CN109985070A - A kind of Cicada flower extract and its application in the preparation of antioxidant medicine - Google Patents

Abstract

The invention discloses a kind of golden cicada flower extract and its application in anti-oxidation medicine is being prepared, is belonging to pharmaceutical technology field.The property for spending different polysaccharide components different to ethyl alcohol alcohol precipitation concentration using golden cicada, isolates and purifies polysaccharide, provides a kind of method for effectively extracting golden cicada flower polysaccharide component, to find there is the golden cicada flower active constituent of accurate antioxidant activity to provide source.The present invention is isolated from golden cicada flower water extract with the active micromolecular polysaccharide of high-efficiency antioxidant, and the impurity such as partially protein are removed by multiple alcohol precipitation, and the golden cicada flower polysaccharide of different molecular structures is efficiently separated, keep desired polysaccharide purity relatively high, to develop the golden cicada flower polysaccharide for being directed to antioxidant activity, polysaccharide utilization rate is improved.

Description

A kind of Cicada flower extract and its application in the preparation of antioxidant medicine

technical field

The invention belongs to the technical field of medicine, and specifically relates to the preparation of a part with antioxidative activity from the Chinese medicinal material Cicada japonica and the application in the preparation of antioxidative drugs.

Background technique

Antioxidant is the abbreviation of antioxidant free radicals. Studies have shown that cancer, aging or other diseases are mostly related to the production of excess free radicals. Looking for natural and efficient antioxidants is the development trend of modern medicine and health care products.

Cordyceps cicadae (Cordyceps cicadae) is a dry complex formed by the ergot fungus Cordyceps cicadae parasitized after cicada nymphs. . The name of cicada flower was first seen in "Leigong Paozhi Lun", which is the first Cordyceps fungus used as a medicinal. Epilepsy, night crying and heart palpitations... The most amazing effect of medical workers' cloud-based medicine. Modern research shows that Cicada flower has the functions of immune regulation, anti-oxidative aging, sedative and sleeping, relieving gout, invigorating kidney and strengthening kidney, anti-tumor, anti-fatigue and stress, regulating lipid metabolism, etc. Clinically, Cicada flower is used to treat chronic kidney disease. The effect is remarkable. Its active ingredients mainly include polysaccharides, nucleosides, mannitol, ergosterol, etc., and studies have shown that the content of Cordyceps acid in cicadas is about 2.2 times that of Cordyceps sinensis, and the content of adenosine is about 4 times that of Cordyceps sinensis. The total amino acid content is more than twice that of it, vitamin E, folic acid, riboflavin, and B vitamins are all higher than those of Cordyceps sinensis, and the content of heavy metals is much lower than that of Cordyceps sinensis. The clinical study of Cicada by Professor Chen Yiping and others found that it has a significant effect in protecting renal function and delaying chronic renal failure; Song Jiemin and others found that Cicada can significantly increase the level of serum hemolysin and the phagocytic activity of macrophages, indicating that it has The role of promoting immune function; a number of pharmacological studies have shown that cinnabar polysaccharide is a good free radical scavenger or free radical reaction inhibitor, which can achieve its anti-oxidation and anti-aging by improving the hematopoietic function and immune function of organisms effect. Therefore, through the extraction and analysis of the active parts of Cicada flower, it is expected to develop new drugs with high-efficiency antioxidant activity.

The research on the active substances of Cicada flower by modern technology mainly focuses on the extraction of a certain overall active part of Cicada flower. Part, patent CN201410046280.X is for the n-butanol part of the water extract of Cicada flower, patent CN201410232441.4 extracts active substances such as alcohol precipitation crude polysaccharide of the water extract of Cicada flower, and studies its neuroprotective and anti-aging activities However, the above invention is not enough for the separation and refinement of the active parts of Cicada flower, and the effective utilization rate of polysaccharides is not high.

SUMMARY OF THE INVENTION

The key technical problem to be solved by the present invention is to utilize the different properties of different polysaccharide components of Cicada chinensis to ethanol alcohol precipitation, so as to fractionate and purify the polysaccharide, and provide a method for effectively extracting the polysaccharide components of Cicada chinensis. Provides the source of the precise antioxidant activity of Cicada flower active ingredients.

