TWI389701B - Extracts of aquilaria hulls and use thereof in the treatment of cancer - Google Patents

Description

Extract of acacia husk and its use in the treatment of cancer

The present invention relates to an ethyl acetate layer of a methanol extract of a genus Aquilaria for killing cancer cells and treating/preventing cancer and uses thereof. In particular, the extract is from Aquilaria agallocha Roxb. and has a large amount of cucurbitacin.

Aquilaria is an angiosperm that is taxonomically classified into Thymelaceaceae . Fifteen species of agarwood have been reported and both produce agarwood. The taxonomy of these species is not fully understood and the taxonomicians do not identify all species. Species include Aquilaria malaccensis , A. agallocha , A. baillonii , A. crassna , A. hirta , and Roche agarwood ( A. rostrata), Belleville sinensis (A. beccariana), cumin sinensis (A. cummingiana), Fei aloes (A. filaria), Karnofsky aloes (A. khasiana), small fruit sinensis (A. microcarpa), Grignard A. grandiflora , A. chinesis or A. sinensis , A. borneensis and A. bancana . Agarwood is native to Asia (from northern India to Vietnam and Indonesia). It is particularly found in the rainforests of Indonesia, Thailand, Cambodia, Laos, Vietnam, Malaysia, Northern India, the Philippines, Borneo and New Guinea. Agarwood is an evergreen tree that grows to a height of 30-40 meters and a diameter of 60 cm. It has white flowers and a fragrant flower. The healthy wood of Agarwood is white, soft, uniform in texture and does not emit odor when newly cut. Under certain pathological conditions, the heartwood becomes saturated with a resin and eventually hardens to a very hard state. The finest agarwood is hard, almost black and sinks when placed in water. In general, agarwood with a lighter shade is considered a defective product because of its small amount of resin.

Natural cucurbitacin constitutes a group of triterpenoids known to us as a result of bitterness and toxicity. It is characterized by a tetracyclic cucurbityl nucleus skeleton, that is, 19-(10→9β)-shift-10a-lanolin-5-ene (also known as 9β-methyl-19-northene-5-ene). It has multiple oxidizing functional groups at different positions. Traditionally, cucurbitacin has been arbitrarily divided into twelve categories and incorporated into cucurbitacin AT. Cucurbitacin is known to be cytotoxic and has anticancer activity in the art. However, the application potential of cucurbitacin is substantially impeded by its non-specific cytotoxicity, and therefore only very limited use is imposed under strict medical control. Cucurbitacin initially based Cucurbitaceae bitter component to the separation, and later found to be present in cruciferous (family Brassicaceae), Scrophulariaceae (Scrophulariaceae), Begoniaceae (Begoniaceae), elaeocarpaceae glycosylated or non-glycosylated form ( Elaeocarpaceae), tetramelaceae (Datiscaceae), from spray Branch (Desfontainiaceae), onion flower Branch (Polemoniaceae), primulaceae (Primulaceae), Rubiaceae (Rubiaceae), Sterculiaceae (Sterculiaceae), Rosaceae (Rosacease) and In plants of Thymelacaeceae (Jian Chao Chen et al., Nat. Prod. Rep., 2005, 22, pp. 386-399). Sonja Sturm and Hermann Stuppner used high pressure liquid chromatography-mass spectrometry (HPLC-MS) to analyze cucurbitacin in medicinal plants and determined via the drug Citrullus colocynthis (Cucurbitaceae) and Bryonia cretica ssp. The cucurbitacin in dioica ( Glycine ), Gratiola officinalis (Scrophulariaceae), Picrorhiza kurroa (Scrophulariaceae) and Iberis umbellate (Crucifera) proves its applicability Sex (Phytochem. Anal. 11, 121-127 (2000)). HPLC method was developed for simultaneous determination of four bioactive cucurbitacin in Cucubita pepo cv Dayangua (Cucurbitaceae) (Journal of Chinese Medicinal Materials, 2007, Vol. 30, No. 4, pp. 418-420) And Chin Hosp Pharm J, 2007, Dec, Vol. 27, No. 12, pp. 1694-1969).

However, none of the prior art provides information on cucurbitacin from agarwood or its husks and its anti-cancer activity.

The present invention provides an acrepe husk extract for killing cancer cells and treating/preventing cancer, which comprises an ethyl acetate layer of a methanol extract obtained by methanol extraction followed by partitioning with ethyl acetate and water.

The invention also provides a method of killing cancer cells comprising administering to an individual an effective amount of an extract of the invention, thereby killing cancer cells.

The invention also provides a method of treating/preventing cancer comprising administering to an individual an effective amount of an extract of the invention, whereby the cancer can be treated and/or prevented.

