Preparation method of a bamboo leaf extract, its product and application

Abstract

The present invention relates to a preparation method of a bamboo leaf extract and its product and application. The preparation method comprises the following steps: mixing a bamboo leaf raw material with an ethanol aqueous solution, subjecting the mixture to a microwave-ultrasound combined treatment, and then subjecting the mixture to reflux extraction, concentrating the extract under reduced pressure, purifying and eluting the concentrate with a macroporous resin, subjecting the eluate to a flocculation treatment, and then drying to obtain the bamboo leaf extract. The extract prepared by the preparation method has clear active ingredients, excellent DPPH free radical scavenging ability and prostaglandin and capsaicin receptor inhibition ability, and no pesticide residues, and can be used as an active ingredient of skin care products and medical auxiliary products, and has an anti-oxidation, anti-inflammatory and soothing effect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and relates to a preparation method of a Bambusa chinensis leaf extract, a product and an application thereof. Background Art

[0002] Lophatherum gracile Brongn. is a perennial herbaceous plant of the genus Lophatherum in the Poaceae Barnhart family. It is also known as pheasant rice, golden chicken rice, magic body grass, and bamboo leaf roll heart. It is mainly distributed in Yunnan, Jiangsu, Anhui, Zhejiang, Hunan, Guangdong, Guangxi, Sichuan and other regions in China, India, Myanmar, Malaysia, New Guinea Island and Japan. It mainly grows on hillsides, woodlands or forest edges, and roadside shades below 1200m above sea level. The dried stems and leaves of Lophatherum gracile are traditional Chinese medicinal herbs in my country. They are recorded in Shennong's Herbal Classic and Compendium of Materia Medica. They belong to the herb family. Li Shizhen said: "Pale bamboo leaves are found everywhere in the fields. In spring, the seedlings are several inches tall, with thin stems and green leaves, just like the stems and leaves of thin bamboos grown from bamboo rice falling to the ground. The roots have dozens of whiskers in a nest, and the whiskers produce seeds, just like Ophiopogon japonicus, but they are harder. They can remove irritability and heat, promote urination, and clear the mind." The "Pale bamboo leaves" have been included in all editions of the "Pharmacopoeia of the People's Republic of China" since 1977. It tastes sweet and light, and is cold in nature. It has the effects of clearing heat and purging fire, removing irritability, and promoting urination. It is often used to treat fever, thirst, mouth sores, and painful urination.

[0003] At present, the extraction and purification process of the effective ingredients in the leaves of Bambusa chinensis is mainly water extraction and alcohol extraction. For example, CN103262943A discloses a preparation method of a Bambusa chinensis leaf extract, which comprises taking the stems and leaves of Bambusa chinensis leaf, crushing them, adding an ethanol solution, refluxing extraction, filtering, concentrating, and spray drying to obtain an alcohol-soluble extract of Bambusa chinensis leaf; taking the filter residue, adding distilled water, soaking and extracting, filtering or centrifuging, concentrating, and spray drying to obtain a water-soluble extract of Bambusa chinensis leaf; mixing the obtained alcohol-soluble extract of Bambusa chinensis leaf and the water-soluble extract of Bambusa chinensis leaf in a mass ratio of 1:1-3 to obtain the product; CN102302685A discloses a method for preparing a Bamboo Leaf Extract, wherein the dried Bamboo Leaf material is crushed and then extracted with an ethanol aqueous solution to obtain a crude extract, and then purified by a macroporous adsorption resin column to obtain the Bamboo Leaf Extract; CN102940597A discloses a method for preparing a Bamboo Leaf Extract, wherein the Bamboo Leaf raw material is added to water, heated and refluxed for extraction, filtered to obtain a mother liquor, the mother liquor is vacuum concentrated, centrifuged to remove insoluble matter, the separated liquid is collected, the separated liquid is subjected to alcohol precipitation extraction, filtered and concentrated, and spray dried to obtain the Bamboo Leaf Extract. The above-mentioned Bamboo Leaf Extract has complex components and cannot achieve accurate quality control, thereby making it difficult to ensure its safety and efficacy.

[0004] Therefore, it is of great significance to develop a bamboo leaf extract with clear active ingredients, antioxidant, anti-inflammatory and soothing effects, which can be used in cosmetics and medical auxiliary products. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a Bambusa chinensis leaf extract, a product thereof and an application thereof, and in particular to provide a method for preparing a Bambusa chinensis leaf extract with clear effective ingredients and having antioxidant and anti-inflammatory and soothing effects, a product thereof and an application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a Bambusa chinensis leaf extract, the preparation method comprising the following steps:

[0008] (1) mixing the raw material of bamboo leaves with ethanol aqueous solution and then subjecting it to microwave-ultrasonic combined treatment;

[0009] (2) subjecting the treated solution to reflux extraction to obtain an extract;

[0010] (3) concentrating the extract under reduced pressure to obtain a concentrated solution;

[0011] (4) Purifying and eluting the concentrated solution with a macroporous resin and collecting the eluate;

[0012] (5) The eluate is flocculated and then dried to obtain a Bambusa chinensis leaf extract.

