TWI520742B - Use of soluble protein extract from antrodia cinnamomea in the preparation of medicinal composition of anti-human immunodeficiency virus - Google Patents

Description

Antrodia camphora soluble protein extract for the preparation of anti-human Use of a pharmaceutical composition for immunodeficiency virus

The present invention relates to a soluble protein extract of Antrodia camphorata, in particular to a soluble protein of Antrodia camphorata having anti-human immunodeficiency virus activity and a pharmaceutical composition thereof for preparing an anti-human immunodeficiency virus.

The human immunodeficiency virus (HIV-1) has a long empty window and is not easy to diagnose as early as possible. In recent years, it has spread rapidly to the whole world.

The currently used agents for the treatment of HIV-1 virus can be divided into four categories, namely, nucleoside reverse-transcriptase inhibitors (NRTIs) and nonnucleoside reverse-transcriptase inhibitors. Inhibitors; NRTIs), protease inhibitors (PIs), and entry inhibitors. All of the above four types are treated by inhibiting the characteristics of HIV-1 virus, and today's treatment of HIV-1 virus uses highly active antiretroviral therapy (HAART), that is, using three kinds of antiretroviral viruses. Drug, usually a nucleoside Nucleicside analogues and protease inhibitors or non-nucleotide reverse transcriptase inhibitors.

However, after taking a large amount of antiretroviral drugs, the half-life of the infected cells decreases, and the HIV-1 virus accelerates the infection of the new cells, causing the patient to have a highly variable HIV-1 virus and stay in the brain. In the testis, intestine, liver, kidney and other tissues, the virus concentration cannot be detected in the blood, and the current drug cannot control the replication of the virus.

Nowadays, the treatment of HIV-1 virus infection uses the high-efficiency antiretroviral method, but it cannot cure the HIV-1 virus, and the virus has high variability. It is necessary to find a more effective therapeutic substance or method.

Therefore, one aspect of the present invention provides a pharmaceutical composition for extracting a soluble protein extract of Antrodia camphorata for preparing an anti-human immunodeficiency virus, which is obtained by protein precipitation of a lysate obtained by culturing a mycelium of Antrodia camphorata The steps are followed by a gel filtration step. The soluble protein extract of Antrodia camphorata contains lacquer oxidase and a plurality of inseparable proteins, which can effectively inhibit the reverse transcriptase activity of human immunodeficiency virus.

According to the above aspect of the present invention, there is provided a use of a soluble protein extract of Antrodia camphorata for the preparation of a pharmaceutical composition against human immunodeficiency virus. In one embodiment, the mycelium of Antrodia cinnamomea TTChang &WNChou; accession number BCRC 35398 is cultured in a dark-protected liquid culture step, and the lysate is subjected to a protein precipitation step and a gel filtration step to obtain a soluble extract of Antrodia camphorata. Protein extract. The soluble protein extract of Antrodia camphorata contains lacquer oxidase and a plurality of inseparable proteins with a molecular weight of 30 kDa to 100 kDa, which can inhibit the reverse transcriptase activity of human immunodeficiency virus.

According to an embodiment of the invention, the light-proof liquid culture step does not include a thermal stress induction step, a continuous light induction step, a metal ion induction step, or an induction step of glucose deficiency.

According to an embodiment of the invention, the above-mentioned light-proof liquid culture step can be carried out, for example, for 10 days to 14 days.

According to an embodiment of the invention, the human immunodeficiency virus is a human immunodeficiency virus type 1 (HIV-1).

According to an embodiment of the present invention, the pharmaceutical composition may comprise 0.11 U/L to 5.27 U/L of lacquer oxidase, and the pharmaceutical composition inhibits at least 46% of reverse transcriptase activity. In another embodiment, the pharmaceutical composition described above may comprise from 0.26 U/L to 5.27 U/L of lacquer oxidase, and the pharmaceutical composition inhibits at least 56% of reverse transcriptase activity. In still another example, the pharmaceutical composition described above may comprise from 0.53 U/L to 5.27 U/L of lacquer oxidase, and the pharmaceutical composition inhibits at least 79% of reverse transcriptase activity. In yet another embodiment, the pharmaceutical composition described above may comprise from 1.1 U/L to 5.27 U/L of lacquer oxidase, and the pharmaceutical composition inhibits at least 90% of reverse transcriptase activity.