The invention also provides a method of inducing apoptosis in a cancer cell comprising administering to the individual an effective amount of an extract of the invention, thereby causing apoptosis of the cancer cell.

The present invention finds that the ethyl acetate layer of the methanol extract of the genus Aquilaria has a large amount of cucurbitacin and other active ingredients and these compounds are effective for killing cancer cells, treating/preventing cancer and inducing apoptosis of cancer cells. In addition, the ethyl acetate layer of the methanol extract of the genus Aquilaria is non-toxic.

As used herein, "a" can mean one or more. As used herein in connection with the scope of the claims, the word "a" may mean one or more than one.

As used herein, "Agarwood husk" refers to the woody husk of the fruit of the agarwood tree.

"Treatment," as used herein, refers to any type of treatment that confers benefits to a patient suffering from a disease, including improving a patient's condition (eg, one or more symptoms), delaying the progression of the disease, preventing or delaying the onset of the disease, and the like.

The present invention provides an acrepe husk extract for killing cancer cells and treating/preventing cancer, which comprises an ethyl acetate layer of a methanol extract obtained by methanol extraction followed by partitioning with ethyl acetate and water. The ratio can be 1:0, 10:1, 10:2, 10:3, 10:4, 10:5, 10:6, 10:8, 1: by cerium oxide column chromatography. The ethyl acetate layer was further divided by 1 and a 0:1 hexane-ethyl acetate mixture and (ii) a mobile phase of methanol to obtain 13 kinds of partitions. The present invention has found that the partitioning portion 3 (10:2) specifically contains β-phytosterol, the dividing portion 7 (10:5) specifically contains ellagdin, and the dividing portion 8 (10:5) specifically contains cucurbitacin E, The dividing portion 9 (10:5) specifically contains cucurbitacin I and cucurbitacin E glycoside, and the dividing portion 11 (1:1) specifically contains cucurbitacin I glycoside, and the dividing portion 12 (0:1) specifically contains scutellarin 5-O-glycoside and mangiferin. --phytol, saponin, cucurbitacin I, cucurbitacin E, mangiferin and its derivatives have anticancer activity. The preferred embodiment is shown below: beta-phytosterol

Saponin

Cucurbitacin E

Cucurbitacin I

Cucurbitacin E glucoside

Cucurbitacin I glucoside

Physicin-5- O -glucoside

Mangiferin

In one embodiment, the present invention provides an Aquilaria husk extract for killing cancer cells comprising a mobile phase extracted by methanol, partitioned with ethyl acetate and water, and using a mixture of hexane-ethyl acetate The fraction obtained by lysing by gelation chromatography. The ratio of the mixture of hexane and ethyl acetate is preferably 10:5 or 10:6. Preferably, the 10:5 partition of hexane and ethyl acetate can be further separated to obtain ellagdin, cucurbitacin E, cucurbitacin I and cucurbitacin E glycoside. According to another embodiment of the present invention, cucurbitacin E and cucurbitacin I are present by dissolving the precipitate in a 10:5 partition of hexane and ethyl acetate by means of a gel column chromatography or a silica gel cartridge. The mobile phase of CHCl ₃ was extracted from the obtained solution, and the fraction obtained by desorbing cucurbitacin E and cucurbitacin I by methanol was used.

According to a preferred embodiment of the present invention, the agarwood husk is obtained from the agarwood husk, agarwood (synonym name: Agallochum malaccense (Lam.) Kuntze), and Aquilariella malaccensis (Lam.) Tiegh ), Aquilaria sinensis , A. sinensis (Lour.) Gilg; common name: agarwood, eaglewood, gharu, aloeswood, 15 One of the species of the genus Aquilaria. Agarwood is an evergreen plant produced in India, China, and Tibet. It is usually described as aloe wood or agar wood. Traditionally, bark, roots and heartwood have been used as folk medicines for the treatment of inflammation, arthritis, vomiting, heart conditions, cough, asthma, leprosy, anorexia, headache and gout due to their medicinal properties. However, without any prior art teachings or indications, the agarwood husk extract is effective for killing cancer cells and treating/preventing cancer.

