KR20150014033A - Food composition for liver activity contaning Water extract of Cordyceps militaris and its manufacturing method - Google Patents

Abstract

The present invention relates to an inhibition effect of a water extract of Cordyceps militaris (CMWE) which is a novel anti-cancer functional substance on hepatocellular carcinoma cells, which is investigated through various experiments. The present invention provides biopharmaceuticals as antitumor agents. A method for preparing the CMWE comprises the steps of: extracting and purifying a functional substance from Cordyceps militaris; cultivating human hepatoma cell line (HepG2) and human liver cell line (Chang) which is normal cells; treating the human liver cell line with hepatotoxicity inducing substance, cultivating the same, and measuring cell viability; analyzing using high performance liquid chromatography (HPLC); and analyzing using flow cytometry and performing western blotting to investigate biochemical mechanisms of protection effects of CMWE on liver cell damages.

Description

TECHNICAL FIELD The present invention relates to a food composition for improving liver function and a method for producing the same,

       The present invention relates to the efficacy of water extracts that enhance liver function from the pupae of Cordyceps sinensis.

The pupa of Cordyceps is very diverse, among which the cocoon, the large pupa, is found in the large pupa of the dead cocoon in the ground and forms several stools. The size of the host is 28 ~ 35mm X 21 ~ 26mm, and the size of 35 ~ 45mm is orange. The club head is divided into a dark orange color, a head of 20 ~ 30mm in size and a bag of 18 ~ 12mm in size to support it. The boundary is clear. The hair is densely distributed in the semitendinous shape, and the size is 590 ~ 550um X 320 ~ 350um. The acorns are 390 ~ 425 μm, and there are thread-shaped aspergillus spores. The ascospores are composed of a large number of cells separated by the septum and develop into a circular secondary spore and germinate.

It is a kind of medicinal mushroom that grows into a living insect body at the time of high temperature and humidity mainly, and kills the host insect and grows a fruiting body on the insect epidermis. The generation of the Chinese cabbage fungus begins with the season when the temperature and humidity become high, especially when the spore of the Chinese cabbage fungus is scattered in the summer and it attaches to the body of living insects (egg, larva, pupa, adult fish) and enters the respiratory, digestive, The spores attached to the smooth skin, which is easy to grasp, take out the germ tube and enter into the body of the insect and take up the nutrients, and many mycelium grows, so the host insect is killed and becomes like a mummy. Dead insects are filled with white sclerotia, and insects with mummies form insects in the body of dead insects when exposed to moderate temperature and humidity after wintering while keeping the insect form intact. By repeating this process, Cordyceps is continuing to produce and propagate. The length of the mushroom is more than a few millimeters to 10 cm depending on the type of Cordyceps, and the color of the mushroom is also very yellow.

The pupa of Cordyceps sinensis is composed of 10.84% moisture, 8.4% fat, 25.32% crude protein, 28.9% carbohydrate and 4.1% ash, and the fat content is 13% saturated fatty acid and 82.2% unsaturated fatty acid. Vitamin B12 contains 0.29mg per 100g.

Cordycepin, an active ingredient in the pupae Cordyceps, is 3'-deoxyadenosine, found in Cordyceps sinensis in 1950 and is found only in Cordyceps militaris and Cordyceps sinensis, It is known that there is anti-cancer, anti-inflammation and antibacterial activity.

In addition, polysaccharides (anticancer, anti-inflammatory, antioxidant, immunity enhancer) and ergosterol (anticancer) are known to contain.

In addition, Cordisepic acid, which contains about 7% of cordyceps, is a substance called D-mannitol, which enters the blood and purifies the head while reducing the internal pressure of the head and diuretic action. Polysaccharides play a very important role in defending the human body from diseases. They increase immunity, protect the heart and liver, inhibit cancer, and contribute to anti-aging. Cordyceps is a precious, long-lived and long-lasting mysterious drug in the literature, which protects the lungs, strengthens the kidneys, prevents hemostasis, coolens the follicles and stops coughing, enhances the natural healing power like immunity and inflammation suppression. .

Thus, Cordyceps militaris has a wide variety of pharmacological actions. Cordyceps is shown to have good therapeutic power against cold, chronic cough, asthma, seizures, anemia, weakness, male sexual dysfunction and hypertension by enhancing physical strength and shows excellent effect for fatigue recovery .

