KR20030016087A - Anticancer composition containing 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isaria japonica YASUDA - Google Patents

Abstract

PURPOSE: A new anticancer composition containing 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol separated from a methanol extract of fruit bodies of Isaria japonica Y ASUDA is provided which induces the death of cancer cells through the apoptosis of various cancer cells containing leukemia, breast cancer, liver cancer, cervical cancer. CONSTITUTION: Fruit bodies of Isaria japonica Y ASUDA are extracted in methanol and subjected to HPLC chromatography to separate -acetyl-12,13-epoxyl-9-trichothecene-3,15-diol(AEDT) of formula(1). The anticancer composition contains an effective amount of the ACDT for treatment of a cancer patient. For an example, I. japonica, coucher No. CHO-6133 is inoculated into a potato glucose culture, cultured at 25d eg.C for 10 days to produce a bacteria colony having a diameter of 5cm. The colony is inoculated into agar plugs, subjected to a second culture after sterilizing at 121 deg.C for 20min and then cultured at 25 deg.C for 8 days to produce fruit bodies.

Description

Anti-cancer agent containing 4-acetyl-12,13-epoxy-9-tricortesin-3,15-diol isolated from the fruiting plant of Snowflake Cordyceps herbaceous acid {Anticancer composition containing 4-acetyl-12,13-epoxyl-9-trichothecene- 3,15-diol isolated from the fruiting bodies of Isaria japonica YASUDA}

In the present invention, 4-acetyl-12,13-epoxy-9-tricortesin-3,15-diol (hereinafter referred to as _AETD ) is newly isolated from the methanol extract of the fruiting body of Isaria japonica Y _ASUDA . It relates to a new anticancer composition containing.

Although cancer treatment has opened the way for considerable advances in medical technology, biotechnology and genetic engineering, cancer is still recognized as a disease that cannot be cured yet.

Although many methods such as chemotherapy, radiation therapy, and incision have been developed as such cancer treatment methods, radiation therapy and incisions are mainly effective when the initial diagnosis of cancer is performed. However, if progression to terminal cancer is seen, cancer should be treated with chemotherapy, but the cure rate is quite low.

Therefore, the clinical field requires a new anticancer agent that is more effective than conventional anticancer agents, but in order to develop such an anticancer agent, development of a new target of the anticancer agent is required.

Recently, various genes and proteins involved in apoptosis have been studied as targets of such anticancer agents. The initiation of this study began with the discovery that anticancer agents used for chemotherapy over the past few years can damage genes in cancer cells, leading to apoptosis, or programmed cell death.

Although the mechanisms by which tumor cells can be killed through apoptosis are controversial, finding a substance that can induce apoptosis has been recognized as a method for identifying new anticancer drugs.

Apoptosis is a form of cell death that appears in all cells, and when apoptosis occurs, distinct morphological and biochemical characteristics are manifested. This apoptosis apoptosis is usually caused by the action of several genes in the end-of-life normal cells, as important as the production of new cells from undifferentiated stem cells. Cells that undergo apoptosis through apoptosis do not have any effect on adjacent cells by inflammatory reactions or the like, and only the cells are destroyed by performing the planned death. The physiological process of apoptosis is distinguished from necrotic cell death, which causes an inflammatory response and also destroys surrounding cells after severe cell damage, swelling and lysis.

Apoptosis is a planned apoptosis that effectively kills virus-infected cells and cells that have been transformed into tumors by natural mutations when the cells that have to be present briefly during life's development disappear. .

Therefore, the development of a substance capable of inducing apoptosis may be characterized by effectively killing only tumor cells without inducing death of normal cells through inflammatory reactions.

In this regard, the present inventors also conducted a lot of research to find a new substance that can induce apoptosis, and as a result it was newly isolated from the methanol extract of fruiting body of the Snow Cordyceps, 4-acetyl-12,13-epoxy-9- Tricotesin-3,15-diol (AETD) has been found to cause apoptosis of cancer cells, especially leukemia cells, and have come to complete the present invention.

Accordingly, it is an object of the present invention to provide a cancer therapeutic composition containing AETD as an active ingredient as a component of the methanol extract of fruiting body of Snow Cordyceps.

