CN117180161A

CN117180161A - Willow leaf extract and extraction method and application thereof

Info

Publication number: CN117180161A
Application number: CN202311401209.4A
Authority: CN
Inventors: 张志锋; 赵日杂; 王月
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2023-10-26
Filing date: 2023-10-26
Publication date: 2023-12-08

Abstract

The application discloses a willow leaf extract and an extraction method and application thereof, and belongs to the technical field of plant extraction. According to the application, through adjusting the extraction process, the willow leaf can be fully extracted, the effective components in the willow leaf are fully extracted, and the yield of the extract can be effectively ensured. The extract obtained by the extraction method provided by the application contains various whitening active ingredients, and can effectively relieve oxidative stress and inflammatory reaction, so that pigment deposition is reduced, and further the whitening effect is realized.

Description

Willow leaf extract and extraction method and application thereof

Technical Field

The application belongs to the technical field of plant extraction, and particularly relates to a willow leaf extract and an extraction method and application thereof.

Background

The herba Salicis Babylonicae, also called herba Violae, herba Cymbopogonis Citrari, is dry whole herb of Salicaceae genus Salicaceae plant Salicaceae. The willow leaf is a perennial herb plant, has more than 200 parts worldwide, is mainly produced in the sand of a stream bed, the sunny and wet places of a ditch and a lake side and in the bushes with the altitude of 1000-4000 meters, and is widely distributed in places such as Sichuan, guizhou, yunnan, jilin, liaoning, jiangsu, zhejiang and the like.

The willow leaf is widely applied to folk medicines, such as prostate, urinary tract, bladder and gastrointestinal diseases or inflammations, epilepsy and the like. The whole herb of the flowers, roots and the whole herb with roots of the willow herb can be used as medicines, wherein the whole herb has the effects of clearing heat and detoxicating, promoting diuresis and stopping diarrhea, promoting digestion and regulating qi and activating blood circulation to set bone, and is mainly used for treating fracture, traumatic injury, furuncle and carbuncle swelling and traumatic hemorrhage; flower, bitter, slightly sweet and cool, has the effects of clearing heat and relieving pain, and regulating menstruation and astringing leucorrhea, and is used for treating toothache, acute conjunctivitis, sphagitis, irregular menstruation and excessive leucorrhea; root, bitter and flat, has the effects of regulating qi, removing food retention, promoting blood circulation, relieving pain, removing toxic substance and detumescence, and is used for amenorrhea, gastralgia, food stagnation and distention. Currently, willow leaves are mostly used for hemostasis, menstrual disorder, prostatitis and sleep problems, and in addition, young leaves of willow leaves are often used as food and feed.

The whitening mechanism mainly comprises a plurality of ways of directly whitening and resisting oxidation by reducing melanin content, wherein the ways of inhibiting melanin formation are more common. Melanin is a macromolecule commonly found in the human body, particularly in the eyes, skin and pigment tissues; the density of melanin determines the color of the tissue. Melanin can protect skin from ultraviolet rays and free radicals, but also can cause skin aging and pigmentation, and cause excessive melanin production to cause problems of darkness, color spots and the like of the skin. Skin aging is the result of complex interactions between epidermis, dermis, subcutaneous fat and extracellular substrates, leading to pigmentation, wrinkling, cramping and other activities caused by oxidative stress, and furthermore, skin aging leads to reduced secretion of hormones, leading to reduced activity and function of skin cells. Tyrosinase is an indispensable enzyme in the melanogenesis process. Therefore, compounds having high antioxidant activity are expected to inhibit pigmentation by affecting biosynthesis of melanin and tyrosinase to promote whitening and delay aging.

At present, the whitening products on the market mainly comprise chemical products, biological products and natural plants, wherein the natural plant whitening agent is derived from natural plants, has good safety, rich resources, is easy to obtain and has low cost, so the whitening products become one of the main lines of research in recent years, but no related report of using the willow leaf extract in cosmetics is seen.

Disclosure of Invention

Aiming at the prior art, the application provides a willow leaf extract, an extraction method and application thereof, which are used for solving the technical problem of low extraction rate of active ingredients of willow leaf and developing new application of the willow leaf extract.