In addition, it has antimicrobial action and has an inhibitory action against Mycobacterium tuberculosis, Streptococcus pneumoniae, Low hemorrhagic septicemia bacillus, Gypsum gypsum bacterium, Ganoderma lucidum, and Bacillus thuringiensis.

 Among the pharmacological actions of Cordyceps, one of the interesting things is anticancer action. Recently, the results of joint research between Tohoku University and Toho University of Pharmacy in Japan show that Cordyceps sinensis has an anticancer effect. According to this report, it is reported that the number of cancer cells after injection of Cordyceps (Bee Cordyhakho) extract once every day for 5 days is decreased to 24 in the stomach of cancer mouse (experimental rat). In other words, it shows that Cordyceps sinensis inhibits the proliferation of cancer cells by about 76%.

 Among the pharmacological effects of Chinese caterpillar fungus, the anti-cancer component of the seeds is produced during the growth of Chinese caterpillar fungus, and it has no side effects at all and shows excellent resistance against bacterial or viral infection. It is a breakthrough discovery for cancer patients because it means suppression of cancer cells themselves and suppression of reproductive rate.

Recently, in Korea, RDA and SNU research institute jointly investigated pharmacological effects, and it was found out that the anticancer effect, immunity enhancement and anti-fatigue action were excellent. The survival days of the mice were 36 days, which was 202.6% longer than that of the control group (17.8 days). The anticancer effect was 1.5 times higher than that of cresstine, which is currently being used as an anticancer drug in clinical practice. In addition, the rate of cancer cell suppression in solid cancer-induced rats was 84%. The extracts of Cordyceps mellifera were administered in the abdominal cavity of rats for 3 days, and the results of immune enhancement were found to be very strong. In particular, it showed about two times stronger immunity enhancement than Zymosan used as an immunostimulating or anticancer drug. According to a recent report, anticancer effects among various substances contained in natural products have been proved by many studies and experiments. Natural products are more reliable and reliable materials due to the substances obtained from nature, not artificial to modern people. In recent years, the use of natural materials has increased in all aspects of medicine, as well as food and cosmetics.

        Korean Patent Laid-Open Publication No. 10-2013-0040655 relates to a novel use of insect glycosaminoglycan, and more particularly relates to a composition for prevention and treatment of fatty liver comprising an insect glycosaminoglycan derived from an insect as an active ingredient, The present invention relates to a composition for preventing or treating hyperlipemia, and the insect glycosaminoglycan can be used for the purpose of prevention or treatment / improvement of fatty liver or hyperlipemia. It has been disclosed that glycosaminoglycan exhibits an effect that can be advantageously used in the development of a therapeutic agent for a new fatty liver or hyperlipemia.

However, none of the above-mentioned patent documents suggest or suggest that the water extract of Cordyceps sinensis extracts contributes to protection of liver function.

      Accordingly, an object of the present invention is to extract and purify a functional substance that enhances liver function from a pupa of Cordyceps sinensis.

Another object of the present invention is to provide a liver protecting effect of Water extract of Cordyceps militaris (CMWE).

The above object of the present invention is achieved by a method for extracting and purifying a functional substance from a pupa of Cordyceps sinensis; Culturing HepG2 (Human hepatoma cell line) and Chang (Human liver cell line) which are normal cells; Measuring cell viability by treating human normal cells with a hepatotoxic agent; High performance liquid chromatography (HPLC); Flow cytometry analysis and Western blotting were performed to identify the biochemical mechanism of protective effect against hepatocyte injury.

The water extract of Cordyceps militaris (CMWE) according to the present invention is an extremely useful invention in the biopharmaceutical industry because it has an excellent effect of inhibiting liver cancer cells and protecting the liver.