The first aspect of the present invention relates to a polysaccharide extract of Cicada flower and a preparation method thereof. The extract has high purity and high antioxidant activity. The technical scheme of the present invention is as follows: a preparation method of the polysaccharide extract of Cicada flower is carried out according to the following steps:

Step 1) degreasing the Cicada flower medicinal material;

Step 2) Add the degreasing Cicada flower medicinal material, add distilled water for reflux extraction, and concentrate under reduced pressure to obtain a crude extract of Cicada flower polysaccharide.

Step 3) Add 95% ethanol to the crude extract of Cicada flower polysaccharide in step 2), so that the final mass concentration of ethanol reaches 30-40%, and after standing for several hours under low temperature conditions for alcohol precipitation, the obtained precipitation is freed from residual ethanol and then frozen. After drying, the primary alcohol precipitation product CP1 and the primary supernatant CS1 are obtained.

Step 4) Add 95% ethanol to the first-level supernatant CS1 obtained in step 3), so that the final quality of ethanol reaches 40-50% (volume concentration), and repeat the operation of step 3) to obtain the secondary alcohol precipitation product CP2 and secondary alcohol precipitation products. Supernatant CS2.

Step 5) Repeat step 4), successively make the final mass concentration of solution ethanol reach 50%-60% (volume concentration), 60%-70% (volume concentration), 70%-80% (volume concentration), and obtain the golden cicada respectively. The anthocyanidin precipitation products CP3, CP4, CP5.

In the step 1), the degreasing is: drying the medicinal material of Cicada flower, pulverizing it through a No. 2 sieve in the pharmacopoeia, leaching the powder of Cicada flower with petroleum ether, carrying out degreasing treatment, and drying for subsequent use. Preferably, the solid-liquid ratio of Cicada flower to petroleum ether is 1:(8-3).

In the reflux extraction condition in the step 2), the mass ratio of Cicada flower to distilled water is 1:10～15, the extraction time is 2～3 hours, the extraction temperature is 80～100 ℃, the extraction times is 2～4 times, and the extraction time is 2～4 times. Concentrate to 1/10-1/5 of the original volume.

The low temperature conditions in the step 3) are 0°C-10°C, and the time for standing alcohol precipitation is 12-48 hours.

The method for removing residual ethanol by precipitation in the step 3) is to add water to reconstitute the precipitation, and then remove the ethanol solvent by rotary evaporation under reduced pressure until the solution has no obvious ethanol odor.

2. The second aspect of the present invention relates to the use of the graded alcohol-precipitated Cicada flower polysaccharide for the preparation of antioxidant drugs.

In order to detect the antioxidant activity of the polysaccharide product obtained by the invention, each graded polysaccharide obtained by the invention was added to the basal medium of Drosophila at a ratio of 1% (w/v), and fed with physiological senescent fruit Fly and hydrogen peroxide oxidative stress in Drosophila, respectively observe the effect of samples on the lifespan of Drosophila at different doses, and analyze the in vivo malondialdehyde (MDA), catalase (CAT) after feeding physiologically aging Drosophila for 30 days. and glutathione peroxidase (GSH-px) and other peroxidation indicators, the results show that 65%-75% graded alcohol-precipitated Cicada flower polysaccharide has significant antioxidant activity in the in vitro model, and has a certain dose Dependence, indicating that the alcohol-precipitated Cicada polysaccharide has a good potential in the preparation of anti-aging drugs.

3. The third aspect of the present invention relates to a pharmaceutical composition composed of the polysaccharide extract of Cicada japonica and pharmaceutically acceptable excipients.

The active parts of Cicada can be used alone or in combination of several parts, and further combined with auxiliary materials, and the dosage forms include: tablets, capsules, pills, granules, suspensions, drop pills, oral liquid preparations and the like.

The carriers or excipients described in the present invention include conventional carriers and excipients in pharmacy, such as solvents, disintegrating agents, flavoring agents, preservatives, coloring agents, binders and the like.

The following examples and pharmacological activity experiments are further detailed descriptions of the present invention, and the examples listed below are not intended to be limiting in any way.

