CN114028298A

CN114028298A - Preparation method and application of pueraria extract nanosuspension for eye care

Info

Publication number: CN114028298A
Application number: CN202111550426.0A
Authority: CN
Inventors: 赵文英; 朱庆书; 朱焕廷; 刘蓓; 陈蕾
Original assignee: Qingdao University of Science and Technology
Current assignee: Qingdao University of Science and Technology
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-02-11

Abstract

The invention discloses a preparation method of kudzu root extract nanometer suspension for eye care, which comprises the following key steps: pulverizing dried radix Puerariae, sieving, adding deionized water, transferring to a pressure extractor, and extracting for a certain time under heat and pressure; after the extraction is finished, directly spraying the extracting solution into a beaker to obtain the pueraria extract nanometer suspension. The method for extracting the effective components of the kudzuvine root and preparing the nano suspension is completed in one step, and the preparation method is simple, convenient, green and safe. Compared with the radix Puerariae extract prepared by conventional extraction method, the radix Puerariae extract nanometer suspension has good transdermal absorption property and blood circulation promoting and blood stasis removing effects. The nanometer suspension can be directly added into eye care cosmetics such as cream, lotion, facial mask, various aqueous products (essence, nutritious water, and soothing water) or added after freeze drying, and can fully exert the medicinal components of radix Puerariae for improving microcirculation, and prevent or relieve black eye, pouch, and crow's feet.

Description

Preparation method and application of pueraria extract nanosuspension for eye care

The technical field is as follows:

the invention relates to a preparation method of a pueraria extract nanometer suspension and application of the pueraria extract nanometer suspension in eye care cosmetics, and belongs to the field of manufacturing and application of daily chemical raw materials.

Background art:

the eye is the most senescent and problematic site in the human body for the following reasons: firstly, the skin around the eyes is only 0.33-0.36 mm thick (corresponding to 1/3 on the face), and the eyes are the parts with the least distribution of sebaceous glands and sweat glands, so that the skin around the eyes is easy to dry and lack of water, and fine wrinkles are generated; secondly, the blinking frequency of each person reaches 2.4-2.8 thousands of times per day, and the eyes are easy to feel acerb and fatigue; thirdly, the capillary vessels around the eyes are very abundant in distribution, if the eyes are used excessively, poor circulation of the eyes and lymph is caused, and edema can occur due to poor circulation, so that blood can be accumulated in the eyes, and finally, the eyes are deeply disgorged, pain and eye bags are deeply died, dark circles are formed in the eyes, crow's feet and the like are generated. For the first two reasons causing eye aging, we are generally unable to do so, so improving microcirculation, re-waking the activity of fibrous connective tissue cells around the orbit, accelerating the metabolism of redundant fat and water in the eye, and relieving visual fatigue are the most important means for solving various problems of the eye.

Kudzu root is root of Pueraria lobata (willla) ohwi, which is a perennial bean plant, and isoflavone components represented by puerarin are main effective components in kudzu root. Modern pharmacological research considers that the drug effect components of the puerarin tablet have various effects of promoting blood circulation to remove blood stasis, improving microcirculation and the like, for example, the puerarin tablet or injection used clinically has good curative effect on treating diabetic eye diseases, and is widely researched and valued by the medical field. However, the puerarin isoflavone derivatives generally have the characteristics of short half-life period, P-gp efflux function, first-pass metabolic effect of intestinal enzymes and the like, have low oral bioavailability and need large-dose administration; in addition, the development of external transdermal absorption preparations is limited due to the characteristics of low water solubility and poor membrane permeability.

Nanosuspensions (nanosuspensions) are submicron colloidal dispersions of pure drug nanoparticles having a particle size of less than 1 μm, with a small amount of surfactant or other carrier as suspending agent. After the medicine is prepared into the nano suspension, the particle size of the medicine can be reduced, the specific surface area of the medicine can be increased, and the solubility of the insoluble medicine can be increased, so that the bioavailability of the medicine is improved.

The invention content is as follows:

the invention aims to provide a kudzuvine root extract nanosuspension which can prevent or relieve various problems of eyes by improving microcirculation of the eyes. The prepared suspension has high transdermal absorption rate, can effectively enhance the effect of the radix puerariae on improving microcirculation, and has no irritation to skin. Can be used as main ingredient for eye care cosmetic for preventing or relieving black eye, pouch, and crow's feet.

The purpose of the invention is realized by the following technical scheme:

(1) pulverizing dried radix Puerariae, sieving with 40 mesh sieve, and adding deionized water at a ratio of 1: 15-100;

(2) then transferring the mixture to a pressure extractor, pressurizing to 0.5MPa, controlling the temperature at 100 ℃ and 130 ℃, and keeping the temperature and the pressure for 10-60 min;

(3) after the extraction is completed, the extract is directly sprayed into a beaker through a sampling tube. Obtaining the kudzu root extract nanometer suspension.

(4) The obtained nanometer suspension can be directly added into cream, lotion, facial mask, and various aqueous products (essence, nutritious water, and soothing water) or added after freeze drying.