In order to achieve the above purpose, the technical scheme adopted by the application is that the application provides an extraction method of a willow leaf extract, which comprises the following steps:

s1: mixing fresh willow leaf with acetic acid solution according to a feed liquid ratio of 1 g:5-10 mL, and pulping to obtain slurry;

s2: mixing the slurry with sodium chloride solution, and carrying out ultrasonic oscillation for 10-20 min to obtain a mixture;

s3: adding a methanol-hydrochloric acid mixed solution into the mixture, heating and refluxing for 30-60 min at 45-100 ℃, filtering, and collecting filtrate;

s4: adding an organic extractant into the filtrate, standing for 30-45 min, centrifuging, collecting an upper organic layer, and concentrating until the volume is not changed, thus obtaining a crude product;

s5: purifying the crude product by using XDA-5 macroporous resin column as purification column and ethanol solution as eluent, collecting eluent, concentrating, and drying.

The beneficial effects of the application adopting the technical scheme are as follows: according to the application, when the willow leaf is extracted, the willow leaf is firstly mixed with acetic acid for pulping, and the acetic acid can not only reduce the pH value of a willow leaf cell system and enable the cell wall to be dissociated to promote the release and extraction of active ingredients, but also promote the stability of the active ingredients such as polyphenol substances and reduce the sensitivity degree and the possibility of activity loss.

After the slurry is obtained, sodium chloride solution is added into the slurry, and the sodium chloride solution can regulate the osmotic pressure of the willow cells, promote water molecules to flow out from the inside of the cells, shrink the cells and release intracellular components, thereby improving the extraction rate of the active ingredients; in addition, sodium chloride can also improve solubility of components such as quercetin, so that the effective components are maximally dissolved in the extraction solvent. After the sodium chloride solution is added, the cell wall structure is influenced by the ultrasonic wave, namely the micro-bubble cracking and the shock wave effect generated by the ultrasonic wave, so that the cell wall is cracked, and the release of target components is facilitated; ultrasound can also increase mass transfer rate of substances in the liquid phase, allowing rapid release of target components from plant tissue into the solvent by increasing convection and diffusion of the liquid phase.

The cell walls of the salix leaf vegetables are basically completely destroyed after the salix leaf vegetables are treated by the acetic acid and sodium chloride solution, and then a methanol-hydrochloric acid mixed solution is added, wherein the hydrochloric acid can change the pH value of the solution, so that the ionic form or molecular structure of the active ingredients is changed, the solubility of the active ingredients is improved, the active ingredients are more soluble in the solvent, and certain unstable compounds can be converted into stable forms, so that the content of the active ingredients is ensured; moreover, the hydrochloric acid can further destroy the cell tissues, which is beneficial to release of the effective components in the cells; the methanol is used as a solvent to better dissolve the active ingredients in the willow leaf, and the active ingredients in the willow leaf are completely dissolved in the methanol in combination with the pretreatment, and then the cosmetic-grade willow leaf extract can be obtained through the purification treatment.

On the basis of the technical scheme, the application can be improved as follows.

Further, the volume concentration of the acetic acid solution is 5 to 10%.

Further, the concentration of the sodium chloride solution is 0.1-0.5 wt%, and the volume ratio of the slurry to the sodium chloride solution is 1:1-3.

Further, the power of ultrasonic oscillation is 200-300W, and the frequency is 25-40 KHz.

Further, the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 3-5:1.

Further, the volume ratio of the mixture to the methanol-hydrochloric acid mixed solution is 1:1-3.

Further, the organic extractant is ethyl acetate, and the filtrate is mixed with ethyl acetate in equal volume.

Further, the XDA-5 macroporous resin column takes XDA-5 macroporous resin as a filler, and the specification is 65 x 100mm; the volume fraction of ethanol solution used for elution was 70%.

The application also discloses the willow leaf extract extracted by the extraction method.

The willow leaf is rich in rutin, apigenin, quercetin and other components, and the components can be effectively extracted by adopting the extraction process. Wherein apigenin is a natural plant flavonoid compound, has an antioxidation effect, can effectively remove free radicals, lighten oxidative stress and inhibit excessive production of melanin; meanwhile, the apigenin has anti-inflammatory property, can reduce the damage of inflammatory reaction to skin, helps to ease and alleviate some pigmentation conditions caused by sun exposure, environmental pollution and the like, and achieves the effect of skin whitening. Rutin also has antioxidant and anti-inflammatory properties, and it also reduces the activity of the maillard cells and inhibits the overproduction of melanin, thereby helping to alleviate some of the mild pigmentation problems. In addition to the antioxidant and anti-inflammatory effects, the quercetin can effectively inhibit the activity of tyrosinase, and can reduce the synthesis of melanin by inhibiting tyrosinase, so that the quercetin is helpful for reducing color spots and brightening skin.