FIG. 1 is a graph showing the characteristics of the CMWE extract of Cordyceps sinensis extract by HPLC.
FIG. 2 is a graph showing the content of cordycepin and adenosine contained in CMWE extracted from the pupa of Cordyceps sinensis.
Figure 3 is a graph showing the standard curves of pure adenosine and cordycepin.
4 is a graph showing the effect of CMWE on Chang cell (normal).
5 is a graph showing the effect of CMWE on HepG2 (cancer cell).
FIG. 6 is a graph showing damage of hepatocyte (Chang cell) according to various SNP (sodium nitroprusside) concentrations.
7 is a graph showing damage rates of hepatocytes (Chang cells) according to various SNP (sodium nitroprusside) concentrations.
8 is a graph showing damage rates of hepatocytes (Chang cells) at a SNP of 600 uM.
FIG. 9 is a graph showing damage of hepatocyte (Chang cell) according to various ethanol concentrations.
FIG. 10 is a graph showing the calculation results of hepatocyte injury rate according to various SNP concentrations. FIG.
11 is a graph showing damage rates of hepatocytes (Chang cells) at 500 uM ethanol.
12 is a graph showing damage of liver cells according to various AAP (Acetaminophen) concentrations.
FIG. 13 is a graph showing damage rates of hepatocytes (Chang cells) according to various AAP (Acetaminophen) concentrations.
14 is a graph showing damage rates of hepatocytes (Chang cells) in 7.5 uM AAP.
15 is a graph showing damage rates of hepatocyte (Chang cell) in AAP of 15 uM.
16 is a graph showing the effect of CMWE on Chang cell (normal).
17 is a graph showing the protective effect of CMWE when Chang cell (normal) is treated with an inhibitor AAP.
18 is a graph showing the protective effect of CMWE when the inhibitor SNP is treated in Chang cell (normal).
19 is a graph showing the results of analysis of flow cytometry using a propidium iodide staining method in a Chang liver cell treated with a pupa extract (CMWE) and 7 mM AAP.
20 is a graph showing the effect of the extract of Cordyceps sinensis extract on cell cycle of Chang liver cells treated with AAP.
21 is a graph showing the effect of the pupae extract (CMWE) on the activity of PARP.
FIG. 22 is a graph showing the effect of the pupae extract (CMWE) on ERK activity.
23 is a graph showing the effect of the pupae extract (CMWE) on the activity of JNK and p38.

Hereinafter, the configuration of the present invention will be described in more detail by way of examples,

It is to be understood that the scope of the present invention is not limited to these embodiments.

< Example  1> From Pupae Cordyceps  Extraction and purification of functional materials

100 g of the dried pupa of Cordyceps sinensis was added to 1 L of distilled water, ground by ultrasonication, and boiled at 100 ° C for 3 hours. The extract was filtered through a Whatman 2 filter paper, and then the volume was reduced to 1 L in a rotary evaporator (52 ° C.). The extract was mixed with 2 × volume of cold 94% alcohol (-20 ° C.), left at -20 ° C. overnight, And then lyophilized in 200 mL of distilled water (CMWE).

As shown in FIG. 1, when HPLC was used at 260 nm, several peaks were observed in 8 to 10 minutes. Therefore, it was confirmed that Polar was contained in a considerable amount, and as a result of HPLC analysis, CMWE exhibited physiological activity It was confirmed that it contains Cordycepin, a substance.

In addition, the structural characteristics of CMWE using HPLC, and the presence of cordycepin and adenosine in CMWE were examined. As a result, CMWE was found to contain both cordycepin and adenosine because several peaks appeared at 3 to 10 minutes (FIG. 2) . The contents of cordycepin and adenosine contained in the CMWE are shown in Table 1.

[Table 1]

< Example  2> Human liver cancer cells HepG2 ( Human hepatoma cell line ) And normal cells Chang ( Human liver cell line ) Culture

HepG2 (human hepatoma cell line) and Chang (human liver cell line), which are normal cells, were purchased from Korean Cell Line Bank. Cell culture was performed in DMEM (Dulbecco's modified Eagle's Medium) supplemented with 10% fetal bovine serum (HyClone Labs, Logan, UT) , 100units / mL penicilling, 100ug / mL into the streptomycin 37 ℃, 5% CO ₂ incubator.

< Example  3> Cell Viability  - MTT assay

Normal human hepatocyte Chang cell line is seeded with 1 × 10 ⁴ cells per well on a 96-well plate using DMEM supplemented with 10% FBS, and starvation is performed with 1% FBS after 24 h.

After 24 h, 1% FBS was added, and CMWE was treated at 0, 12.5, 25, 50, 100 ㎍ / ㎖ and cultured for 30 min. Sodium Nitroprusside (SNP) 600μM and Acetaminophen (AAP) 15mMf are treated respectively.

After incubation, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide) reagent was added to the final concentration and the formazan dye formed was dissolved in 2-propanol . Cell viability was measured by measuring absorbance at 570 nm.