[0013] The preparation method of the bamboo leaf extract involved in the present invention creatively adopts microwave-ultrasonic combined treatment, macroporous resin purification and enrichment, and combines flocculation treatment to effectively remove large polar impurities therein. The prepared extract has clear effective ingredients, excellent DPPH free radical scavenging ability and prostaglandin and capsaicin receptor inhibition ability, and has no pesticide residues. It can be used as an active ingredient in skin care products and medical auxiliary products, and plays an antioxidant, anti-inflammatory and soothing role.

[0014] Preferably, the light bamboo leaf raw material in step (1) is the light bamboo leaf powder obtained by sieving through 70-90 mesh (for example, 70 mesh, 72 mesh, 74 mesh, 77 mesh, 80 mesh, 83 mesh, 86 mesh, 88 mesh, 90 mesh, etc.) after being crushed. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0015] The volume fraction of the ethanol aqueous solution in step (1) is 20%-60%, for example, 20%, 21%, 23%, 26%, 30%, 35%, 40%, 45%, 50%, 60%, etc. Other specific points within this numerical range can be selected and will not be described in detail here.

[0016] The present invention creatively finds that the use of 20%-60% by volume ethanol aqueous solution for reflux extraction can effectively improve the antioxidant and anti-inflammatory soothing abilities of the Bambusa truncatula extract.

[0017] The mass ratio of the ethanol aqueous solution to the light bamboo leaf raw material is (3-15):1, wherein the specific point values ​​in (3-15) can be independently selected from 3, 5, 5.2, 5.4, 5.7, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here one by one.

[0018] Preferably, the temperature of the combined treatment in step (1) is 30-70°C, for example, 30°C, 40°C, 41°C, 42°C, 45°C, 48°C, 50°C, 52°C, 54°C, 56°C, 60°C, 70°C, etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0019] The power of the microwave treatment in the combined treatment is 40-400 W, for example, 40 W, 50 W, 55 W, 60 W, 65 W, 70 W, 75 W, 80 W, 85 W, 90 W, 95 W, 100 W, 150 W, 200 W, 250 W, 300 W, 400 W, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0020] The time of microwave treatment in the combined treatment is 10-30 min, for example, 10 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 30 min, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0021] The power of the ultrasonic treatment in the combined treatment is 50-600 W, for example, 50 W, 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 200 W, 250 W, 300 W, 350 W, 400 W, 450 W, 500 W, 600 W, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0022] The time of ultrasonic treatment in the combined treatment is 3-15 min, for example, 3 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0023] The present invention creatively finds that when microwave-ultrasonic combined treatment is carried out, the use of 40-400 W microwave power and 50-600 W ultrasonic power can effectively improve the antioxidant and anti-inflammatory soothing ability of the light bamboo leaf extract compared to other powers. The increase in microwave power can increase the temperature inside the plant cells and accelerate the rupture of the cell wall. However, too high microwave power will cause the heating speed to be too fast, which will lead to thermal damage or side reactions, thereby affecting the extraction effect; the shorter the time required for the collapse of the cavitation bubble with the increase in ultrasonic power, the stronger the cavitation effect generated by ultrasound per unit time, which is beneficial to the extraction of light bamboo leaves. However, too high ultrasonic power will produce a buffering effect, reduce the rate of energy transfer and cause the cavitation effect to weaken, and may also cause the degradation or isomerization of the effective ingredients. The present invention also finds that microwave treatment for 10-30 min, ultrasonic treatment for 3-15 min, and the temperature of the combined treatment of 30-70 ° C can further improve the antioxidant and anti-inflammatory soothing ability of the light bamboo leaf extract.

[0024] Preferably, the number of reflux extractions in step (2) is 1-3 times, for example, 1 time, 2 times, 3 times, and the time of each extraction is independently 1-3 h, for example, 1 h, 1.2 h, 1.4 h, 1.7 h, 2.0 h, 2.4 h, 2.8 h, 3 h, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0025] Preferably, the reduced pressure concentration in step (3) is carried out at a temperature of 40-70°C (e.g., 40°C, 45°C, 46°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, etc.), and other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0026] The reduced pressure concentration is to 1 / 5-1 / 15 of the extract, for example, 1 / 5, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 12, 1 / 14, 1 / 15, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0027] Preferably, the macroporous resin in step (4) comprises AB-8 or D101 macroporous resin.