Compared with the prior art, the invention has the beneficial effects that: extracting isoflavone components from radix Puerariae by subcritical water technology, and directly spraying radix Puerariae extractive solution into blank beaker. The extraction and the preparation of the nanoparticles are completed in one step, and the nanosuspension with the particle size within the range of 100-1000nm can be prepared by controlling the factors such as the extraction temperature, the time, the injection speed and the like. The nanometer suspension differs from the traditional nanometer particle in that the traditional nanometer particles such as nanometer liposome, lipid nanometer particle, polymer nanometer capsule, nanometer ball, polymer micelle and the like belong to matrix skeleton type, and are solid colloidal particles formed by wrapping the drug by high molecular polymer, and the nanometer suspension and the traditional nanometer particle have differences in concept and preparation process. In addition, the preparation process for preparing the nano suspension is different from the conventional nano suspension method, and because the kudzu root extract contains the components such as saponin, polysaccharide and the like, a surfactant, a suspending agent and the like are not required to be additionally added, so that the effect components are less influenced by an exogenous additive.

The invention has the main advantages that: the preparation process is simple and the cost is low; the kudzu root extract is added into the cosmetic matrix in a nanoparticle form, so that the skin permeation rate of the active ingredients is high, and the microcirculation improving effect is better.

Description of the drawings:

FIG. 1 is the in vitro transdermal cumulative release rate versus time curve for the nanosuspensions prepared in example 3 (1. extraction by conventional method, 2. subcritical water extraction);

FIG. 2 shows the results of the blood circulation-promoting and blood stasis-removing experiments of the nanosuspensions prepared in example 3 (1. propranolol group, 2. nanosuspension group, 3. water-decoction extract group, 4. physiological saline group).

The specific implementation mode is as follows:

the present invention will be described in further detail with reference to examples. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified. Any method, process, product, etc. that complies with the principles and novel and inventive features disclosed herein and which complies with the claims or the description set forth herein falls within the scope of the present invention.

Example 1

(1) Crushing the dried kudzu root medicinal material, sieving the crushed kudzu root medicinal material by a 40-mesh sieve, and adding deionized water according to the proportion of 1: 50;

(2) then transferring to a pressurizing extractor, pressurizing to 0.5MPa, controlling the temperature at 120 ℃, and keeping the temperature and pressure for 10 min;

Example 2

(2) then transferring to a pressurizing extractor, pressurizing to 0.5MPa, controlling the temperature at 130 ℃, and keeping the temperature and pressure for 20 min;

Example 3

(2) then transferring to a pressurizing extractor, pressurizing to 0.5MPa, controlling the temperature at 120 ℃, and keeping the temperature and pressure for 20 min;

Example 4

(1) Crushing the dried kudzu root medicinal material, sieving the crushed kudzu root medicinal material by a 40-mesh sieve, and adding deionized water according to the proportion of 1: 30;

(2) then transferring to a pressurizing extractor, pressurizing to 0.5MPa, controlling the temperature at 115 ℃, and preserving heat and pressure for 30 min;

Example 5

(2) then transferring to a pressurizing extractor, pressurizing to 0.2MPa, controlling the temperature at 125 ℃, and keeping the temperature and pressure for 40 min;

Example 6

(1) Crushing the dried kudzu root medicinal material, sieving the crushed kudzu root medicinal material by a 40-mesh sieve, and adding deionized water according to the proportion of 1: 15;

Determination of total flavone content in kudzu vine root nano suspension

Measuring the content of flavonoid components in the extract by spectrophotometry. Dissolving puerarin as standard substance in 60% ethanol, diluting to constant volume, and measuring absorbance at 253 nm. The absorbance values were plotted against the concentration, and a standard curve a of 0.0142C +0.0706(R2 of 0.9981) was linearly regressed to calculate the pueraria flavonid content of examples 1-6 according to the standard curve, and the results are shown in table 1.

Particle size determination of kudzu root nano suspension

Examples 1-6 sample particle sizes were measured using a ZS90 malvern laser particle sizer. The results are shown in Table 1.

TABLE 1 EXAMPLES 1-6 AND WATER-DECOCATED EXTRACT FLUORINE SOLUTION FLAVONOID COMPONENT CONTENT AND PARTICULAR DISTRIBUTION

The extraction rate and the particle size distribution are comprehensively evaluated, and the kudzuvine root nano suspension is prepared under the process conditions of the example 3.

Skin allergy test (example 3)

According to a conventional method, 4 adult healthy rabbits are taken, the hair of the ear quilt with the area of 4cm multiplied by 4cm is removed by 8 percent sodium sulfide before the test, and the injury is determined after 24 hours of observation. The treated rabbits were randomly divided into 2 groups. The group 1 is an experimental group (smearing nanometer suspension), the group 2 is a blank group (giving distilled water), and then the skin allergy conditions of the rabbits are observed in 0h, 24h, 48h and 72h respectively.

The experimental result shows that after the drug is administrated, the ear skin of the rabbit has no change such as erythema, edema and the like, and the rabbit has no general allergic reaction such as asthma, unstable standing or shock and the like. The nanosuspension is shown to have no allergic reaction when being applied to the skin.