The willow leaf extract extracted by the application contains a plurality of active components with anti-inflammatory and antioxidant effects, and can effectively relieve pigmentation caused by oxidative stress and inflammation, thereby producing good whitening effect.

The application also discloses application of the willow leaf extract in preparing cosmetics. The prepared cosmetic has excellent whitening effect and can be used as a whitening cosmetic.

The beneficial effects of the application are as follows:

1. according to the application, through adjusting the extraction process, the willow leaf can be fully extracted, the effective components in the willow leaf are fully extracted, and the yield of the extract can be effectively ensured.

2. The extract obtained by the extraction method provided by the application contains various whitening active ingredients, and can effectively relieve oxidative stress and inflammatory reaction, so that pigment deposition is reduced, and further the whitening effect is realized.

Detailed Description

The following describes the present application in detail with reference to examples.

Example 1

A willow leaf extract is extracted by the following process:

s1: mixing fresh willow leaves with acetic acid solution with volume concentration of 8% according to the feed liquid ratio of 1g to 8mL, and pulping to obtain slurry;

s2: mixing the slurry with a sodium chloride solution with the concentration of 0.3wt% according to the volume ratio of 1:2, and carrying out ultrasonic oscillation for 15min to obtain a mixture; the power of ultrasonic oscillation is 250W, and the frequency is 30KHz;

s3: adding a methanol-hydrochloric acid mixed solution into the mixture, heating and refluxing for 45min at 60 ℃, filtering, and collecting filtrate; the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 4:1, and the volume ratio of the mixture to the methanol-hydrochloric acid mixed solution is 1:2;

s4: adding an equal volume of ethyl acetate into the filtrate, shaking for 2min, standing for 40min, centrifuging, collecting an upper organic layer, and concentrating until the volume is not changed, thus obtaining a crude product;

s5: purifying the crude product by using XDA-5 macroporous resin column as purification column and 70% ethanol solution as eluent, collecting eluent, concentrating, and drying.

Example 2

A willow leaf extract is extracted by the following process:

s1: mixing fresh willow leaves with acetic acid solution with volume concentration of 5% according to a feed liquid ratio of 1g to 5mL, and pulping to obtain slurry;

s2: mixing the slurry with a sodium chloride solution with the concentration of 0.1wt% according to the volume ratio of 1:1, and carrying out ultrasonic vibration for 20min to obtain a mixture; the power of ultrasonic oscillation is 200W, and the frequency is 25KHz;

s3: adding a methanol-hydrochloric acid mixed solution into the mixture, heating and refluxing at 45 ℃ for 60min, filtering, and collecting filtrate; the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 3:1, and the volume ratio of the mixture to the methanol-hydrochloric acid mixed solution is 1:1;

s4: adding an equal volume of ethyl acetate into the filtrate, shaking for 2min, standing for 30min, centrifuging, collecting an upper organic layer, and concentrating until the volume is not changed, thus obtaining a crude product;

Example 3

A willow leaf extract is extracted by the following process:

s1: mixing fresh willow leaves with acetic acid solution with volume concentration of 10% according to the feed liquid ratio of 1g to 10mL, and pulping to obtain slurry;

s2: mixing the slurry with a sodium chloride solution with the concentration of 0.5wt% according to the volume ratio of 1:3, and carrying out ultrasonic oscillation for 10min to obtain a mixture; the power of ultrasonic oscillation is 300W, and the frequency is 40KHz;

s3: adding a methanol-hydrochloric acid mixed solution into the mixture, heating and refluxing for 30min at 100 ℃, filtering, and collecting filtrate; the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 5:1, and the volume ratio of the mixture to the methanol-hydrochloric acid mixed solution is 1:3;

s4: adding an equal volume of ethyl acetate into the filtrate, shaking for 2min, standing for 45min, centrifuging, collecting an upper organic layer, and concentrating until the volume is not changed, thus obtaining a crude product;