[0028] The mass ratio of the macroporous resin to the light bamboo leaf powder raw material is (2-7):1, wherein the specific point values ​​in (2-7) can be independently selected from 2, 3, 3.1, 3.3, 3.6, 4, 4.2, 4.4, 4.6, 5, 7, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0029] The purification and elution steps include: after adsorption, standing for 1-15 hours, first eluting 1-6 BV with water, then eluting 3-10 BV with 3%-25% ethanol aqueous solution, and then eluting 4-12 BV with 60%-95% ethanol aqueous solution.

[0030] The specific elution procedure, i.e., eluting with water first and then with ethanol-water solution, combined with a specific concentration of ethanol-water solution, can effectively improve the antioxidant and anti-inflammatory soothing capabilities of the Bambusa truncatula extract.

[0031] The 1-15 h can be 1 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 15 h, etc. The 1-6 BV can be 1 BV, 2 BV, 3 BV, 4 BV, 5 BV, 6 BV, etc. The 3-10BV can be 3 BV, 4 BV, 5 BV, 6 BV, 7 BV, 8 BV, 10 BV, etc. The 4-12 BV can be 4 BV, 5 BV, 6BV, 7 BV, 8 BV, 9 BV, 10 BV, 12 BV, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0032] The 3%-25% ethanol aqueous solution can select an ethanol aqueous solution with a concentration of 3%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, etc., and the 60%-95% ethanol aqueous solution can select an ethanol aqueous solution with a concentration of 60%, 70%, 75%, 80%, 85%, 90%, 95%, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0033] Preferably, the flocculation treatment in step (5) uses a flocculant, and the amount of the flocculant used is 0.05-1.3% of the eluent, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.7%, 0.8%, 0.9%, 1%, 1.3%, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0034] The flocculant is a combination of gelatin, ZTC1+1 and citric acid.

[0035] The mass ratio of the gelatin, ZTC1+1 and citric acid is 1:(1-5):(1-12), wherein the specific point values ​​in (1-5) can be independently selected from 1, 1.1, 1.3, 1.5, 1.7, 2, 2.3, 2.6, 3, 5, etc., wherein the specific point values ​​in (1-12) can be independently selected from 1, 2, 2.2, 2.4, 2.7, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0036] The present invention also unexpectedly discovered that the antioxidant and anti-inflammatory soothing abilities of the Lophatherum gracile extract are significantly improved after flocculation treatment by using a flocculant composed of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:(1-5):(1-12).

[0037] Preferably, the drying in step (5) comprises freeze-drying.

[0038] The freeze-drying temperature is -90~-40°C, for example, -90°C, -80°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, -40°C, etc. The freeze-drying time is 42-78 h, for example, 42 h, 48 h, 50 h, 52 h, 56 h, 60 h, 64 h, 68 h, 72 h, 78 h, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0039] Other specific point values ​​within the above numerical range can be selected and will not be described in detail here.

[0040] In a second aspect, the present invention provides a Bambusa chinensis leaf extract prepared according to the preparation method of the Bambusa chinensis leaf extract described in the first aspect, wherein the Bambusa chinensis leaf extract has DPPH free radical scavenging effect and prostaglandin and capsaicin receptor inhibitory effect.

[0041] In a third aspect, the present invention provides the use of the Bambusa chinensis leaf extract according to the second aspect in the preparation of antioxidant and / or anti-inflammatory soothing products, in the preparation of prostaglandin inhibitors, and in the preparation of capsaicin receptor inhibitors;

[0042] The products include cosmetics and medical auxiliary products.

[0043] The cosmetics include essence, lotion, emulsion, cream, facial mask or facial cleanser.

[0044] According to the research results of the present invention, the Kochia scoparia seed extract obtained in the above manner has a significant inhibitory effect on prostaglandins. Therefore, the Kochia scoparia seed extract can be used as an in vitro preparation for basic scientific research, such as exploring the mechanism of action of prostaglandin-related physiological activities, rather than being ultimately used for antioxidant and / or anti-inflammatory relief.

[0045] Similarly, according to the research results of the present invention, the Kochia scoparia seed extract obtained in the above manner has a significant inhibitory effect on capsaicin receptors. Therefore, the Kochia scoparia seed extract can be used as an in vitro preparation for basic scientific research, such as exploring the mechanism of action of capsaicin receptor-related physiological activities, rather than being ultimately used for antioxidant and / or anti-inflammatory relief.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The preparation method of the bamboo leaf extract involved in the present invention creatively adopts microwave-ultrasonic combined treatment, macroporous resin purification and enrichment, and combines flocculation treatment to effectively remove large polar impurities therein. The obtained extract has clear effective ingredients, and the total content of glucose isomaltol and monomer compound 1 therein is greater than 20%. It has excellent DPPH free radical scavenging ability and prostaglandin and capsaicin receptor inhibition ability, so it can be used on sensitive skin and eczema skin, and has no pesticide residues. It can be used as an active ingredient in skin care products and medical auxiliary products, and plays an antioxidant, anti-inflammatory and soothing role. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a liquid chromatogram of the Bambusa longituba extract obtained in Example 1;