The application of the soluble protein extract of Antrodia camphorata, which excludes the use of an induced environment, induces the production of lacquer oxidase by the mycelium of A. angustifolia, and the soluble protein extract of A. angustifolia can be obtained by the protein precipitation step and the gel filtration step. The obtained Astragalus sinensis soluble protein extract Containing lacquer oxidase and a plurality of inseparable proteins, it can effectively inhibit the reverse transcriptase activity of human immunodeficiency virus.

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; A graph of values at different incubation times.

Fig. 2 is a graph showing changes in dry weight of Antrodia camphorata mycelium according to an embodiment of the present invention.

Fig. 3 is a graph showing the laccase oxidase activity of the Antrodia camphorata broth in different pH values of McIlvaine buffer solution according to an embodiment of the present invention.

Fig. 4 is a graph showing the oxidase activity of the Antrodia camphorata broth in a McIlvaine buffer solution (pH 3) at different culture times according to an embodiment of the present invention.

Fig. 5 is a photograph showing the soluble protein of the Antrodia camphorata broth according to an embodiment of the present invention, which was subjected to colloidal gel electrophoresis and activity staining with polypropylene guanamine.

Fig. 6 is a photograph showing the soluble protein of the Antrodia camphorata broth according to an embodiment of the present invention, which was subjected to polyacrylamide colloidal electrophoresis and general staining.

Fig. 7 is a photograph showing the soluble protein of the Antrodia camphorata broth according to an embodiment of the present invention, which was subjected to polyacrylamide colloidal electrophoresis and silver staining.

Fig. 8 is a bar graph showing the oxidase activity of the soluble protein extract of Antrodia camphorata with different dilution ratios of the extract of the Antrodia camphorata lycoprotein by ammonium sulfate precipitation and molecular sieve filtration according to an embodiment of the present invention.

Fig. 9 is a photograph showing the soluble protein extract of Astragalus membranaceus by the ammonium sulfate precipitation and the molecular sieve filtration step and the different dilution ratios, which are subjected to polyacrylamide colloidal electrophoresis and activity staining according to an embodiment of the present invention.

Fig. 10 is a bar graph showing the activity of Antrodia camphorata broth in vitro against HIV-1 according to an embodiment of the present invention.

Figure 11 is a bar graph showing the activity of soluble protein extracts of different dilution ratios of Astragalus membranaceus broth after ammonium sulfate precipitation and molecular sieve filtration in vitro for anti-HIV-1 activity according to an embodiment of the present invention.

As described above, the present invention provides a soluble protein extract of Antrodia camphorata obtained by culturing a mycelium obtained from the mycelium of Antrodia camphorata, followed by a protein precipitation step and a gel filtration step.

The term "Arctium sinensis" as used herein refers to Antrodia cinnamomea TTChang & WNChou, a unique species of Taiwan (Accession No. BCRC 35398). An endemic species of Taiwan is known as a brown rot fungus, which is named for its ability to parasitize on burdock trees. In the past, many research reports pointed out that Antrodia camphorata has a high degree of attention because it has many physiologically active ingredients and a wide range of therapeutic effects. However, Antrodia camphorata belongs to the brown rot fungus. In the past research, it was considered that this bacterium should not secrete laccase oxidase and decompose lignin. The inventor of the present invention discloses that the hyphae of Antrodia camphorata can be induced by using specific inducing conditions such as a thermal stress inducing step, a continuous light inducing step, a metal ion inducing step or an inducing step of glucose deficiency, etc., in the inventor of the Republic of China. The body produces paint oxidase. However, in the present case, the above various induction steps are discarded, and only a specific liquid culture solution, for example, an acidic culture solution containing glucose, sucrose, yeast extract, protein mash, inorganic salts or the like (for example, pH<6.0) is used, and cultured in the dark for 5 days. After 14 days or 10 days to 14 days, a broth containing a soluble protein extract of Antrodia camphorata can be obtained.

As used herein, "fermented liquor" refers to an acidic fermentation broth (eg, pH < 4.7) obtained by culturing the Antrodia camphorata in the dark. In general, the acidic fermentation broth can sequentially obtain a soluble protein extract of Antrodia camphorata through a protein precipitation step and a gel filtration step, wherein the obtained extract of Astragalus sinensis soluble protein contains substantially only soluble protein, and does not contain polysaccharides. A component other than a soluble protein such as a lipid. In other embodiments, after the liquid-free liquid culture step is completed, the acidic broth may be selectively subjected to solid-liquid separation by conventional filtration or the like to remove the mycelium of Antrodia camphorata.