The invention separates small molecular polysaccharides with high-efficiency antioxidant activity from the water extract of Cicada japonica, and removes some impurities such as proteins through multiple alcohol precipitation, and the polysaccharides of different molecular structures are effectively separated, so that the target polysaccharide can be effectively separated. The purity is relatively high, in order to study the polysaccharide of Cicada flower for antioxidant activity and improve the utilization rate of polysaccharide.

Description of drawings

Fig. 1 Cicada polysaccharide CP5 prolongs the lifespan of physiologically aging Drosophila (n=100). Randomly divided into 4 groups: blank control group, high 1%, medium 0.5% and low 0.1% dose groups of Cicada flower polysaccharide, with 100 animals in each group. The survival of the flies was observed and recorded daily, and the medium was changed every 3 days until all the flies died. Data were analyzed with the Log-rank test.

Figure 2 Cicada cicada CP4 polysaccharide can inhibit the damage caused by oxidative stress of hydrogen peroxide in Drosophila (n=100). Randomly divided into 4 groups: blank control group, high 1%, medium 0.5% and low 0.1% dose groups of Cicada flower polysaccharide, with 100 animals in each group. The survival of the flies was observed and recorded daily, and the medium was changed every 3 days until all the flies died. Data were analyzed with the Log-rank test.

Fig. 3 The effect of Cicada polysaccharide CP4 on the activity of antioxidant enzymes in Drosophila. Randomly divided into 4 groups: blank control group, high 1%, medium 0.5% and low 0.1% dose groups of Cicada flower polysaccharide, with 100 animals in each group. The medium was changed every 3 days, and the content of MDA, GSH-PX and CAT enzyme activity in Drosophila were measured after the 30th day of feeding. Data are presented as mean ± SD. Data were analyzed by ANOVA followed by Dunnett's t-test. Compared with blank control group, *P<0.05, **P<0.01, ***P<0.001. (A) CP4 treatment reduces malondialdehyde (MDA) levels in Drosophila. (B) CP70-60 treatment significantly increased catalase (CAT) levels in Drosophila. (C) CP4 treatment enhanced the activity of glutathione peroxidase (GSH-px) in Drosophila flies in a dose-dependent manner in both female flies, while the middle doses in (B) and (C) were most pronounced in male flies .

Detailed ways

Example 1 The effect of 70% ethanol-fractionated alcohol-precipitated polysaccharide fractions on physiological aging Drosophila:

(1) Preparation of 70% ethanol-graded alcohol-precipitated polysaccharide fraction (CP5) of Cicada flower:

Dried Cicada flower medicinal materials, crushed through the No. 2 sieve of the pharmacopoeia, and extracted the Cicada flower powder with petroleum ether with a liquid-material ratio of 5:1, and carried out degreasing treatment. recycling below. Weigh 50 g of Cicada flower powder and extract it twice with 500 mL of distilled water at 90°C for 2 hours each time. After the extract is concentrated under reduced pressure, add 3/7 times the volume of 95% ethanol to make the final concentration of ethanol. After reaching 30%, it was allowed to stand at 4°C for 12 hours. The polysaccharide after centrifugal precipitation is reconstituted with water, concentrated under reduced pressure until there is no alcohol smell, and freeze-dried to obtain the CP1 fraction of the crude polysaccharide of Cicada japonica. According to the remaining volume of the supernatant, continue to add 1/6 times the volume of absolute ethanol to make the final concentration of ethanol reach 40%. The method is the same, freeze-drying, and obtain 40% ethanol-fractionated alcohol-precipitated polysaccharide fraction (CP2). The final concentration of ethanol was sequentially made to reach 50%, 60%, 70% and 80% to obtain each graded Cicada flower polysaccharide (CP3, CP4, CP5 and CP6) and remaining water fractions. Among them, the polysaccharide CP5 component is the polysaccharide part precipitated by 70% ethanol of Cicada flower.

(2) Cultivation of Drosophila:

Drosophila, wild-type Oregon K melanogaster, was provided by the School of Pharmacy, Jiangsu University. Drosophila were placed in a 50 mL culture flask containing 5 mL of medium in a biochemical incubator (Bongxi Instrument Technology (Shanghai) Co., Ltd.) at 25°C, 60±5% humidity, and 24h day-night alternation. Drosophila medium, a standard corn meal agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China; http://www.hopebiol.com/), stored at room temperature, protected from light and under dry conditions .

