparation method of natural plant extract with whitening effect, characterized in that, take cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract with a weight ratio of 1-3:1-3:1-3, mix them evenly to obtain a mixed extract; The preparation method of the cactus extract comprises the following steps: crush the cactus and mix it evenly with water to obtain a cactus slurry; then add chloroform to the cactus slurry for extraction to obtain a chloroform extract; concentrate and dry the chloroform extract to obtain the cactus extract; The preparation method of the ophiopogon japonicus extract comprises the following steps: extract ophiopogon japonicus with ethyl acetate to obtain an extract, and concentrate and dry the extract to obtain the ophiopogon japonicus extract; The preparation method of the sophora flavescens leaf extract comprises the following steps: extract sophora flavescens leaf with chloroform to obtain an extract, and concentrate and dry the extract to obtain the sophora flavescens leaf extract; Load the mixed extract onto a silica gel column, first elute with 2-4 times the column volume of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:34-36 and discard the eluate; then elute with 3-6 times the column volume of a mixed organic solvent composed of chloroform and methanol with a volume ratio of 100:54-56 and collect the eluate, concentrate and dry the collected eluate to obtain the natural plant extract.

2. The preparation method of the natural plant extract according to claim 1, characterized in that, The weight ratio of cactus extract, ophiopogon japonicus extract and sophora flavescens leaf extract is 1:1:

1.

3. The preparation method of the natural plant extract according to claim 1, characterized in that, The dosage ratio of cactus, water and chloroform is 1 kg:3-5 L:3-5 L.

4. The preparation method of the natural plant extract according to claim 1, characterized in that, The extraction of ophiopogon japonicus and sophora flavescens leaf is carried out by maceration extraction for 1-3 days.

5. The preparation method of the natural plant extract according to claim 4, characterized in that, The dosage ratio of ophiopogon japonicus to ethyl acetate is 1 kg:5-10 L, and the dosage ratio of sophora flavescens leaf to chloroform is 1 kg:6-12 L.

6. The preparation method of the natural plant extract according to claim 5, characterized in that, The dosage ratio of ophiopogon japonicus to ethyl acetate is 1 kg:6 L, and the dosage ratio of sophora flavescens leaf to chloroform is 1 kg:8 L.

7. Application of the natural plant extract prepared by the method according to any one of claims 1-6 in the preparation of skin care products with whitening effect.