As used herein, "Astragalus sinensis soluble protein extract" refers to a product comprising a natural lacquer oxidase and a plurality of inseparable proteins after a gel filtration step using, for example, a molecular sieve of a specific specification, wherein the lacquer oxidase has an enzyme activity. And the molecular weight of the plurality of inseparable proteins is from 30 kDa to 100 kDa. The molecular sieve of the above specific specification can be selected from various commercially available molecular sieves, thereby separating proteins having a molecular weight of 30 KDa to 100 KDa.

The "Astragalus sinensis soluble protein extract" of the present invention has reverse transcriptase activity which inhibits human immunodeficiency virus, and particularly has reverse transcriptase activity which inhibits human type 1 human immunodeficiency virus (HIV-1). When the content of laccase oxidase in the soluble protein extract of Antrodia camphora is higher, the first type The inhibitory effect of the reverse transcriptase activity of the immunodeficiency virus (HIV-1) is better. In one example, a soluble protein extract of Antrodia camphorata containing 0.11 U/L to 5.27 U/L of lacquer oxidase can inhibit at least 46% of HIV-1 reverse transcriptase activity. In another illustration, a soluble protein extract of Antrodia camphorata containing 0.26 U/L to 5.27 U/L of lacquer oxidase can inhibit at least 56% of HIV-1 reverse transcriptase activity. In yet another illustration, a soluble protein extract of Antrodia camphorata containing 0.53 U/L to 5.27 U/L of lacquer oxidase can inhibit at least 79% of HIV-1 reverse transcriptase activity. In yet another illustration, a soluble protein extract of Antrodia camphorata containing 1.1 U/L to 5.27 U/L of lacquer oxidase can inhibit at least 90% of HIV-1 reverse transcriptase activity. Therefore, the Astragalus sinensis soluble protein extract can be used for the preparation of a pharmaceutical composition against human immunodeficiency virus.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Preparation of Antrodia camphorata broth 1 solid state culture

Antrodia camphorata (Access Code BCRC 35398) used in this example was purchased from the Food Industry Research and Development Institute (FIRDI) BioResource Collection and Research Center (BCRC).

Before extensively cultivating the mycelium of Antrodia camphorata, the mycelium of Antrodia camphorata can be cultured in a solid medium or a liquid culture solution having a conventional culture formula. In this embodiment, the solid medium may be a suitable solid medium M25, including, for example 2 weight percent (wt%) of malt extract ^{(Bacto TM malt extract; BD,} NJ, USA), 2wt% of glucose, the protein 0.1wt% peptone ^{(Bacto TM peptone; BD, NJ} , USA) and 2wt% of agar ^{(Bacto TM agar; BD, NJ} , USA). Antrodia camphorata (BCRC 35398) was cultured on M25 solid medium and cultured at 26 ° C for 30 days in the dark. After 30 days of culture, the mycelium of Antrodia camphorata was covered with a culture plate, and the appearance was pale reddish brown (not shown), and it had a milky aroma.

2 liquid culture

Next, a solid culture block containing a mycelium having a size of 1.5 cm × 1.5 cm was cut out from the above M25 solid medium, and the M25 liquid culture solution was added after chopping. In this embodiment, M25 may comprise a liquid culture medium of 3wt% glucose, 0.5wt% of sucrose, 1.5wt% of yeast extract ^{(Bacto TM yeast extract; BD,} NJ, USA), 1.3wt% of peptone protein, 0.05wt% of magnesium sulfate _{(magnesium sulfate; MgSO 4; JTBaker} ), 0.05wt% of potassium dihydrogen phosphate _{(potassium phosphate, monobasic; KH 2} PO 4; JTBaker) 0.05wt% of phosphate and potassium (potassium phosphate, dibasic ; K ₂ HPO ₄ ; JTBaker), having a pH of 5.5. Then, the M25 liquid culture solution containing the mycelium of Antrodia camphorata was subjected to a small amount of light-proof liquid culture, which was shake-cultured at 26 ° C for about 7 days at a rotation speed of 100 rpm to 110 rpm to obtain a small amount of liquid broth of the Astragalus membranaceus mycelium.

Thereafter, the M25 liquid culture solution containing the mycelium of Antrodia camphorata was inoculated to a fresh M25 liquid culture solution at a ratio of 10% (v/v). Of course Thereafter, a large amount of light-proof liquid culture was carried out, which was shake-cultured at a rotation speed of 100 rpm to 110 rpm at 26 ° C for 1 day to 14 days to obtain a large amount of liquid broth of the Astragalus membranaceus mycelium.