Acute toxicity test (example 3)

According to a conventional method, 4 adult healthy rabbits are taken, and the hair on two sides of the vertebral column of the rabbit, which is about 3cm multiplied by 3cm, is removed before administration. The treated rabbits were randomly divided into 2 groups, and the experimental group rubbed the skin with sterilized abrasive paper until the skin was damaged by bleeding; the control group was untreated and was intact skin. Two groups of rabbits were treated with the nanosuspensions separately. After 24h the drug was wiped off with warm water, observed, and then the spraying of the same drug was continued for 7 consecutive days. The animals were observed and recorded daily for changes in body weight, skin, hair, eyes and mucous membranes, respiration, limb movements and other toxic manifestations. The results show that the nano-suspension of the kudzu root extract has no irritation to normal intact skin and broken skin of rabbits within 24, 48 and 72 hours after multiple times of skin irritation.

Nanosuspension in vitro transdermal experiments (example 3)

With Na₂S removing the abdominal hair of a healthy mouse, cleaning, cutting off the neck, killing, immediately cutting off the abdominal skin, removing subcutaneous adipose tissues and mucus tissues by a blade, cleaning by normal saline, and storing in a refrigerator for later use.

Nanosuspensions were prepared as in example 3. And taking the rat skin out of the refrigerator, recovering to room temperature, repeatedly washing the rat skin with normal saline, and lightly wiping the rat skin with filter paper. The skin was held between the delivery and receiving chambers using Franz diffusion cells (receiving cell volume 5.3mL, internal diameter 1cm, effective area 0.785cm2) with stratum corneum facing the delivery chamber. Precisely measuring appropriate amount of the nanosuspension, adsorbing the nanosuspension with filter paper (the area is slightly smaller than the area of the supply chamber), and making close contact with skin. 5ml of Phosphate Buffered Saline (PBS) (pH 7.4, L0mmol/L) is added into a receiving chamber, the temperature of a water bath is (32 +/-0.5) DEG C, the constant speed is 300r/min, 5ml of fresh medium is sampled at regular time in lh, 2h, 4h, 6h, 8h, 10h, 12h and 24h respectively and is immediately supplemented back, the filtration is carried out by a 0.45 mu m microporous membrane, and the concentration of the drug in the receiving solution is determined by an ultraviolet-visible spectrophotometry method. The in vitro permeability of the water decoction extract is determined by the same method. The experimental results are shown in figure 1.

Evaluation of the microcirculation improving effect of nanosuspension (example 3)

Taking one bullfrog, breaking the frog brain and spinal cord, fixing the frog on a frog board, cutting a cut at the ventral side, pulling out a section of small intestine, unfolding mesentery, and fixing the mesentery on the frog board by using a pin. Onto the surface of the container, the wilsonii solution is added dropwise to prevent drying. Observing the blood flow condition in arterioles, venules and capillaries under a 100-time microscope, and distinguishing the flow speed, direction and characteristics of the arterioles, venules and capillaries; dripping 0.01% adrenaline on the mesenteric blood vessel, and observing the change of the caliber of the blood vessel and the blood flow speed; after the change, the blood vessel is washed by the wilford liquid, and then the kudzuvine root nanometer suspension is dripped on the mesenteric blood vessel to observe the change of the caliber of the blood vessel and the blood flow speed. The results are shown in Table 2.

Table 2 nanosuspension improving microcirculation experiment results

After the radix puerariae nanometer suspension is added, the blood flow rate in mesentery circulation is accelerated, which shows that the blood circulation can relax blood vessels and promote the microcirculation of frog mesentery.

Evaluation of efficacy of nanosuspension for promoting blood circulation and removing blood stasis (example 3)

Mouse hypoxia tolerance test. 32 mice were divided into four groups, namely a blank control group (physiological saline), a positive control group (propranolol), an administration group 1 (nanosuspension), and an administration group 2 (water decoction extract). Taking adult mice with weight close to that of the adult mice, performing intragastric administration for 0.2ml/10g, respectively putting the adult mice into jars filled with soda lime after administration for 30min, and sealing the jars. The activity of the observed mice is compared, and the time of twitch appearance and survival time of the mice are recorded. The experimental results are shown in figure 2.

Through statistical analysis (t test), the p of the nano suspension group and the p of the normal saline group are less than 0.001, the difference is very obvious, the kudzu root nano suspension has the effect of prolonging the survival time of mice, the effect is close to that of propranolol serving as a positive control medicament, the p of a water decoction extract administration group and the p of the normal saline group is more than 0.1, and the effect of prolonging the survival time of the mice is not obvious.

Claims

1. A preparation method of pueraria extract nanometer suspension is characterized by comprising the following process steps: pulverizing dried radix Puerariae, sieving, adding deionized water at a mass ratio of 1: 10-100, transferring into a pressure extractor under extraction pressure of 0.5MPa, at extraction temperature of 100-.

2. The pueraria lobata extract nanosuspension of claim 1, wherein the nanosuspension is added to a cream, an emulsion, a mask, a water product (a smoothing toner, a nutritive water, a soothing water) or the like for eye care directly or after freeze-drying.