(3) Effects of CP5 on physiologically aging Drosophila:

400 male Drosophila flies that emerged within 8 hours were collected. Randomly divided into 4 groups: 0% control group and 1%, 0.5%, 0.1% CP5-treated dose groups, with 100 animals in each group, divided into 4 tubes, with 25 animals in each tube. Drosophila in the control group were fed with basal medium, while those in the CP5 dose group were fed with medium mixed with 70% ethanol-fractionated alcohol-precipitated polysaccharide. Dosing normally, with medium changes every 3 days. The survival of the fruit flies was observed and recorded every two days until all the fruit flies died, and the survival time was observed. Plot lifespan curves and calculate the average lifespan, half time to death, and maximum lifespan of the flies. The longest lifespan was calculated as the average lifespan of the longest surviving 10% of the Drosophila population (Figure 1).

Table 1 Lifespan parameters of physiologically aging Drosophila fed with Cicada polysaccharide CP5

NOTE: Maximum lifespan was calculated as the mean lifespan of the longest surviving 10% of flies in each group (*P<0.05, **P<0.01, ***P<0.001 compared to blank control).

In the Drosophila physiological aging model, it can be intuitively concluded that each dose of the polysaccharide CP5 prolongs the half death time, average lifespan and maximum lifespan of Drosophila. Among them, the middle-dose group had the most significant effect. Compared with the control group, it could prolong the half-death time of fruit flies by 31.03%, the average lifespan by 29.98%, and the maximum lifespan by 25.58%. It shows that CP5 has the effect of prolonging the lifespan of Drosophila (Table 1).

Example 2 The effect of 65% ethanol-graded alcohol-precipitated polysaccharide fractions on H ₂ O ₂ oxidative stress aging Drosophila:

(1) Preparation of 65% ethanol-graded alcohol-precipitated polysaccharide part of Cicada flower:

Dried Cicada flower medicinal materials, crushed through the No. 2 sieve of the pharmacopoeia, leached the Cicada flower powder with petroleum ether with a liquid-material ratio of 8:1, and carried out degreasing treatment. recycling below. Weigh 50 g of Cicada flower powder and extract it twice with 600 mL of distilled water at 80 °C for 3 hours each time. After the extract is concentrated under reduced pressure, 95% ethanol is added to make the final concentration of ethanol reach 35%. Under the condition of ℃, let stand for 24 hours. The polysaccharide after centrifugal precipitation is reconstituted with water, concentrated under reduced pressure until there is no alcohol smell, and freeze-dried to obtain the CP1 fraction of the crude polysaccharide of Cicada japonica. According to the remaining volume of the supernatant, continue to add 1/6 volume of 95% ethanol to make the final concentration of ethanol reach 45%. The method is the same, freeze-drying, and obtain 45% ethanol-precipitated Cicada flower polysaccharide fraction (CP2). The above operations were repeated to make the final concentration of ethanol reach 55%, 65%, and 75% in turn to obtain each graded Cicada flower polysaccharide (CP2, CP3, CP4, CP5) and remaining water fractions. Among them, the polysaccharide CP4 component is the 65% ethanol-graded alcohol-precipitated polysaccharide part of Cicada flower.

(2) Effects of 65% ethanol-graded alcohol-precipitated polysaccharide fractions of Cicada officinalis on H ₂ O ₂ oxidative stress aging Drosophila:

400 male Drosophila flies that emerged within 8 hours were collected. Randomly divided into 4 groups: control group and 1%, 0.5%, 0.1% CP4 dose groups, 100 animals in each group, divided into 4 tubes, 25 animals in each tube. Drosophila in the control group were fed with basal medium, while those in the CP4 dose group were fed with medium supplemented with 65% ethanol-fractionated alcohol-precipitated polysaccharide fractions. On the 30th day of normal administration, the fruit flies were removed, and after starvation adaptation for 2 hours, the fruit flies were transferred to a clean and sterilized culture tube, and a piece of circular filter paper was placed at the bottom of the tube. _{% H2O2} solution infiltration.

The number of dead flies was recorded every 3 h until all flies died. Plot lifespan curves and calculate the mean lifespan, half time to death and maximum lifespan of the flies (Figure 2).