However, the mycelium and the mashing solution of different culture days are separately collected by a conventional method (for example, filtration, solid-liquid separation, etc.). The obtained mycelium was placed in an oven at 60 ° C, dried and weighed. The resulting broth was further measured for pH and lacquer oxidase activity.

Please refer to FIG. 1 , which is a graph showing the pH value of the Antrodia camphorata broth in different culture time according to an embodiment of the present invention. As is apparent from the results of Fig. 1, the pH of the mashing solution tends to decrease as the culture time increases. After 1 day of culture, the pH of the broth was similar to the original pH of the M25 medium (pH 5.5), but as the mycelium grew in a large amount, the glucose of the medium was consumed, so that the pH of the broth was cultured for 2 days. After 9 days, it gradually decreased to about pH 4.3. However, after 10 to 14 days of culture, the pH of the broth was increased to about pH 4.6.

Please refer to FIG. 2, which is a graph showing the dry weight of the mycelium of Antrodia camphorata according to an embodiment of the present invention at different culture times. As can be seen from the results of Fig. 2, as the culture time increases, the dry weight of the mycelium also increases day by day.

Example 2: Assessing the biological activity of the extract of Astragalus membranaceus 1. Evaluate the preferred pH of the paint oxidase activity test

In this example, the laccase oxidase activity of the broth of Example 1 was tested using McIlvaine buffer solutions of different pH values, wherein the McIlvaine buffer solution (McIlvaine buffer) was composed of different ratios of citric acid solution and disodium hydrogen phosphate. The (Na ₂ HPO ₄ ) solution was formulated with buffer solutions of different pH values.

First, take 50 mL of B. chinensis broth for 14 days, and use M25 broth as a blank group, and add 50 mL of McIlvaine buffer solution of pH 3.0, pH 4.0, pH 5.0, pH 6.0 and pH 7.0 respectively. ). Subsequently, 100 mL of the original concentration of 1 mM 2,2-diazide-bis(3-ethyl-benzothiazoline-6-sulfonic acid) [2,2'-azino-bis (3-ethylbenzo-thiazoline-6-) was added. Sulfonic acid); ABTS] was used as a coloring substrate. After 120 minutes in the dark, the OD reader was used to detect the OD ₄₀₅ value of the sample solution.

Please refer to FIG. 3, which is a graph showing the laccase oxidase activity of the Antrodia camphorata broth in different pH values of McIlvaine buffer solution according to an embodiment of the present invention. From the results of Fig. 3, it was found that the lysate was reacted with ABTS for 30 minutes in the McIlvaine buffer of pH 3.0, and there was a color reaction, and the color reaction continued as the reaction time increased. On the other hand, the remaining pH solution did not react. Therefore, the following example measures the activity of the oxidase enzyme in the mash by a McIlvaine buffer at pH 3.0.

2. Evaluate the oxidase activity of the extract of A. nigrum

Take M25 medium as a blank group, take 50 mL of the broth for 1 day to 14 days, add 50 mL of McIlvaine buffer solution (pH 3), and add 100 mL of ABTS with the original concentration of 1 mM as a coloring matrix, protected from light. after 30 minutes, the absorbance immediately using ELISA reader detection of the sample solution OD _405.

Please refer to FIG. 4, which is a graph showing the oxidase activity of the Antrodia camphorata broth in a McIlvaine buffer solution (pH 3) at different culture times according to an embodiment of the present invention. The enzyme activity per unit (unit; U) referred to in the present invention is defined as the enzyme which produces 1 μmol of ABTS oxidation product per minute of catalytic activity. As for the ELISA reader detecting the absorbance of the sample solution, the activity of the oxidase enzyme can be calculated according to the following formula (I), wherein ΔAbs represents the difference between the absorbance values of the sample mash and the blank group (M25 medium), ε Represents the Mohr extinction coefficient (36,000 M ^-1 cm ^-1 ), b represents the optical path length of the spectrophotometer (cm), and t represents time (minutes). The result is shown in Fig. 4.

From the results of Fig. 4, it was found that paint oxidase activity (0.2 U/L) was detected in the fermentation broth from the seventh day. As the culture time increases, the lacquer oxidase activity in the mash also increases, and can increase to about 2.0 U/L by the 14th day.