< Experimental Example 1 > Normal hepatocytes ( Normal cell )sign Chang cell In Pupae Cordyceps  extract CMWE Effect measurement

       In order to investigate the effect of CMWE on the normal cell, Chang cells, pupae Cordyceps sinensis extract (CMWE) was cultured in normal cells at 100, 200, 400, 800, and 1000 ug / Chang cells to measure the effect of CMWE.

As shown in FIG. 4, when cultured for 48 hours and 72 hours, it was found that CMWE had no effect on normal cells, and CMWE showed no inhibitory effect on normal hepatocytes. Therefore, it was confirmed that normal cells were protected at 800 and 1000 ug / ml.

In addition, cell viability was slightly lower in the 72 hr culture than in the 48 hr culture.

< Experimental Example  2> Pupae Cordyceps  extract CMWE Of liver cancer cells HepG2 - cell ) Inhibition effect measurement

The concentration of CMWE was 100, 200, 400, 800, and 1000 ug / mL in HepG2 cells. As a result, the viability of the cancer cells decreased in 48 hours and 72 hours , And it was confirmed that the addition of CMWE concentration of 400 ug / ml or more was effective in HepG2 cell as a hepatic cancer cell (Fig. 5).

      < Experimental Example  3> normal cells by inhibitor ( Chang cell ) And the IC50 of each inhibitor and Pupae Cordyceps  extract CMWE Measuring the protective effect of

In order to find out whether a typical liver function inhibitor (SNP, Acetoaminophen, ethanol) could protect the Chang cell from damaging the pupa, CMWE of the pupa, we measured the IC 50 which damages the normal cell.

       In order to compare the damage of normal cells (Chang cells) by SNP, experiments were conducted to determine the concentration of SNP as an inhibitor,

When the concentration of SNPs of 0, 250, 500, 750, and 1,000 uM was treated in normal cells ( ¹ x 10 ⁴ cells / well), the cell viability decreased by 50% at 619.3 uM 7).

        As a result, the cell viability was reduced by 50% at a SNP of 619.3 uM. Therefore, it was confirmed that 600 uM of SNP affects the hepatocyte (Chang cell). Thus, it was reaffirmed to give a liver survival rate of approximately 50-60% at a SNP of 600 uM (Fig. 8).

         In order to compare the damage of normal cells (Chang cells) by ethanol, the concentration of ethanol as an inhibitor was determined.

Cells treated with Chang cells (1 × 10 ⁴ cells / well) at concentrations of 0, 100, 200, 300, 400 and 500 μM were reduced to 50% cell viability at 514.5 μM (Figs. 9 and 10).

     As a result of the above experiment, it was confirmed that 50% cell viability was decreased at 514.5 uM SNP, and therefore it was confirmed that 600 uM SNP affects the hepatocyte (Chang cell). Thus, at 500 uM ethanol concentration, liver cell damage was reaffirmed to give a liver survival rate of 70-80% (Fig. 11).

         In order to compare the damage of normal cells (Chang cells) by AAP (Acetaminophen), we conducted an experiment to determine the concentration of AAP as an inhibitor. As a result,

When the AAP concentration of 0, 5, 10, 20 and 40 μM was treated with Chang cells (1 × 10 ⁴ cells / well), the cell viability was reduced by 50% at the level of 10 μM 12 and FIG. 13).

As a result of the above experiment, 50% cell viability was reduced in 10 uM AAP. Therefore, it was confirmed that hepatocyte (Chang cell) affects 7.5 uM and 15 uM AAP. Thus, in 7.5 uM AAP, hepatocyte (Chang cell) was confirmed to have a hepatocyte survival rate of approximately 70% (Fig. 14) and 15 uM AAP hepatocyte (Chang cell) hepatocyte survival rate was approximately 20% 15).

< Experimental Example  3> Pupae Cordyceps  sample( CMWE )of Liver protection  Action mechanism

        In order to investigate the effect of CMWE, a pupa extract of Chinese pupa, on the normal cell, Chang cells, pupa extracts of CMWE were cultured at 0, 12.5, 25, 50, 100 and 200 ㎍ / The effect of CMWE was confirmed by treatment with Chang cells, which is a normal cell (Fig. 16).

The results showed that CMWE did not give any inhibitory effect on normal hepatocytes. The results showed that CMWE was not toxic to normal cells at 12.5 and 25 ㎍ / ㎖ , It was confirmed that the cells had a protective action against normal cells. In conclusion, CMWE showed no inhibitory effect on normal hepatocytes (Fig. 16).