[0049] Figure 2 is a liquid chromatography comparison chart of the monomer compound 2 in the light bamboo leaf extract prepared in Example 1 and the glucose isomaltol standard;

[0050] Figure 3 is the H NMR spectrum of the monomer compound 1 in the extract of the leaves of the bamboo obtained in Example 1;

[0051] Figure 4 is the carbon NMR spectrum of the monomer compound 1 in the extract of the leaves of the bamboo obtained in Example 1;

[0052] Figure 5 is the nuclear magnetic resonance DEPT 135 spectrum of the monomer compound 1 in the extract of the leaves of the bamboo obtained in Example 1;

[0053] Figure 6 is the H NMR spectrum of the monomer compound 2 in the extract of the leaves of the bamboo obtained in Example 1;

[0054] Figure 7 is the carbon NMR spectrum of the monomer compound 2 in the extract of the leaves of the bamboo obtained in Example 1;

[0055] Figure 8 This is the nuclear magnetic resonance DEPT 135 spectrum of the monomer compound 2 in the extract of the leaves of the loblolly bamboo obtained in Example 1. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0057] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0058] The gelatin involved in the following examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; ZTC1+1 was purchased from Beijing Zhengtiancheng Clarification Technology Co., Ltd.; and chitosan was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0059] Example 1

[0060] This embodiment provides a method for preparing a Bambusa longituba extract:

[0061] (1) Take 50 g of Bambusa chinensis leaves, grind them into 80 mesh fine powder, mix them with 0.5 kg of 30% ethanol aqueous solution, and then carry out microwave-ultrasonic treatment at 50°C, with microwave power of 100 W, microwave time of 20 min, and ultrasonic power of 250 W, ultrasonic time of 8 min. The Bambusa chinensis leaves powder mixture was refluxed and extracted twice, each time for 2 h, and the extracts were combined;

[0062] (2) The combined extract was concentrated to 25 g at 60°C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 220 g of D-101 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined supernatant of the centrifugation was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 2 h. First, 2 BV was eluted with water, then 4 BV was eluted with 10% ethanol aqueous solution, and then 8 BV was eluted with 80% ethanol aqueous solution. The eluate was collected;

[0063] (3) Add 0.5% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:1:5 to the eluent, stir at 50°C for 30 min, let stand and cool, then filter, and freeze-dry at -65°C for 60 h to obtain a Bambusa obesa leaf extract.

[0064] Example 2

[0065] This embodiment provides a method for preparing a Bambusa longituba extract:

[0066] (1) Take 50 g of Bambusa chinensis leaves, grind them into 70 mesh fine powder, mix them with 0.25 kg of 50% ethanol aqueous solution, and then carry out microwave-ultrasonic treatment at 40°C, with microwave power of 300 W, microwave time of 15 min, and ultrasonic power of 100 W, ultrasonic time of 10 min. The treated Bambusa chinensis leaves powder mixture was refluxed and extracted 3 times, each time for 1 h, and the extracts were combined;

[0067] (2) The combined extract was concentrated to 25 g at 45 °C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 150 g of AB-8 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined supernatant of the centrifugation was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 6 h. First, 3 BV was eluted with water, then 8 BV was eluted with 5% ethanol aqueous solution, and then 5 BV was eluted with 90% ethanol aqueous solution. The eluate was collected;

[0068] (3) Add 0.1% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:3:10 to the eluent, stir at 50°C for 30 min, let stand and cool, then filter, and freeze-dry at -50°C for 72 h to obtain a Bambusa obesa leaf extract.

[0069] Example 3

[0070] (1) Take 50 g of Bambusa longituba leaves, grind them into 90 mesh fine powder, mix them with 0.4 kg of 20% ethanol aqueous solution, and then carry out microwave-ultrasonic combined treatment at 60°C, with microwave power of 50 W, microwave time of 25 min, ultrasonic power of 500 W, ultrasonic time of 5 min, and reflux extraction of the Bambusa longituba leaf powder mixture once, each time for 3 h, to obtain an extract;

[0071] (2) The extract was concentrated to 25 g at 65 °C under reduced pressure, and the concentrate was centrifuged at 3000 rpm. The supernatant was set aside. The precipitate was re-dissolved in 30 g of water and centrifuged again. The supernatant obtained by centrifugation was combined with the supernatant of the first centrifugation and set aside. 250 g of D-101 macroporous resin was loaded into the column. After elution with pure water until there was no alcohol smell, the combined supernatant of the centrifugation was taken for sample adsorption at a sample adsorption rate of 0.5 BV / h. After the sample was loaded, it was allowed to stand for 12 h. First, 5 BV was eluted with water, then 3 BV was eluted with 15% ethanol aqueous solution, and then 10 BV was eluted with 70% ethanol aqueous solution. The eluate was collected;

[0072] (3) Add 1% of the solution of a flocculant consisting of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:2:4 to the eluent, stir at 50°C for 30 min, let stand and cool, then filter, and freeze-dry at -80°C for 48 h to obtain a bamboo leaf extract.