Example 3: Preparation of Antrodia camphora soluble protein extract 1. Ammonium sulfate precipitation step and gel filtration step

Next, the broth obtained in Example 1 was subjected to an ammonium sulfate precipitation step to precipitate and concentrate the soluble protein. In this embodiment, the large amount of the cultured broth can be placed in an ice bath of crushed ice so that the liquid level of the mash is not higher than the height of the crushed ice. Next, the previously dried ammonium sulfate is slowly added to the pre-cooled mash and stirred for 30 minutes, wherein the weight of the solid ammonium sulphate (g) is added per liter of the broth, and the protein of the broth is The final concentration at 0 °C varies, and the specific amount of ammonium sulfate added is well known to those of ordinary skill in the art to which the present invention pertains, so it is not to be understood. Thereafter, the mixture was centrifuged at 6500 rpm for 30 minutes at 4 ° C, and the supernatant was centrifuged for the first time. The first centrifugation of the supernatant is followed by the addition of ammonium sulfate and the above stirring and centrifuging steps are repeated, and the second centrifuged protein precipitate is taken out, and the second centrifuged protein precipitate is dissolved in the second deionized water to a second. The solution was returned and stored at 4 °C.

The second solution was subjected to a gel filtration step using a molecular sieve (Amicon Ultra, 100,000 MWCO, 4 mL), wherein the gel filtration step was carried out by centrifugation at 3000 rpm for 10 minutes at 4 ° C, and the filtrate was collected.

Thereafter, the filtrate was subjected to a gel filtration step using molecular sieves (Amicon Ultra, 30,000 MWCO, 4 mL), and centrifuged at 3000 rpm for 10 minutes at 4 °C. This step is repeated five times.

Then, leaving 250 μL of the precipitate in the molecular sieve, and dissolving it into a third solution in 750 μL of secondary deionized water, and storing it at 4 ° C, wherein The three-time solution is a soluble protein extract of Antrodia camphorata.

2. Polyacrylamide guanamine colloidal electrophoresis (SDS-PAGE)

First, the formulation of 10% separating gel was 4.87 mL of secondary deionized water, 2.5 mL of Tris-HCl (pH 8.8) with a concentration of 1.5 M, and 2.5 ml of acrylamide/bis (original concentration of 40%). 37.5: 1), 100 μL of original concentration 10% (w/v) SDS solution, 25 μL of original concentration of 10% ammonium persulfate (APS) and 2.5 μL of TEMED to prepare a total volume of 10 ml; stacking gel containing 2.44 ml of secondary deionized water, 1 mL of Tris-HCl (pH 6.8) with a original concentration of 0.5 M, 472 μL of acrylamide/bis (37.5:1) with a native concentration of 40%, and a raw concentration of 40 μL of 10% (w/ v) SDS solution, 25 μL of APS with a native concentration of 10% and TEMED of 2.5 μl, with a total volume of 4 mL.

After that, place the plastic table in the electrophoresis tank, inject a proper amount of 1x tank buffer, and mix the protein sample (broth or burdock soluble protein extract) with 2x loading buffer and inject the gel. Next, the colloid was first electrophoresed at a voltage of 80 volts to allow the samples to be stacked at the same height. The colloid is then electrophoresed using a voltage of 100 volts to separate the sample according to the molecular weight of the protein.

After SDS-PAGE was completed, the colloid was immersed in Fixation Buffer I (containing 50 volume percent of secondary deionized water, 40 volume percent of methanol, 10 volume percent of acetic acid) for 10 minutes. Next, using 25 mL of ABTS with a concentration of 10 mM, the band can be clearly seen, and the result is as shown in the fifth. The figure shows.

Please refer to Fig. 5, which is a photograph showing the gelatin electrophoresis and activity staining of A. nigrum broth after 4 to 14 days according to an embodiment of the present invention. From the results of Fig. 5, it was found that the dye was stained with a 10 mM ABTS as a coloring substance, and the band was observed after about 15 minutes. Next, the results of Fig. 5 show that the lacquer oxidase secreted to the extracellular space by the mycelium of Antrodia camphorata has oxidative activity starting from the seventh day, and the lacquer oxidase activity is stronger as the culture time increases.