1 is a diagram showing the apoptosis induction effect of AETD on human leukemia cells (Human Leukemia Cells, HL-60) (in Figure 1, A is the form of leukemia cells 4 hours after treatment with 10 nmol / L AETD) In particular, B shows the result of DNA fragmentation test in treated cells when 10 nmol / L AETD was treated for 6 hours.).

Figure 2 is a result of measuring the cell ratio of the sub-G ₁ group corresponding to apoptotic cells using a flow cytometer.

Figure 3a shows the treatment of the caspase family protein inhibitor Z-VAD-fmk, caspase-1 specific activity inhibitor Ac-YVAD-CHO and caspase-3 specific activity inhibitor Z-DEVD-fmk DNA fragmentation test results.

Figure 3b is the result of measuring the caspase-3 activity over time after AETD treatment.

In order to achieve the above object, the anticancer agent composition according to the present invention is a substance for inducing apoptosis of cancer cells, 4-acetyl-12,13-epoxy-9-tricortesin-3,15- It is characterized by containing a diol (AETD).

[Formula 1]

The effective amount of AETD as an active ingredient in the anticancer agent composition according to the present invention may be appropriately selected depending on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient and the condition of the disease, but is not specifically limited. In general, AETD may be formulated to be administered at a concentration of 0.01 to 10 mg / kg body weight. Of course, the dose of the active ingredient may be an amount outside the above range.

Formulations for administering AETD within the above ranges may have conventional forms, and may be used, for example, in the form of pills, capsules, drinks, injections, pharmaceuticals, and the like. They can be administered orally or various parenteral routes of administration for leukemia, and contain various excipients, carriers or diluents which are suitable for the dosage form and are well known to those skilled in the art and can be readily selected by those skilled in the art. can do.

Hereinafter, the present invention will be described in more detail.

Isaria fruiting body has long been found to have an excellent effect on the treatment of cancer, and many studies are still underway to isolate the substance having cancer treatment from the cordyceps.

In particular, the fruiting body of snow Cordyceps sinensis has been used in the treatment of numerous diseases including cancer, but little is known about its medicinal properties and biological components.

However, in the present invention, the AETD component isolated from the methanol extract of the E. coli fruiting body using bioasssy-guided fractionation causes apoptosis in various cancer cells, thereby inhibiting the growth of cancer cells and inducing cell death. Has been found to have cancer treatment efficacy.

On the other hand, cells that perform apoptosis in the past are typically (1) morphological changes including chromatin condensation and fission are observed under an optical microscope, and (2) DNA fragmentation is shown by agarose gel electrophoresis. Biochemically, DNA fragmentation by flow cytometry, terminal transferase-induced dUTP subtitle end-labeling (TUNEL) staining differences, and changes in kespace activity have been found to occur.

Also in the present invention, it was confirmed that AETD causes apoptosis of various cancer cells using propidium iodide-staining (PI-staining) staining and flow cytometric analysis, and IC ₅₀ concentration (10) induction of apoptosis of AETD by measuring nuclear morphological changes in nmol / L), the ladder pattern of internucleosomal DNA fragmentation, and caspase-3 activity The activity was confirmed again.

As a result, AETD can induce apoptosis by apoptosis in various cancer cells, leukemia cells, breast cancer cells, liver cancer cells, cervical cancer cells, and is particularly effective against leukemia cells.

On the other hand, the present invention provides a method for producing AEDT. That is, the manufacturing method of the AEDT according to the present invention,

1) obtaining a snow Cordyceps sinensis fruit body from Snow Cordyceps sinensis;

2) obtaining a methanol extract of the snow Cordyceps sinensis fruiting body obtained in step 1);

3) characterized in that the method comprising the step of separating the AEDT by performing HPLC chromatography from the methanol extract.

Hereinafter, although an Example and drawing demonstrate this invention in detail, this invention is not limited by this.

Experimental examples of the following Examples are the results obtained by performing each experiment at least three times, using a statistical analysis using Student's t-test. The limit of significance was set based on the p value <0.05.

Reference Example 1 Material

The reagents and samples used in the examples below were obtained as follows.

RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco / BRL (Grand Island. NY.U.S.A). Dimethy sulfoxide (DMSO), 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide, trypan blue, DAPI and propidium ioda Propidium iodide (PI) was purchased from Sigma Chemical, St. Louis, Mo., USA. Z-VAD-fmk, Z-DEVD-fmk and Ac-YVAD-CHO were also purchased from CalBiochem, San Diego, Calif., U.S.A. All solvents were of analytical grade and were purchased from Merck (Darckstadt, Germany).

Reference Example 2 Cell Culture

Including HL-60 (Accession No. CCL-240), U937 (Accession No. CRL-2367), Hella (HeLa; Accession No. CCL-13), MCF-7 (Accession No. HTB-22) and HepG2 (Accession No. 77400) Various human tumor cell lines were distributed from the Depositary of the American Type Culture Collection (ATCC; Rockville. MD. USA), and unless otherwise noted, all human tumor cell lines were added with 10% Uteaser serum (FBS). In the RPMI-1640 culture medium, the culture was maintained at 37 ° C. in a humid atmosphere with 95% air and 5% carbon dioxide (CO ₂ ). Tumor cells were cultured with aliquots in new culture medium every 2-3 days to maintain exponential growth.

In addition, the cell numbers of the various cells mentioned in the following Examples were measured by a standard procedure using a hemacytometer used to count white blood cells, and cell viability was measured using trypan blue.

Reference Example 3 Preparation of Human Polymorphonuclear Leukocytes (PMNLs)

The PMNLs used in the experiments below were centrifuged with Histopaque 1119-1077 (Sigma, Diagnostics, St. louis, MO, USA, Protocol No 1119) to collect blood collected by venifuncture from healthy young men. Obtained through separation.

Then, the isolated PMNLs were washed with phosphate-buffered saline and then resuspended in RPMI-1640 culture to obtain PMNLs cell solution with a concentration of 13,105 cells / ml.

Reference Example 4 Collection and Cultivation of Snow Cordyceps Sinensis

The species of Cordyceps sinensis used in the present invention was used as a species isolated from Moaksan, Jeollabuk-do, Korea, and was identified as Snowflower Cordyceps by Professor Woo Deok-Hyun of Korea.

Identified Snow Cordyceps sinensis ( I. japonica , voucher No. CHO-6133) was inoculated in potato glucose medium and cultured at 25 ° C. for about 10 days until the colonies had a diameter of 5 cm. The fungal colonies formed were gray.

The obtained bacterial colonies were stored at a temperature of about 4 ° C., used as stock cultures, and used for the following test cultures. Then, 500 ml culture medium was filled into a 1-l flask, sterilized at 121 ° C. for 20 minutes, and then cooled to 15 ° C., and then inoculated with four 5 mm agar plugs obtained from the storage culture. Second culture was performed. The flask culture was incubated at 140 ° C. for 3 days at 140 rpm and then for 4 days at 160 rpm. At this time, the culture medium was used consisting of 200g / ℓ of potatoes and 30g / ℓ of sugar. All cultures were performed in the dark.

Then, a third fermentation was carried out in 100 g silkworm pupa. The non-divided medium was sterilized at 121 ° C. for 40 minutes, and the sterilized non-divided medium was cooled to 15 ° C., and then inoculated with 5 ml of spore solution. Then, it was incubated for 8 days in the dark at 25 ℃. After incubation, the culture conditions were changed to 60% humidity and further incubated in the dark for 10 days, followed by further incubation for 30 days under the culture conditions maintained at 20 ° C., 80% humidity and 2000 lux brightness, and then the fruiting body of Snow Cordyceps Got it. Then, methanol extracts were isolated from the fruiting bodies of the Snow Caterpillar sect obtained using conventional methods in the art, in order to perform subsequent chemical and biological experiments.

Example 1 Isolation and Structure Determination of AETD

The 226 g of air dried Snow Cordyceps sinensis fruit body obtained by the method of Reference Example 4 was extracted with methanol (MeOH) for 48 hours. The methanol extract was concentrated and then suspended in water. Then successively further separated with n-hexane, CH ₂ Cl ₂ , ethyl acetate (ethyl acetate, EtOAc) to obtain 247.5 mg of EtOAc soluble fraction. Then, the concentration of the methanol solution in which the EtOAc soluble fraction was diluted with distilled water was gradientd from 30% to 100% (v / v), and C18 flash column chromatography was performed. Then, 36.8 mg of the fractions eluted in 30% and 40% methanol were mixed and then preparative ceramic HPLC (preparative) for at least 40 minutes using a 15 to 35% gradient of CH ₃ CN solution diluted with distilled water. reversed-phase HPLC), and then further HPLC for 10 minutes with 100% CH ₃ CN solution to give 12.3 mg of compound 1.