< Experimental Example  4> Inhibitor ( AAP , SNP ) In normal cells Chang cell ) And Bundata The extract CMWE Protective effect of

       We investigated whether CMWE, a chrysanthemum Cordyceps sinensis extract, could protect hepatocytes when normal hepatocytes were damaged by inhibitors (APP, SNP).

When APP was treated with normal cells (1 × 10 ⁴ cells / well), AAP showed a decrease of cell viability of 50% at 10 μM level. Therefore, in order to test the protective effect of CMWE, a hepatocyte injury caused by AAP, and CMWE, a 12.5, 25.50, and 100 μg / ml of CMWE, The effect of the action was tested.

  C (control-treated hepatocytes) and C '(hepatocytes treated with 10 μM AAP) were higher than hepatocyte C' treated with 10 μM AAP, compared to the groups treated with various CMWEs, AAP was shown to have a protective effect when treated with hepatocytes (Fig. 17).

In the above experiment, when treated with Chang cells (1 × 10 ⁴ cells / well), the SNP decreased by 50% at the level of 500 μM. Therefore, hepatocyte injury by SNP and pupa extract of Cordyceps sinensis To investigate the protective effect of CMWE, CMWE was treated with 12.5, 25.50 and 100 μg / ml, respectively, and treated with 600 μM of SNP as an inhibitor.

  When CMWE was treated with 12.5 and 25 μg / ml, the SNP was treated at 600 μM when C (control - untreated hepatocytes) and C '(hepatocytes treated with 600 uM SNPs) were compared with various CMW treated groups The results were higher than those of group C 'treated with 600 uM of SNP in one group. These results showed that when the hepatocyte was damaged by the inhibitor SNP in the pupa chrysanthemum extract (CMWE), it had a protective effect (FIG. 18).

 < Example  4> Identification of Biochemical Mechanism of Protection Effect on Hepatocyte Injury

        Flow cytometry analysis and Western blotting method using propidium iodide staining for cell signaling substances involved in apoptosis and survival in order to elucidate the biochemical mechanism of the protective effect on the hepatocyte injury by the pupa extract of Chinese pupa (CMWE) Experiments were conducted.

< Experimental Example  1> Flow cytometry  analysis

Propidium iodide (PI) staining was performed to measure cell cycle changes of Chang liver cell by AAP (Acetoaminophen). First, Chang liver cells were cultured in 100 mm dish at 70 ~ 80%, and then cultured with pupa extract (CMWE) and AAP for 24 hours. Cells were washed twice with PBS and then trypsin / EDTA and centrifuged at 450 xg for 5 min. Cells were plated at 1 × 10 ⁶ cells / well, and 5 ml of 1% (w / v in PBS) paraformaldehyde solution was added and left in the ice bath for 15 min. Cells were harvested by centrifugation at 450 xg for 5 min, washed twice with 5 ml PBS. After centrifugation, 0.5 ml of PBS was added to the cells, and 5 ml of ice-cold 70% (v / v) ethanol was added thereto. The cells were centrifuged at 450 xg for 5 min to remove ethanol, washed with 1 ml of PBS, and then suspended with 0.1 ml of PBS. 50 μg / ml of PI was added to the cell suspension, DNase-free RNase was added to a final concentration of 1 μg / ml, and the mixture was covered with foil to block light and stained for 30 min. The PI-stained cells were filtered to take single cells and analyzed 1x10 ⁴ cells with FACS Calibur (Becton Dickinson, San Jose, Calif.).

        As shown in FIG. 19 and FIG. 20, when 7 mM AAP was applied to normal hepatocytes, the G2 / M group decreased and the Sub-G1 group increased in the cell cycle, Is known to be a characteristic feature of apoptosis process. In addition, the Sub-G1 phase increased by AAP tended to decrease in a concentration-dependent manner by the concurrent administration of CMWE. These results are shown in more detail in Fig. 20B. In Fig. 20B, the Sub-G1 period of the Control group was 2.35%, which was increased to 12.37% by the administration of 7 mM AAP. The concentration of CMWE decreased to 11.86%, 10.4%, and 7.8%, respectively, when the final concentration of CMWE was 50, 100, 200 / .