In addition, after the completion of the above SDS-PAGE, the colloid additionally uses a buffer containing Coomassie® Brilliant-Blue (100 mL of buffer containing 0.25 g of Coomassie® Brilliant-Blue powder, 53 mL of secondary deionized water, 40 mL of methanol, 7mL of acetic acid) was stained, and then faded to a clearing buffer with 53% by volume of secondary deionized water, 40% by volume of methanol, and 75% by volume of acetic acid. The result is shown in Figure 6. Show.

Please refer to FIG. 6 , which is a photograph showing the molecular weight of the lacquer oxidase of about 60 kDa, and the molecular weight of the enamel oxidase is about 4 k14, according to an embodiment of the present invention. The molecular weights of the two inseparable proteins are about 35 kDa and 45 kDa, respectively.

From the results of Fig. 6, it is known that as the culture time increases, the concentrations of two inseparable proteins (molecular weights of about 35 kDa and 45 kDa) also increase. However, at the position corresponding to the molecular weight of 60 kDa, the band of lacquer oxidase (molecular weight about 60 kDa) as shown in Fig. 5 was not observed. One of the reasons may be that the oxidative enzyme of the Coomassie Brilliant-Blue is decomposed by the lacquer oxidase. So Can't color.

In addition, considering that the above film is dyed with Coomassie® Brilliant-Blue, the sensitivity is low, so the silver dye with higher sensitivity is selected for analysis. After the above SDS-PAGE is completed, the colloid is immersed in the above fixed buffer I for 30 minutes, and then fixed buffer II (containing 85 volume percent of secondary deionized water, 10 volume percent of methanol, and 5 volume percent of acetic acid) Fix the gel for 15 minutes and repeat this step again.

Silver staining analysis was then performed using the Bio-Rad Silver Stain kit. First, the oxidizer buffer was used for 5 minutes, and the colloid was repeatedly washed with a second deionized water to be transparent. Next, the colloid was treated with silver reagent for 20 minutes, and the colloid was washed with secondary deionized water for 1 minute, and the colloid was placed in the developer until the band was clearly visible. Subsequently, the colloid was immersed in a stop solution (containing 95% by volume of secondary deionized water, 5 volume percent of acetic acid) for 5 minutes to complete silver staining, and the results are shown in Fig. 7.

Please refer to Fig. 7, which is a photograph showing the 4 to 14 days of Antrodia camphorata broth after being subjected to polypropylene guanamine colloidal electrophoresis and silver staining according to an embodiment of the present invention. As can be seen from the results of Fig. 7, as the culture time increases, the protein concentration in the broth is higher, and the protein composition is more complicated.

Example 4: Evaluation of paint oxidase activity of extracts of Astragalus membranaceus soluble protein

The soluble protein extract of Antrodia camphorata, which was precipitated by ammonium sulfate precipitation and molecular sieve filtration, was serially diluted 2 times, 5 times, 10 times, 20 times, 50 times and 100 times, respectively, as a protein sample solution. Next, 1 mM of ABTS was added to each group of protein sample solutions as a coloring substrate, and after 30 minutes in the dark, the OD ₄₀₅ absorbance of the sample solution was detected by an ELISA reader, and the results are shown in FIG.

Please refer to FIG. 8 , which is a bar graph showing the oxidase activity of the soluble protein extract of Antrodia camphorata with different dilution ratios according to an embodiment of the present invention, wherein the undiluted soluble protein extract of Antrodia camphorata is diluted 2 times. The sample of 5 times was not listed because of the excessive color after the reaction, and the absorbance value could not be measured. From the results of Fig. 8, it can be seen that the oxidative enzyme activity can be measured by diluting the soluble protein extract of Antrodia camphorata by 10 times, 20 times, 50 times, 100 times and 150 times.

In addition, the soluble protein extract of Antrodia camphorata was diluted 10 times, 20 times, 50 times, 100 times, 200 times and 300 times, respectively, and then subjected to polypropylene guanamine colloid electrophoresis, and 10 mM ABTS was used as a coloring substrate. The activity was stained, and the results are shown in Fig. 9.

Please refer to FIG. 9 , which is a photograph showing the soluble protein obtained by the ammonium sulfate precipitation step of the extract of the soluble protein extract of Antrodia camphorata by polyacrylamide colloidal electrophoresis and activity staining according to an embodiment of the present invention. The soluble protein extract of Antrodia camphorata with a dilution factor of more than 100 times was not observed due to the low activity of the paint oxidase.