In this example, the column used for HPLC chromatography used Alltech HS Hyperprep 100 BDS C18 (1.0 × 25 cm; 8 μm particle size; 2 mL / min; UV detection at 210 nm).

The structure of the isolated compound 1 was determined by MS and NMR data reported in the literature (Roush WR, J. Am. Chem. Soc., 107, 3354-3355), 4-acetyl-12, 13-epoxy- It was identified as 9-tricortesin-3,15-diol (AEDT) and the purity of this material was greater than 95%.

NMR spectra of isolated AEDTs were recorded on CD ₃ OD using a Jeol Eclipse-500 MHz spectrometer, and chemical changes were referenced to the corresponding residual solvent signal (3.30 / 49.0).

In addition, the results of mass spectrometry (MS) analysis of the separated AEDT were obtained by micro mass Quattro LC by electron spray ionization.

[Formula 1]

Experimental Example 1 Measurement of Cell Viability Inhibition Effect of AETD on Leukemia Cells by MTT Test

The survival inhibition effect of various cancer cells of AETD was measured by measuring the amount of mauve formazan produced by reducing MTT by the mitochondrial dehydrogenase of dense cells. The cancer cell lines were tested by the following method using leukemia cell lines of HL-60 and U-937, cervical cancer cell lines of HeLa cells, breast cancer cell lines of MCF-7 cells, and liver cancer cell lines of HepG2 cells. Normal cells of PMNLs were used.

That is, cells with exponential growth were suspended in RPMI 1640 medium to a density of 1 × 10 ⁶ cells / ml and treated with various concentrations of AETD. Then, after 4 hours of further incubation, 1 mg / ml of MTT solution was added to the culture, and further incubated for 2 hours. The resulting MTT-formazan product was dissolved in an equal volume of lysis buffer (20% SDS solution with 50% N, N-dimethylformamide, pH4.7). Then, after further incubation for 20 to 24 hours, the amount of formazan was measured by measuring the absorbance at 570 nm. The results are shown in Table 1 below.

Cell line IC50 (nmol / ℓ) HL-60 (CCL-240, ATCC company name) U-937 (CRL-2367, ATCC company name) HeLa (CCL-13, ATCC company name) MCF-7 (HTB-22, ATCC company name) HepG2 (77400, ATCC company name) PMNLs (Normal polymorphonuclear leukocytes) 10224553170> 1000 * The result is the result of MTT analysis 6 hours after AETD treatment, IC50 value is a statistical hydrotherapy calculated by repeating the experiment for 3 wells per each experimental bacteria. * IC50 value is 50% of each The concentration of AETD when cells die.

As shown in Table 1 above, as a result of confirmation by MTT analysis, AETD showed anticancer activity against not only leukemia cells but also various cancer cells such as cervical cancer, breast cancer and liver cancer, and most sensitively to leukemia cells. It can act as an anticancer agent, but it was confirmed that it is not toxic to general cells at all.

Experimental Example 2 Nuclear Staining Using DAPI / PI

HL-60 cells at a concentration of 1 × 10 ⁶ cells / ml, with or without 10 nmol / L AETD in RPMI 1640 medium containing 10% fetal bovine serum, 6 The cells were cultured in 6-well dishes. After 4 hours, 1 ml of cell suspension was centrifuged at 1000 rpm for 3 minutes to precipitate cells. Thereafter, 100% of formalin buffered with 4% neutral buffer was fixed to the cell precipitate. Immobilized 50 μl of cell suspension was transferred to a slide and dried at room temperature. The immobilized cells were washed with PBS, then dried and stained for 1 minute with DNA-specific fluorochrom DAPI (4,6-Diamidino-2-phenylindole) / PI. Attached cells were washed with PBS, dried and mounted with 90% glycerol (making a study specimen or slide). Slides were observed using a fluorescence microscope. The results are shown in FIG.