[0073] Example 4

[0074] This embodiment provides a method for preparing a Bambusa damicornis leaf extract, which differs from Embodiment 1 only in that the mass fraction of the ethanol aqueous solution in step (1) is 10%, and other conditions remain unchanged.

[0075] Example 5

[0076] This embodiment provides a method for preparing a Bambusa damicornis leaf extract, which differs from Embodiment 1 only in that the mass fraction of the ethanol aqueous solution in step (1) is 70%, and other conditions remain unchanged.

[0077] Example 6

[0078] This embodiment provides a method for preparing a Bambusa chinensis leaf extract, which differs from Embodiment 1 only in that after the sample is loaded in step (2), the sample is allowed to stand for 2 h, 2 BV of the sample is first eluted with water, 4 BV of the sample is then eluted with a 10% ethanol aqueous solution, and 8 BV of the sample is then eluted with a 50% ethanol aqueous solution, and the eluate is collected, while other conditions remain unchanged.

[0079] Example 7

[0080] This embodiment provides a method for preparing a Bambusa chinensis leaf extract, which differs from Embodiment 1 only in that the flocculant in step (3) is replaced by gelatin and ZTC1+1 in a mass ratio of 1:1, and the usage amount is still 0.5%, and other conditions remain unchanged.

[0081] Example 8

[0082] This embodiment provides a method for preparing a Bambusa scabra extract, which differs from Embodiment 1 only in that the flocculant in step (3) is replaced by gelatin and citric acid in a mass ratio of 1:5, and the usage amount is still 0.5%, and other conditions remain unchanged.

[0083] Example 9

[0084] This embodiment provides a method for preparing a Bambusa scabra extract, which differs from Embodiment 1 only in that the flocculant in step (3) is replaced by ZTC1+1 and citric acid in a mass ratio of 1:5, and the usage amount is still 0.5%, and other conditions remain unchanged.

[0085] Example 10

[0086] This embodiment provides a method for preparing a Bambusa chinensis leaf extract, which differs from Embodiment 1 only in that the gelatin and other substances in step (3) are replaced by chitosan, and other conditions remain unchanged.

[0087] Comparative Example 1

[0088] This comparative example provides a method for preparing a Bambusa chinensis leaf extract, which differs from Example 1 only in that in step (1), microwave-ultrasonic combined treatment is not performed, but is replaced by microwave treatment at 50°C 200 W for 20 min, and other conditions remain unchanged.

[0089] Comparative Example 2

[0090] This comparative example provides a method for preparing a Bambusa damicornis leaf extract, which differs from Example 1 only in that in step (1), microwave-ultrasonic combined treatment is not performed, but ultrasonic treatment is performed at 50°C 500 W for 8 min, and other conditions remain unchanged.

[0091] Comparative Example 3

[0092] This comparative example provides a method for preparing a Bambusa chinensis leaf extract, which differs from Example 1 only in that in step (1), no microwave-ultrasonic combined treatment is performed. 50 g of Bambusa chinensis leaf is taken, ground into 80 mesh fine powder, mixed with 0.5 kg of 30% ethanol aqueous solution, and refluxed for extraction twice, each time for 2 h. The extracts are combined, and other conditions remain unchanged.

[0093] Comparative Example 4

[0094] This comparative example provides a method for preparing a Bambusa chinensis leaf extract, which differs from Example 1 only in that no flocculation treatment is performed in step (3), and other conditions remain unchanged.

[0095] Test Example 1

[0096] The active ingredient analysis of the Bambusa chinensis leaf extract obtained in Example 1 was performed using HPLC. The results obtained are as follows: Figure 1 As shown, it can be seen that it contains monomer compound 1, monomer compound 2, flavonoid compounds and polyphenol compounds.