Comparative example 1

A willow leaf extract is extracted by the following process:

s1: mixing fresh willow leaves with sodium chloride solution with concentration of 0.3wt% according to a feed-liquid ratio of 1g to 8mL, and pulping to obtain slurry;

s2: ultrasonically oscillating the slurry for 15min to obtain a mixture; the power of ultrasonic oscillation is 250W, and the frequency is 30KHz;

Comparative example 2

A willow leaf extract is extracted by the following process:

s2: adding a methanol-hydrochloric acid mixed solution into the slurry, heating and refluxing for 45min at 60 ℃, filtering, and collecting filtrate; the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 4:1, and the volume ratio of the slurry to the methanol-hydrochloric acid mixed solution is 1:2;

s3: adding an equal volume of ethyl acetate into the filtrate, shaking for 2min, standing for 40min, centrifuging, collecting an upper organic layer, and concentrating until the volume is not changed, thus obtaining a crude product;

s4: purifying the crude product by using XDA-5 macroporous resin column as purification column and 70% ethanol solution as eluent, collecting eluent, concentrating, and drying.

Comparative example 3

A willow leaf extract is extracted by the following process:

s3: adding methanol solution into the mixture, heating and refluxing at 60 ℃ for 45min, filtering, and collecting filtrate; the volume fraction of the methanol solution is 75%, and the volume ratio of the mixture to the methanol solution is 1:2;

Comparative example 4

A willow leaf extract is extracted by the following process:

s1: mixing fresh willow leaves with water according to a feed liquid ratio of 1g to 8mL, and pulping to obtain slurry;

Experimental example 1: yield of the extract

The yield of the willow herb extract was calculated according to the following formula, and the results are shown in table 1.

TABLE 1 extract yield of willow leaf

	Yield (%)
		Example 1	10.24
Example 2	9.60
		Example 3	10.67
Comparative example 1	7.36
		Comparative example 2	7.80
Comparative example 3	8.28
		Comparative example 4	5.94

As can be seen from table 1, the extraction process according to the present application gave an extract yield of 9.6% or more, whereas the extraction process according to the comparative example gave a reduced extract yield, because:

comparative example 1 compared with example 1, the cell wall of the willow herb was not effectively broken without the acetic acid treatment, and the stability of the effective ingredient was not ensured, resulting in a decrease in the yield of the final extract.

Comparative example 2, compared with example 1, the osmotic pressure of the willow cells could not be adjusted during the extraction without sodium chloride treatment, and the active ingredients in the cells could not be released completely, resulting in a decrease in the yield of the final extract.

Comparative example 3 was compared with example 1, only methanol was added during the heat refluxing, but hydrochloric acid was absent, and the cell wall of the willow leaf was not further damaged, so that the total release of the internal active ingredient was not possible, and the yield of the extract was lowered.

Comparative example 4 compared with example 1, the extract was obtained by extracting the willow leaf with only a mixed solution of methanol and hydrochloric acid, the cell wall was hardly destroyed, and the active ingredient in the cell was hardly released, resulting in a lower yield of the extract.

Experimental example 2: inhibition rate of tyrosinase by extract

1. Preparation of the solution

Preparation of Phosphate Buffered Saline (PBS): accurately weighing 71.6g of disodium hydrogen phosphate and 31.2g of sodium dihydrogen phosphate respectively, adding deionized water for full dissolution, respectively transferring into 1000mL volumetric flask to obtain solutions with constant volume of 0.2moL/L, uniformly mixing at a ratio of 51:49, and regulating pH to 6.8.

Preparing a tyrosine solution: accurately weighing 0.01g tyrosinase in a 100mL volumetric flask, and fixing the volume by using PBS solution to obtain the product.

Preparing a dopamine solution: accurately weighing a certain amount of dopamine, adding a small amount of 0.1moL/L hydrochloric acid for pre-dissolving, preparing 1.5g/L dopamine solution by using PBS solution after complete dissolving, and storing at 4 ℃ in a dark place.

Preparation of sample solution: the extracts obtained in examples and comparative examples were prepared as 0.4g/L sample solutions with PBS solution, respectively.