Example 5: Evaluation of anti-HIV-1 activity of Antrodia camphorata broth and its soluble protein extract 1. Assessing the activity of Antrodia camphorata in vitro against HIV-1 in vitro

This example evaluates the anti-HIV-1 activity of the Antrodia camphorata broth by using a commercially available kit, such as the Colorimetric experimental kit of the Reverse Transcriptase Assay produced by Roche.

Briefly, add 5 ng of HIV-1-RTase (in 20 μL of lysis buffer) to a microcentrifuge tube, and add 20 μL of Aconite Bacteria broth for 1 to 14 days, and add 20 μL of polyA×oligo(dT) ₁₅ The reaction was carried out at 37 ° C for 1 hour. Next, the above reaction solution was sucked into an MP module, covered with aluminum foil, and reacted at 37 ° C for 1 hour. Thereafter, the reactant liquid was blotted dry, 250 μL of washing buffer was added to rinse the reaction for 30 seconds, and carefully drained, and this step was repeated 5 times. Then, 200 μL of anti-DIG-POD working solution (200 mU/μl) was added, and the aluminum foil was covered and reacted at 37 ° C for 1 hour. Thereafter, the liquid of the reactant was carefully drained, rinsed with 250 μL of washing buffer for 30 seconds, and carefully drained, and this step was repeated 5 times. Next, 200 μL of ABTS substrate solution was added, oscillated to green at 250 rpm, and OD _{405 was} detected by ELISA reader. Subsequently, M25 medium was used as a control group, and the inhibition rate (%) of HIV-1 reverse transcriptase was calculated according to the following formula (II) to evaluate the activity of anti-HIV-1 in vitro, and the results are shown in Fig. 10. .

In addition, the effective inhibition rate can be further calculated according to the following formula (III): In formula (III), Xm represents the log maximum dose, I represents log (maximum dose/adjacent dose), P represents the sum of positive reaction rates, Pm represents the maximum positive reaction rate, and Pn represents the minimum positive reaction rate.

Please refer to FIG. 10, which is a bar graph showing the activity of Antrodia camphorata broth in vitro against HIV-1 according to an embodiment of the present invention. From the results of Fig. 10, it was found that the M25 medium was used as the control group, and the inhibition rate (%) of HIV-1 reverse transcriptase was from the day of culture until the 14th day of culture, and the inhibition rate was about 20%. Between % and 95%. Among them, the inhibition rate (%) of HIV-1 reverse transcriptase from the culture of 5 days until the culture of 14 days of broth was more obvious, and the inhibition rate was about 60% to 95%.

2. Assessing the anti-HIV-1 activity of extracts of Astragalus membranaceus soluble protein

This embodiment utilizes the same commercially available kit and the same detection method as described above to detect undiluted stock solution, diluted 2 times, 5 times, 10 times, 20 times, 50 times, 100 times by ammonium sulfate precipitation and molecular sieve The inhibition rate (%) of the filtered Astragalus sinensis soluble protein extract against HIV-1 reverse transcriptase was evaluated for its activity against HIV-1, and the results are shown in Fig. 11.

Please refer to FIG. 11 , which is a bar graph showing the activity of the extract of Astragalus sinensis soluble protein against HIV-1 in vitro according to an embodiment of the present invention. From the results of Fig. 11, it can be seen that the M25 medium is used as the control group, and the stock solution, the dilution ratios of 2 times, 5 times, 10 times, 20 times, 50 times, 100 times of the soluble protein extract of Antrodia camphorata have HIV-1 reverse transcriptase. The inhibition rate (%) has an inhibition rate of about 16% to over 90%. Among them, the stock solution, diluted 2 times, 5 times the soluble protein extract of Astragalus sinensis, HIV-1 reverse transcriptase The inhibition rate (%) is more obvious, and the inhibition rate is over 90%. The inhibition rate of HIV-1 reverse transcriptase of the soluble protein extract of A. angustifolia was 10%, and the inhibition rate of HIV-1 reverse transcriptase of the Astragalus membranaceus soluble protein extract was 56%, diluted by 20 times. The inhibition rate of HIV-1 reverse transcriptase by a 50-fold multiple of the soluble protein extract of Antrodia camphorata was also 46%. The inhibition rate of HIV-1 reverse transcriptase of the Astragalus membranaceus soluble protein extract of 100 times the dilution factor was 16%. Further, according to the formula (III), it can be calculated that the effective inhibitory concentration of the extract of the extract of Antrodia camphorata is 5.27 U/L. Therefore, the above-obtained soluble extract of Antrodia camphorata can effectively inhibit the reverse transcriptase activity of human immunodeficiency virus, and is further applied to the preparation of a pharmaceutical composition for anti-human immunodeficiency virus.