[0097] The extract of the leaves of the bamboo shoots in Example 1 was separated and purified by a high-efficiency preparative liquid phase, and the chromatographic column used for the preparative liquid phase was XBridge Prep, the inner diameter of the chromatographic column was 19 mm, the length of the chromatographic column was 150 mm, the filler of the chromatographic column was OBD C18, and the particle size of the filler was 5 μm. The conditions of the preparative liquid chromatography used were: mobile phase A was water, mobile phase B was acetonitrile, and gradient elution was performed. The elution procedure of the gradient elution is: 0-2 min, mobile phase A: 98-98%, mobile phase B: 2-2%; 2-16 min, mobile phase A: 98-70%, mobile phase B: 2-30%; 16-17 min, mobile phase A: 70-10%, mobile phase B: 30-90%; 17-18 min, mobile phase A: 10-10%, mobile phase B: 90-90%; 18-19 min, mobile phase A: 10-98%, mobile phase B: 90-2%; 19-21min, mobile phase A: 98-98%, mobile phase B: 2-2%. The elution flow rate is 10 mL / min, the detection wavelength is 270 nm, and the preparative chromatographic column temperature is 25°C to obtain monomer compound 1 with a purity greater than 99.0%. The nuclear magnetic resonance structure was identified, and the nuclear magnetic resonance hydrogen spectrum is shown as follows Figure 3 The data are shown below, and the carbon NMR spectrum is shown below. Figure 4 The data are shown below, and the NMR DEPT 135 spectrum is shown below. Figure 5 As shown, the structure of monomer compound 1 is determined to be as shown in formula (I).

[0098] 1 H-NMR (500 MHz, DMSO), δ (ppm): 4.5 (1H, d, j=5.9Hz, H-1), 6.48 (1H, d, j=5.6Hz, H-5), 8.24 (1H, d, j=5.6Hz, H-6), 4.7 (1H, d, j=7.8Hz, H-1'), 3.13(1H, dt,j=2.9,7.8Hz, H-2'), 3.2(1H, m, H-3'), 3.14(1H, m, H-4'), 3.08(1H, m, H-5'), 3.44(1H, m, H-6'), 3.66(2H, dd, j=2.6,11.6Hz, H-6').

[0099] 13C-NMR (125 MHz, DMSO), δ (ppm): 56.29 (C-1), 175.11 (C-2), 162.40 (C-3), 141.12 (C-4), 116.92 (C-5), 156. 58(C-6), 103.33(C-1'), 77.45(C-2'), 76.69(C-3'), 74.22(C-4'), 70.26(C-5'), 61.45(C-6').

[0100] Formula (I)

[0101] High-efficiency preparative liquid phase was selected to separate and purify the monomer compound 2 in the extract of Psoralea corylifolia. The chromatographic column used for the preparative liquid phase was XBridge Prep, the inner diameter of the chromatographic column was 19 mm, the length of the chromatographic column was 150 mm, the filler of the chromatographic column was OBD C18, and the particle size of the filler was 5 μm. The conditions of the preparative liquid chromatography used were: mobile phase A was water, mobile phase B was acetonitrile, and gradient elution was performed. The process of the gradient program is: 0-2 min, mobile phase A: 98-98%, mobile phase B: 2-2%; 2-16 min, mobile phase A: 98-70%, mobile phase B: 2-30%; 16-17 min, mobile phase A: 70-10%, mobile phase B: 30-90%; 17-18 min, mobile phase A: 10-10%, mobile phase B: 90-90%; 18-19 min, mobile phase A: 10-98%, mobile phase B: 90-2%; 19-21 min, mobile phase A: 98-98%, mobile phase B: 2-2%. The elution flow rate is 10 mL / min, the detection wavelength is 270 nm, and the preparative chromatographic column temperature is 25 ° C. Monomer compound 2 is obtained with a purity greater than 99.0%. The nuclear magnetic resonance structure is identified, and the nuclear magnetic resonance hydrogen spectrum is as shown Figure 6 The data are shown below, and the carbon NMR spectrum is shown below. Figure 7 The data are shown below, and the NMR DEPT 135 spectrum is shown below. Figure 8 As shown, the monomer compound 2 is determined to be glucose isomaltol, wherein Figure 2 This is a HPLC comparison chart of monomer compound 2 in the extract of light bamboo leaves and glucose isomaltol standard.

[0102] 1H-NMR (500 MHz, DMSO), δ (ppm): 2.37 (3H, s, H-1), 6.44 (1H, d, j=5.6Hz, H-5), 8.14 (1H, d, j=5.6Hz, H-6), 4.74 (1H, d, j=7.8Hz, H-1'), 3.11 (1H, m, H-2'), 3.18(1H, m, H-3'), 3.15(1H, m, H-4'), 3.09(1H, m, H-5'), 3.43(1H, m, H-6'), 3.63(2H, dd, j=3.8, 11.3Hz, H-6').

[0103] 13 C-NMR (125 MHz, DMSO), δ (ppm): 15.72(C-1), 174.70(C-2), 161.82(C-3), 142.26(C-4), 116.69(C-5), 156. 17(C-6), 104.11(C-1'), 77.80(C-2'), 76.84(C-3'), 74.42(C-4'), 70.13(C-5'), 61.40(C-6').