2. Measurement method

Respectively adding PBS solution, sample solution and tyrosinase solution into each test tube according to the reaction composition in Table 2, mixing, reacting in a constant temperature water bath at 37deg.C for 10min, respectively rapidly adding 1mL dopamine solution, mixing, placing into water bath, reacting for 5min, and immediately measuring A after reacting for 5min _λ＝475 A is measured separately _C1 、A _C2 、A _T1 And A _T2 Group 3 were tested in parallel. The inhibition rate of tyrosinase by the extract was calculated as follows:

inhibition ratio = [1- (A) _T1 -A _T2 /A _C1 -A _C2 )]*100％

Wherein, C1 is blank group, C2 is used for blank zeroing, T1 is sample group, and T2 is used for sample zeroing.

TABLE 2 composition of reaction solution

Reaction set	PBS/mL	Stock solution/mL	tyrosinase/mL
				C1	13		2
C2	15
				T1	11	2	2
T2	13	2

The inhibition rate of tyrosinase by the extract is shown in table 3.

TABLE 3 inhibition of tyrosinase by willow leaf extract

	Inhibition ratio (%)
		Example 1	78.25
Example 2	76.56
		Example 3	80.00
Comparative example 1	67.24
		Comparative example 2	70.31
Comparative example 3	72.44
		Comparative example 4	59.30

As can be seen from Table 3, the extract obtained by the extraction process of the application has a high inhibition rate on tyrosinase, and can remarkably inhibit melanin generation, thereby producing good whitening effect.

The extraction process in the comparative example can not completely extract the active ingredients in the willow leaf, and the content of the active ingredients is low, so that the inhibition rate of tyrosinase is lower than that of the example, and the whitening effect is poor.

Experimental example 3: skin whitening effect

70 healthy subjects (35 men and 35 women) were collected, equally divided into 7 groups, then an opaque adhesive tape with a through hole (diameter of the through hole is 2 cm) was attached to the arm of the subject, and then the additional part of each tester was irradiated with ultraviolet rays at a dose 1.5 times the minimum erythema dose, and skin darkening was induced. After the ultraviolet irradiation, the extracts obtained in examples and comparative examples were applied to the irradiated sites, respectively. And after two months, skin color was measured with a colorimeter (commercially available).

The effect of each extract was judged by determining an "L" value indicating the skin brightness and darkness, which was corrected by a standard plate in the present application, and repeated a plurality of times at one portion to uniformly measure the pigmentation portion. The skin color difference (Δl) between the point at which the application of the extract was started and the point at which the application of the extract was completed was calculated according to the following formula:

Δl=luminance L after two months of experiment-luminance L at the start of experiment

The results are shown in Table 4.

TABLE 4 whitening effect of willow leaf extract

	Whitening effect (DeltaL)
		Example 1	2.95
Example 2	2.74
		Example 3	3.02
Comparative example 1	2.13
		Comparative example 2	2.18
Comparative example 3	2.35
		Comparative example 4	1.55

As can be seen from Table 4, the extracts obtained in the examples have excellent whitening effect and are effective in reducing melanin deposition. The extract obtained in the comparative example also has a certain whitening effect, but the whitening effect is greatly reduced compared with the extract in the examples due to the lower content of the active ingredients.

While specific embodiments of the application have been described in detail in connection with the examples, it should not be construed as limiting the scope of protection of the patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Claims

1. The method for extracting the willow leaf extract is characterized by comprising the following steps of:

2. The extraction method according to claim 1, characterized in that: the volume concentration of the acetic acid solution is 5-10%.

3. The extraction method according to claim 1, characterized in that: the concentration of the sodium chloride solution is 0.1-0.5 wt%, and the volume ratio of the slurry to the sodium chloride solution is 1:1-3.

4. The extraction method according to claim 1, characterized in that: the power of the ultrasonic oscillation is 200-300W, and the frequency is 25-40 KHz.

5. The extraction method according to claim 1, characterized in that: the volume ratio of the methanol to the hydrochloric acid in the methanol-hydrochloric acid mixed solution is 3-5:1.

6. The extraction method according to claim 5, characterized in that: the volume ratio of the mixture to the methanol-hydrochloric acid mixed solution is 1:1-3.

7. The extraction method according to claim 1, characterized in that: the organic extractant is ethyl acetate, and the filtrate is mixed with ethyl acetate in an equal volume.

8. The extract of willow leaf obtained by the extraction method according to any one of claims 1 to 7.

9. Use of the willow leaf extract of claim 8 in the preparation of cosmetics.

10. The use according to claim 9, wherein: the cosmetic is a whitening cosmetic.