These results show that AAP-induced cytotoxicity, that is, cell cycle arrest and cell death induced by hepatocyte growth factor (CMWE) in normal hepatocytes, have hepatocyte protective activity.

< Experimental Example  2> Western blotting  analysis

       Chang liver cells, normal hepatocytes, were cultured in a 60 mm dish at 70-80%, and then cultured in a medium containing 1% FBS for 24 hours. Chang liver cell extract (CMWE) was administered at a certain concentration, and 30 minutes later, AAP, a hepatotoxic inducer, was administered. After incubation for 24 hours, the cells were lysed by adding lysis buffer and centrifuged at 15,000 g for 20 minutes to obtain a western blot sample. The protein concentration of the cell lysate was measured by the BCA method, and the cell lysate was subjected to SDS-PAGE to be 30 μg of the protein. After transferring the protein to PVDF membrane, the membrane was blocked with 5% non-fat skim milk, first treated with the desired antibody solution, washed with TBS-T, and attached with secondary antibody. As much as possible, TBS-T unbound antibody was washed and ECL solution (Thermo Scientific, Rockford, Ill.) Was evenly distributed on the membrane in the dark room and sensitized with X-ray film. Western blot analysis was performed using the Image J program developed by NIH (National Institutes of Health, USA). For the analysis by Image J, the average value obtained by repeating 3 times was used.

      As a result, it was confirmed that the amount of PARP activated by 7 mM AAP (3 lane) was increased as shown in FIG. 21, and the amount of PARP activated by the CMWE administration was decreased. In addition, CMWE-induced cordycepin also showed patterns similar to CMWE when administered concurrently with AAP at 10, 20, 50, and 100 μM, respectively, and the amount of activated PARP was decreased in response to cordycepin concentration.

  The reduced results of activated PARP acting at the last stage of apoptosis by the combined administration of CMWE and cordycepin showed that CMWE and cordycepin reduced cytotoxicity at the early stage of cytotoxic signal transduction, PARP activity was decreased.

        In addition, we investigated whether CMWE and cordycepin affect the activity of ERK protein, ERK, which is known as a signal transduction protein involved in cell survival and proliferation. As shown in FIG. 22, the activity of ERK (p-ERK) was decreased by the administration of 7 mM AAP, a cytotoxic inducer. The concurrent administration of CMWE and cordycepin showed a slight increase in the amount of p-ERK, but no significant increase was observed. However, when the ratio of the amount of activated ERK (p-ERK / -actin) to the amount of cells as shown in Fig. 22B was confirmed, it was confirmed that the amount of p-ERK also increased in the concentration of CMWE and cordycepin.

  In other words, the increase of p-ERK, which is involved in the survival and proliferation of the cells by administration of CMWE and cordycepin, despite the induction of cytotoxicity in normal hepatocytes, suggests that CMWE and cordycepin have a physiological activity effect for protecting hepatocytes from cytotoxicity .

         Second, Western blot experiments (FIG. 23) were performed on JNK and p38 proteins activated by physiochemical external stresses such as reactive oxygen species, inflammatory cytokine, and UV in cells.

  As shown in FIG. 23A, when cytotoxicity was induced by 7 mM AAP, the activity of JNK (p-JNK) was increased and the activity of p38 (p-p38) was slightly decreased compared with that of the control group. When combined with AAP and CMWE, the lowered p38 activity did not recover to the level of the control group, but the increased JNK activity was observed to be dependent on the concentration of CMWE.

  As shown in FIG. 23B, when the ratio of activated JNK (p-JNK / -actin) to the same cell was examined, the activity of JNK increased by cytotoxicity was increased by CMWE And the concentration was decreased to the same level as that of the control group.

Claims (4)

       (CMWE), which is characterized by dissolving the precipitate obtained by centrifuging the distilled water, and lyophilizing the precipitate obtained by centrifuging the dried pupa extract of the Chinese pupa, 10 times of distilled water, &Lt; / RTI &gt; A pupa extract of a pupa for improving liver function produced according to the method of claim 1 A pharmaceutical composition for preventing and treating liver cancer, which comprises Cordycepin and Adenosine derived from a pupa chrysanthemum extract prepared according to the method of claim 1. A functional food composition for functional improvement of liver function characterized by comprising cordycepin and adenosine derived from a pupa chrysanthemum extract prepared according to the method of claim 1 as an active ingredient.

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