[0104] The active ingredient analysis of the Lophatherum gracile extracts obtained in Examples 1-3 and Comparative Examples 1-4 was performed using HPLC, and the contents of the monomer compound 1 and the monomer compound 2 were as shown in Table 1.

[0105] Table 1

[0106]

[0107] It can be seen from the data in Table 1 that the extract of the leaves of the loblolly bamboo obtained by the specific extraction process of the present invention is rich in monomer compound 1 and monomer compound 2, and the sum of the contents of the two compounds is greater than 20%.

[0108] By comparing Example 1 with Comparative Examples 1, 2, and 3, it can be found that the ultrasonic step before extraction can significantly increase the content of the two compounds, and the microwave step before extraction can more significantly increase the content of the two compounds; by comparing Example 1 with Comparative Example 4, it can be found that the specific flocculation step can also increase the content of the two compounds.

[0109] Test Example 2

[0110] The antioxidant capacity of the extracts of Bambusa longituba obtained in Examples 1-10 and Comparative Examples 1-4 was tested:

[0111] The DPPH free radical scavenging effect of the extract of Bambusa chinensis leaves was determined with vitamin C as the control. Vitamin C and Bambusa chinensis leaves extract were prepared into solutions with concentrations of 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.0312 mg / mL, and 0.0156 mg / mL, respectively, and 1 mL of each was taken for use. After DPPH was prepared into a 0.51 mg / mL solution, 1 mL was added to the test sample and the control sample respectively. The absorbance was measured after 30 minutes of reaction, and a curve was drawn based on the absorbance and sample concentration.

[0112] Under this condition, the IC values ​​of vitamin C and bamboo leaf extract on DPPH free radical scavenging were 50 The values ​​are shown in Table 2.

[0113] Table 2

[0114]

[0115] It can be seen from the data in Table 2 that the extract of the leaves of the loblolly tree obtained by the specific extraction process of the present invention has a significantly excellent DPPH free radical scavenging ability, that is, an antioxidant effect.

[0116] By comparing Example 1 with Examples 4-5, it can be found that the concentration of ethanol during reflux extraction will have a significant effect on the antioxidant capacity of the extract; by comparing Example 1 with Example 6, it can be found that the concentration of ethanol during elution using a macroporous resin will also affect the antioxidant capacity of the extract; by comparing Example 1 with Examples 7-10, it can be found that gelatin, ZTC1+1 and citric acid as flocculants play a synergistic role, and at the same time, a specific flocculant combination can effectively improve the antioxidant capacity of the extract; by comparing Example 1 with Comparative Examples 1-2, it can be found that microwaves and ultrasound play a synergistic role in the treatment of light bamboo leaf raw materials; by comparing Example 1 with Comparative Example 3, it can be found that microwave-ultrasound combined treatment of light bamboo leaf raw materials significantly improves the antioxidant capacity of the extract; by comparing Example 1 with Comparative Example 4, it can be found that flocculation treatment significantly improves the antioxidant capacity of the extract.

[0117] Test Example 3

[0118] The prostaglandin inhibition test and capsaicin receptor inhibition test were performed on the Bambusa chinensis leaf extracts obtained in Examples 1-10 and Comparative Examples 1-4:

[0119] (1) Test of inhibition of prostaglandins

[0120] A keratinocyte model was selected to measure the inhibitory effect of the extract of Bambusa chinensis leaves on prostaglandins (PGE2). Keratinocytes were selected, and after the cells were revived, the cells were inoculated into 6-well plates when the plating rate reached about 60%, and incubated overnight in a CO2 incubator. When the cell plating rate in the 6-well plate reached 30%-50%, Bambusa chinensis leaves extract (concentration 0.01875 mg / mL) and positive drug dexamethasone (concentration 0.01%) were added to the cells to administer and stimulate the cells. After the administration was completed, the 6-well plates were cultured for 24 hours. The remaining test groups except the blank control group were irradiated with UVB, and the irradiation dose was 300 mJ / cm 2 After washing the cells with PBS, place the 6-well plate in CO 2 After incubation in the incubator for 24 h, the cell culture supernatant was collected and monitored and analyzed according to the operating instructions of the ELISA detection kit. The results are shown in Table 3.