Table 2 The effect of Cicada cicada CP4 polysaccharide on the lifespan of Drosophila acutely injured by H ₂ O ₂ (n=100)

NOTE: Maximum lifespan was calculated as the mean lifespan of the 10% longest surviving flies in each group (*P<0.05, **P<0.01, ***P<0.001 compared to blank control.)

It can be seen from Table 2 and Figure 2 that in the acute oxidative injury of H ₂ O ₂ , the survival time of Drosophila was significantly different between the administration group and the model group. Each dose of Cicada polysaccharide CP4 could prolong the half death time, average lifespan and maximum lifespan of Drosophila flies. Among them, the middle-dose group had the most significant effect. Compared with the control group, it could prolong the half death time of fruit flies by 29.17%, prolong the average lifespan by 28.91%, and prolong the maximum lifespan by 20.0%.

Example 3 The effect of 75% ethanol-fractionated alcohol-precipitated polysaccharide on antioxidant indexes in Drosophila:

(1) Preparation of 75% ethanol-fractionated alcohol-precipitated polysaccharide (CP4):

Dried Cicada flower medicinal materials, crushed through the No. 2 sieve of the pharmacopoeia, extracted the Cicada flower powder with petroleum ether with a liquid-material ratio of 3:1, and carried out degreasing treatment. recycling below. Weigh 50 g of Cicada flower powder, and extract it with 750 mL of distilled water at 100 °C for three times under reflux for 2 hours each time. After the extract is concentrated under reduced pressure, add 1/5 times the volume of anhydrous ethanol to make the final ethanol concentration. 40% under the condition of 10 ℃, let stand for 48h. The polysaccharide after centrifugal precipitation is reconstituted with water, concentrated under reduced pressure until there is no alcohol smell, and freeze-dried to obtain the CP1 fraction of the crude polysaccharide of Cicada japonica. According to the remaining volume of the supernatant, continue to add 1/6 times the volume of absolute ethanol to make the final ethanol concentration reach 50%. The method is the same, freeze-drying, and obtain 50% ethanol-precipitated Cicada flower polysaccharide fraction (CP2). The above operations were repeated to make the final concentration of ethanol reach 65%, 75% and 85% in turn to obtain each graded Cicada flower polysaccharides (CP3, CP4 and CP5) and remaining water fractions. Among them, the polysaccharide CP4 component is 75% ethanol-graded alcohol precipitation polysaccharide of Cicada flower.

(2) Effects of CP4 on antioxidant indexes in Drosophila:

720 male and female Drosophila eclosion within 8 hours were collected. Randomly divided into 4 groups: control group and 1%, 0.5%, 0.1% CP4-treated dose groups, 90 female and 90 male flies in each group, each group is divided into 3 tubes, 30 in each tube, replaced every 3 days a medium. Drosophila in the control group were fed with basal medium, and Drosophila in the CP4 dose group were fed with medium supplemented with 75% ethanol-fractionated alcohol-precipitated polysaccharide from Cicada flower. On the 10th day, 20th day and 30th day, respectively, take one tube of each group of female and male Drosophila. After starvation for 2 hours, add normal saline to grind in ice bath, and centrifuge to obtain Drosophila tissue homogenate. Follow the kit instructions to measure protein content, MDA content, CAT activity and GSH-px content in tissue homogenate (Figure 3).

Table 3 The effect of the polysaccharide CP4 of Cicada flower on the antioxidant capacity of Drosophila

Note: Compared with blank control group, *P<0.05, **P<0.01, ***P<0.001.

It can be seen from the table that after 30 days of feeding with polysaccharide CP4, the content of MDA in the flies in each dose group was significantly lower than that in the control group (P<0.0001); , CAT enzyme activity and GSH-PX content gradually increased, especially in female Drosophila, and the difference between each dose group and the control group was statistically significant (P<0.01 or P<0.01).

The preparation of embodiment 4 Jinchanhua dripping pills

Weigh 400g of polyethylene glycol 4000 respectively, melt it on a water bath, add 70% ethanol of Cicada flower - graded alcohol-precipitated polysaccharide 450g freeze-dried powder, stir evenly, pour it into a heat preservation tube, adjust the constant temperature device, and make the liquid medicine at Drop into the cooled liquid paraffin at 80-90°C (temperature ±4°C), after dripping, pour the pills onto the filter paper to absorb the paraffin oil, then add a small amount of talc powder and mix well to obtain 70% of Cicada flower. 1000 ethanol-graded alcohol-precipitated polysaccharide dropping pills.