In the above embodiment, the number of samples per set of data is at least three (n ≧ 3).

It should be noted that, in the present invention, the soluble protein of Astragalus sinensis according to the present invention is exemplified by a specific concentration of a soluble protein extract of Antrodia camphorata, a specific purified and isolated reagent and conditions, a specific test kit, a specific analytical method or a specific instrument. The present invention is not limited thereto, and the Astragalus sinensis soluble protein extract of the present invention and its application are also not excluded from the spirit and scope of the present invention. Other concentrations of Antrodia camphora soluble protein extract, other purified and isolated reagents and conditions, other test kits, other analytical methods, or other instruments may be used.

It can be seen from the above embodiments of the present invention that the soluble protein extract of Antrodia camphorata and the use thereof of the present invention have the advantages of eliminating the use of an induced environment, It can induce the production of lacquer oxidase from the mycelium of Antrodia camphorata, and the soluble protein extract of Antrodia camphorata can be obtained by the protein precipitation step and the gel filtration step. The obtained soluble extract of Antrodia camphorata contains lacquer oxidase and a plurality of inseparable proteins, which can effectively inhibit the reverse transcriptase activity of human immunodeficiency virus, and is then used for the preparation of a pharmaceutical composition against human immunodeficiency virus.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims (7)

The use of a soluble protein extract of Antrodia camphorata for preparing a pharmaceutical composition against human immunodeficiency virus, wherein the soluble protein extract of Antrodia camphorata is one of mycelium of Antrodia cinnamomea TTChang & WNChou (Accession No. BCRC 35398) on M25 The medium is immersed in one of the immersed liquids in the dark liquid culture step for 5 days to 14 days, followed by a protein precipitation step and a gel filtration step, and the dark liquid culture step does not include a thermal stress induction step. a continuous light-inducing step, a metal ion-inducing step or a glucose-deficient inducing step, wherein the pH of the broth of the liquid culture step is less than pH 4.7 over the period of 5 days to 14 days. The fermented liquid is subjected to a gel filtration step of 30,000 MWCO, and the precipitate obtained by the gel filtration step is dissolved in secondary deionized water to form the soluble protein extract of Antrodia camphorata. The soluble protein extract of Antrodia camphorata comprises a laccase oxidase and a plurality of molecular weights of 30 KDa to 100 KDa. An inseparable protein to inhibit the reverse transcriptase activity of the human immunodeficiency virus. The use of the extract of Antrodia camphorata soluble protein as described in claim 1 for the preparation of a pharmaceutical composition for anti-human immunodeficiency virus, wherein the dark liquid culture step is carried out for 10 days to 14 days. The use of the Astragalus sinensis soluble protein extract as described in claim 1 for the preparation of a pharmaceutical composition against human immunodeficiency virus, wherein the human immunodeficiency virus is the first human immunodeficiency virus (HIV-1). The use of the Astragalus sinensis soluble protein extract according to claim 1 for the preparation of a pharmaceutical composition for anti-human immunodeficiency virus, wherein the pharmaceutical composition comprises at least 0.11 U/L to 5.27 U/L of the lacquer oxidation An enzyme, and the pharmaceutical composition inhibits at least 46% of the reverse transcriptase activity. The use of the extract of Antrodia camphorata soluble protein as described in claim 1 for the preparation of a pharmaceutical composition for anti-human immunodeficiency virus, wherein the pharmaceutical composition comprises at least 0.26 U/L to 5.27 U/L of the lacquer oxidation An enzyme, and the pharmaceutical composition inhibits at least 56% of the reverse transcriptase activity. The use of the extract of Antrodia camphorata soluble protein as described in claim 1 for the preparation of a pharmaceutical composition for anti-human immunodeficiency virus, wherein the pharmaceutical composition comprises at least 0.53 U/L to 5.27 U/L of the paint oxidation An enzyme, and the pharmaceutical composition inhibits at least 79% of the reverse transcriptase activity. The use of the extract of Antrodia camphorata soluble protein as described in claim 1 for the preparation of a pharmaceutical composition for anti-human immunodeficiency virus, wherein the pharmaceutical composition comprises at least 1.1 U/L to 5.27 U/L of the paint oxidation An enzyme, and the pharmaceutical composition inhibits at least 90% of the reverse transcriptase activity Sex.