[0121] (2) Test of the inhibitory effect on capsaicin receptor

[0122] A keratinocyte model was selected to measure the inhibitory effect of the extract of the leaves of the bamboo on the capsaicin receptor (TRPV1). Keratinocytes were selected, and after the cells were revived, the cells were inoculated into 24-well plates until the plating rate reached about 60%, and incubated overnight in CO2 culture. When the cell plating rate in the 24-well plate reached 40%-60%, 0.0031 mg / mL of the extract of the leaves of the bamboo and 15.6 μg / ml of the positive drug trans-4-tert-butyl cyclohexanol were added to the cells for administration and stimulation. After the administration, the 24-well plates were cultured for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min, and the immunofluorescence test of TRPV1 was performed. The photos were taken under a microscope for observation. The results are shown in Table 3.

[0123] Table 3

[0124]

[0125]

[0126] It can be seen from the data in Table 3 that the Bambusa truncatula extract prepared by the specific extraction process of the present invention has a significantly excellent effect of inhibiting prostaglandin and capsaicin receptors, and can be used to prepare anti-inflammatory and soothing products.

[0127] By comparing Example 1 with Examples 4-5, it can be found that the concentration of ethanol during reflux extraction will have a significant effect on the anti-inflammatory and soothing ability of the extract; by comparing Example 1 with Example 6, it can be found that the concentration of ethanol during elution using a macroporous resin will also affect the anti-inflammatory and soothing ability of the extract; by comparing Example 1 with Examples 7-10, it can be found that gelatin, ZTC1+1 and citric acid as flocculants play a synergistic role, and at the same time, a specific flocculant combination can also effectively improve the anti-inflammatory and soothing ability of the extract; by comparing Example 1 with Comparative Examples 1-2, it can be found that microwaves and ultrasound play a synergistic role in the treatment of the light bamboo leaf raw material; by comparing Example 1 with Comparative Example 3, it can be found that the microwave-ultrasound combined treatment of the light bamboo leaf raw material significantly improves the anti-inflammatory and soothing ability of the extract; by comparing Example 1 with Comparative Example 4, it can be found that flocculation treatment significantly improves the anti-inflammatory and soothing ability of the extract.

[0128] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a Bambusa chinensis leaf extract, characterized in that: The preparation method comprises the following steps: (1) Mixing the raw material of bamboo leaves with ethanol aqueous solution and then subjecting it to microwave-ultrasonic combined treatment; (2) refluxing the treated solution to obtain an extract; (3) concentrating the extract under reduced pressure to obtain a concentrated solution; (4) Purifying and eluting the concentrated solution with a macroporous resin and collecting the eluate; (5) flocculating the eluate and then drying it to obtain a Bambusa chinensis leaf extract; The volume fraction of the ethanol aqueous solution in step (1) is 20%-60%; the mass ratio of the ethanol aqueous solution to the light bamboo leaf powder raw material is (3-15):1; the temperature of the combined treatment is 30-70°C; the power of the microwave treatment is 40-400 W, and the time of the microwave treatment is 10-30 min; the power of the ultrasonic treatment is 50-600 W, and the time of the ultrasonic treatment is 3-15 min; The number of reflux extractions in step (2) is 1-3 times, and the time of each extraction is independently 1-3 hours; The macroporous resin in step (4) comprises AB-8 or D101 macroporous resin; the mass ratio of the macroporous resin to the light bamboo leaf powder raw material is (2-7):1; the purification and elution steps comprise: after the adsorption is completed, standing for 1-15 hours, first eluting 1-6 BV with water, then eluting 3-10 BV with 3%-25% ethanol aqueous solution, and then eluting 4-12 BV with 60%-95% ethanol aqueous solution; The flocculation treatment in step (5) uses a flocculant, and the amount of the flocculant used is 0.5%-1.3% of the eluent; the flocculant is composed of gelatin, ZTC1+1 and citric acid in a mass ratio of 1:(1-5):(1-12).

2. The method for preparing the extract of Bambusa obesa L. according to claim 1, characterized in that: The light bamboo leaf raw material in step (1) is the light bamboo leaf powder obtained by crushing and then passing through a 70-90 mesh sieve.

3. The method for preparing the extract of Bambusa obesa L. according to claim 1, characterized in that: The reduced pressure concentration in step (3) is carried out at 40-70°C.

4. The method for preparing the extract of Bambusa obesa L. according to claim 1, characterized in that: The drying in step (5) includes freeze drying; The freeze-drying temperature is -90 to -40°C, and the freeze-drying time is 42 to 78 hours.

5. A Bambusa longituba extract, characterized in that The Bambusa damifolia extract is prepared according to the preparation method of the Bambusa damifolia extract according to any one of claims 1 to 4, and the Bambusa damifolia extract has DPPH free radical scavenging effect and prostaglandin and capsaicin receptor inhibitory effect.

6. Use of the extract of Bambusa longituba leaves according to claim 5 in the preparation of antioxidant and / or anti-inflammatory soothing products; The products include cosmetics or medical auxiliary products; The cosmetics include essence, lotion, emulsion, cream, facial mask or facial cleanser.

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