The preparation of embodiment 5 Jinchanhua capsules

1000g of lyophilized powder of 70% ethanol of Cicada flower - graded alcohol precipitation polysaccharide, mixed evenly with 500g of medicinal starch, dried, and made into capsules by 0.45g each.

The preparation of embodiment 6 Cicada flower tablet

Cicada flower 70% ethanol - 1000g of freeze-dried powder of graded alcohol precipitation polysaccharide, 500g of starch, mixed evenly, granulated with an appropriate amount of ethanol, granulated by a granulator, and pressed into tablets, each 0.35g.

The preparation of embodiment 7 Jinchanhua granules

Cicada flower 70% ethanol - graded alcohol precipitation polysaccharide freeze-dried powder 1500g, starch 1000g, icing sugar 400g, mixed evenly, granulated with an appropriate amount of ethanol, dried, granulated and packaged.

Claims (7)

1. a preparation method of Cicada flower polysaccharide extract, is characterized in that carrying out according to the following steps: Step 1) degreasing the Cicada flower medicinal material; Step 2) adding distilled water to reflux the degreasing Cicada flower medicinal material, and concentrating under reduced pressure to obtain a crude extract of Cicada flower polysaccharide; Step 3) Add 95% ethanol to the crude extract of Cicada flower polysaccharide in step 2), so that the final concentration of ethanol reaches 30-40% (volume concentration), and after standing for several hours under low temperature conditions, alcohol precipitation is removed. Residual ethanol is then freeze-dried to obtain first-grade alcohol precipitation product CP1 and first-grade supernatant CS1; Step 4) Add 95% ethanol to the primary supernatant CS1 obtained in step 3), so that the final concentration of ethanol reaches 40-50% (volume concentration), and repeat the operation of step 3) to obtain the secondary alcohol precipitation product CP2 and secondary alcohol precipitation products. supernatant CS2; Step 5) Repeat step 4), successively make the final ethanol concentration of the solution reach 50%-60% (volume concentration), 60%-70% (volume concentration), 70%-80% (volume concentration), and obtain Cicada flower respectively Alcohol precipitation products CP3, CP4, CP5. 2. the preparation method of a kind of Cicada flower polysaccharide extract according to claim 1, it is characterized in that described step 1) described degreasing is: dry Cicada flower medicinal material, pulverize the No. 2 sieve of pharmacopoeia, petroleum ether immersion The powder of Cicada flower is extracted, subjected to degreasing treatment, and dried for subsequent use; preferably, the solid-liquid ratio of Cicada flower to petroleum ether is 1:(8-3). 3. the preparation method of a kind of Cicada flower polysaccharide extract according to claim 1, it is characterized in that in described step 2), in the reflux extraction condition, the mass ratio of Cicada flower and distilled water is 1:10～15, extracting The time is 2 to 3 hours, the extraction temperature is 80 to 100° C., the extraction times are 2 to 4 times, and the mixture is concentrated under reduced pressure to 1/10-1/5 of the original volume. 4 . The method for preparing a polysaccharide extract of Cicada flower according to claim 1 , wherein the low temperature condition in the step 3) is 0° C.-10° C., and the standing time for alcohol precipitation is 12 to 48 hours. 5 . 5. the preparation method of a kind of Cicada flower polysaccharide extract according to claim 1, it is characterized in that in described step 3), the method for removing residual ethanol by precipitation is to add water to redissolve the precipitation, and the ethanol solvent is removed by rotary evaporation under reduced pressure , until the solution has no obvious ethanol odor. 6. A use of a polysaccharide extract of Cicada japonica for preparing an antioxidant drug. 7. The pharmaceutical composition composed of the polysaccharide extract of Cicada japonica and pharmaceutically acceptable excipients; the active parts of Cicada japonica can be individually or combined with several parts, and further combined with auxiliary materials, and the dosage forms include: tablets, capsules, pills , granules, suspensions, drop pills, oral liquid preparations.