As shown in FIG. 1A, typical morphological features of cell death by apoptosis, such as condensation of nuclear chromatin, nuclear fragmentation and apoptotic bodies, are observed in AETD-treated cells. (The cells marked with * in FIG. 1A are cells in which apoptosis is being performed.).

Experimental Example 3 DNA Fragmentation Analysis

1 × 10 ⁶ cells / ml of HL-60 cells were treated with various concentrations of AEDT for 6 hours. Then, AEDT treated cells were Winsim separated for 3 minutes at 1000rpm, washed with PBS. To the cell precipitate, lysis buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 0.5% SDS, 0.5 mg / ml proteinase K (Sigma Chemical Co., St. louis. MO. USA)) was added and room temperature It was made to react by 30 minutes and dissolved. Then, 25 mg / ml of RNaes A was added, followed by reaction at 37 ° C. for 30 minutes, and then protein precipitation solution was added, followed by centrifugation at 4 ° C. at 1300 rpm for 10 minutes to precipitate all proteins. The supernatant was then extracted with phenol / chloroform (1: 1, v / v) and the aqueous phase was extracted once more with chloroform / isoamyl alcohol (24: 1, v / v). . The DNA was then precipitated by adding two volumes of isopropanol. DNA pellets recovered by centrifugation were washed with 70% ethanol, dried in air and then dissolved in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. After quantification of DNA, 10 μg of DNA was electrophoresed with 1.5% agarose gel at 5V / cm. Then, DNA was stained with EtBr (ethidium bromide) and observed under ultraviolet light. Cells in which apoptosis occurred were identified through a plurality of oligonucleosomal DNA fragments at about 180-200 bp positions. Standard molecular size was measured using a 1kbp DNA size marker, the results are shown in Figure 1b below.

As shown in Figure 1b, as a biochemical feature of apoptosis in cells treated with AETD, it can be confirmed that the internucleosomal region is decomposed into DNA fragments of 80 to 200bp by endogenuous DNase. there was.

Experimental Example 4 Flow Cytometric Analysis

The exponential growth cells were suspended in RPMI 1640 medium to a density of 1 × 10 ⁶ cells / ml, treated with AETD at a concentration of 50 nmol / L, and then cultured at 37 ° C. for 6 hours.

The AETD treated 1 × 10 ⁶ cells / well HL-60 cells were fixed in 80% chilled ethanol for 30 minutes, washed with PBS, and then 0.5% tryptone X-100 containing 1 mg / ml RNase A. The solution was treated with 0.25 ml at 37 ° C. for 30 minutes. Then, 0.25 ml of a PI solution of 50 mg / ml concentration was added, and left in the dark for 30 minutes. Subsequently, the DNA amount of the sub-G1 phase was measured from the sample cells by passing through a FACS Vantage (Becton Dickinson, San jose, CA, USA) flow cytometer to determine the cell number from the sample. The results are shown in Table 2 and FIG. 2.

Cell line (original) Sub-G1 (sub-G1) group DNA amount (%) Culture AETD HL-60 U-937 HeLa MCF-7 HepG2 PMNLs > 2 > 3 > 3 > 3 > 2 > 3 82.11 ± 1.5 ^b 46.23 ± 1.3 ^b 25.31 ± 3.4 ^b 17.24 ± 2.5 ^b 10.17 ± 1.2 ^b 2.03 ± 1.3 ^c a, This data value is obtained by three repeated tests, and represented by the mean ± average error. b, p <0.05 indicates the statistically analyzed value compared to the control group cultured only c. There is no significant difference between groups.

The apoptosis peak (sub-G1 peak, apoptotic peak) of the sub-G1 phase in the cell cycle can be measured by flow cytometry to determine the degree of apoptosis. As a result of this experiment, as shown in FIG. It can be seen that the apoptosis peak of the sub-G1 phase also increases. These results show that AETD induces apoptosis of HL-60 cells, killing HL-60 cells as leukemia cell lines.

In addition, as shown in Table 2, as a result of measuring the DNA content corresponding to the sub-G1 phase for each cancer cell treated with AETD, AETD increases the DNA content corresponding to the sub-G1 phase in all cancer cells These results show that not only leukemia cells, but also cancer cells through apoptosis for most cancer cells, but did not change the content of DNA corresponding to the sub-G1 phase at all for normal cells. .

Experimental Example 5 FIEA Test for the Activity of Caspase-3

The mechanism by which apoptosis occurs is markedly conserved among several species and is caused by a very complex process. However, it is commonly accepted in the art that, for stimulation both inside and outside cells, caspase family proteins play an important role in determining whether apoptosis or survival in cells will be maintained. That is, many studies have shown that the activation of caspase family proteins, especially caspase-3, is most important for cells to perform apoptosis.

Based on this fact, the DNA fragmentation test of Experimental Example 3 was carried out to confirm whether AEDT mediated the activation of caspase-3, causing apoptosis.

In other words, the leukemia cell line of HL-60 was treated with Z-VAD-fmk (50 μM) as an inhibitor of the activity of all caspase family proteins, and Caspase-1 specific inhibitor Ac-TVAD-CHO (50 μM) or caspa Z-DEVD-fmk (50 μM), an aze-3 specific inhibitor, was simultaneously treated with AETD at a concentration of 10 nmol / L and incubated at 37 ° C. for 6 hours. Then, DNA fragmentation test was performed by the method of Experimental Example 3. The results are shown in Figure 3a.

As shown in FIG. 3A, Z-VAD-fmk, an inhibitor of caspase family proteins, and Z-DEVD-fmk, a caspase-3 specific inhibitor, inhibit DNA fragmentation by apoptosis caused by AEDT. It can be seen that. Thus, these results show that AEDT induces apoptosis of tumor cells by apoptosis through activation of caspase-3.

On the other hand, after HL-60 cells were treated with AEDT by the same method as above, the activity of caspase-3 according to the treatment time was measured by the following method.

In other words, the caspase-3 activity was measured using a Fluorometric Immunosorbent Enzyme Assay (FIEA) kit by a method provided by Beohringer Mannheim. At various time intervals 2 × 10 ⁶ cells were treated with AETD and then washed with cold PBS. After stirring gently, the pellet was resuspended in the dissolution solution and incubated for 1 minute at room temperature. Then, after centrifugation, the supernatant was added to the microplate wells coated with anti-kespace-3 and incubated at 37 ° C. for 1 hour. After washing, 1 × substrate was added to the microplate wells. It was added and left at 37 ° C. for 2 hours or overnight. The samples were then analyzed using a fluorescence analyzer (Molecular Device; Orleans, CA, USA) set to an excitation wavelength of 405 nm and an emission wavelength of 510 nm. The results are shown in Figure 3b. As shown in Figure 3b, after the AETD treatment, it was confirmed that 2 to 3 hours, the caspase-3 activity is maximized.

As described above, 4-acetyl-12,13-epoxy-9-tricortesin-3,15-diol (AEDT), newly isolated from the fruiting body of I. japonica , activates caspase-3. To induce death of tumor cells through apoptosis of various tumor cells. Therefore, the composition according to the present invention can be used as a composition containing AEDT as an active ingredient and exhibiting anticancer activity against various tumor cells.

Claims (6)

In an amount effective for the treatment of a patient diagnosed with cancer, it contains 4-acetyl-12,13-epoxy-9-tricortesin-3,15-diol (AEDT) represented by the structural formula Anticancer agent composition. [Formula 1] 2. The anticancer agent composition according to claim 1, wherein the AEDT is separated from the methanol extract of the Snow Cordyceps Sinensis fruiting body. The anticancer agent composition according to claim 1 or 2, wherein the AEDT induces apoptosis of tumor cells. The anticancer agent composition according to claim 1 or 2, wherein the AEDT activates caspase-3 to induce apoptosis. The anticancer composition according to claim 1 or 2, wherein the cancer is selected from the group consisting of leukemia, breast cancer, liver cancer and cervical cancer. 1) obtaining a snow Cordyceps sinensis fruit body from Snow Cordyceps sinensis; 2) obtaining a methanol extract of the snow Cordyceps sinensis fruiting body obtained in step 1); 3) A method for producing an AEDT comprising the step of separating the AEDT by performing HPLC chromatography from the methanol extract.