According to another embodiment of the present invention, the ethyl acetate layer of the methanol extract of the agaric husk can be separated by centrifugal partition chromatography (CPC). The ethyl acetate layer of the agaric husk is dissolved in a solvent system containing chloroform-methanol-water (the ratio is preferably 6:7:4, v/v/v), and the solvent is subjected to centrifugal partition chromatography to separate the cucurbit Prime. According to the present invention, cucurbitacin I glucoside, cucurbitacin E glucoside, cucurbitacin I and cucurbitacin E can be obtained. According to another embodiment of the present invention, nine kinds of divisions are obtained, wherein the division portion 1 is obtained within 0-14 minutes, and the division portion 2 containing 82.49% mangiferin is obtained within 15-18 minutes, within 19-22 minutes Obtaining the dividing portion 3, obtaining the dividing portion 4 containing 73.54% of cucurbitacin I glycoside in 23-26 minutes, obtaining the dividing portion 5 containing 63.24% cucurbitacin E glycoside in 27-38 minutes, obtained in 39-63 minutes The partitioning portion 6 containing 80.09% cucurbitacin I obtains the partitioning portion 7 containing β-phytosterol in 64-65 minutes, and obtains the dividing portion 8 containing 76.87% cucurbitacin E in 66-75 minutes and is in the last 76 The division section 9 is obtained within 80 minutes. Molecular sieves (gel filtration or gel permeation) chromatography can be further used to increase the purity of the above compounds.

The extract of the invention may be formulated for administration in the treatment of cancer. In the manufacture of the pharmaceutical formulations of the invention, the extracts of the invention are typically admixed, inter alia, with a pharmaceutically acceptable carrier. Of course, the carrier must be acceptable in the sense of being compatible with any other ingredients in the formulation and must be innocuous to the patient. The carrier can be a solid or a liquid or both, and is preferably formulated with the extract as a unit dosage formulation. The extracts of the present invention may be incorporated into the formulations of the present invention, and the formulations of the present invention may be prepared by any of the well known pharmaceutical techniques, including essentially mixing the components comprising one or more complexing agents as appropriate.

Formulations of the invention include those suitable for oral, rectal administration, topical administration, buccal administration, parenteral administration (eg, subcutaneous administration, intramuscular administration, intradermal administration, inhalation administration, or Intravenous administration) and transdermal administration of the formulation, but the most appropriate route in any given situation will depend on the nature and severity of the condition being treated and the nature of the particular active product employed.

The formulations suitable for oral administration may be in the form of discrete units, such as capsules, blister, troches, or lozenges, each containing a predetermined amount of active compound; in powder or granule form; solutions in aqueous or nonaqueous liquids Or in the form of a suspension; or in the form of an oil-in-water or water-in-oil emulsion. The formulations may be prepared by any suitable pharmaceutical process comprising the step of bringing the extract of the invention into association with a suitable carrier, which may contain one or more complexing agents as described above.

In general, the formulations of the present invention are prepared by uniformly and intimately mixing the active compound with a liquid or finely divided solid carrier or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules containing the active compound and optionally one or more compounding agents. The compressed lozenge can be prepared by compressing the compound in a free-flowing form, such as a powder or granule, optionally mixed with a binder, lubricant, inert diluent and/or surface active/dispersing agent, in a suitable machine. Molded tablets can be made by molding in a suitable machine a powdered compound moistened with an inert liquid binder.

In addition to the extracts described herein, the present invention also provides effective methods of treatment and uses. For example, the present invention provides a method of killing cancer cells and a method of treating/preventing cancer. According to an embodiment of the present invention, there is provided a method of killing cancer cells and treating/preventing cancer, wherein the method comprises administering to the individual an effective amount of the extract of the present invention so as to treat and/or prevent cancer. According to another embodiment of the invention, a method of inducing apoptosis in a cancer cell comprises administering to the individual an effective amount of an extract of the invention to treat and/or prevent cancer. Also provided is the use of an extract of the invention for killing cancer cells and treating/preventing cancer. According to an embodiment of the invention, the cancer system referred to herein is selected from the group consisting of colorectal cancer, gastric cancer, prostate cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer, colon cancer, cervical cancer, and hepatocellular carcinoma.

The subject can be treated and typically a human subject by using the extract of the invention, but the method of the invention can also be applied to other individuals for veterinary use, especially for mammalian individuals, including but not limited to horses, cows, dogs , rabbits, sheep and their analogues. The therapeutically effective dose of any particular compound will vary to some extent between different compounds and between patients, and will depend on the condition and route of delivery of the patient. As a general recommendation, a dose of from about 50 mg/kg to about 1,000 mg/kg will have therapeutic efficacy, and higher doses may be used for oral and/or aerosol administration. The dosage is preferably from about 50 mg/kg to about 800 mg/kg, from about 50 mg/kg to about 600 mg/kg, from about 50 mg/kg to about 500 mg/kg, from about 50 mg/kg to about 300 mg/ Kg, from about 100 mg/kg to about 300 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 150 mg/kg to about 500 mg/kg, from about 200 mg/kg to about 500 mg/kg or 250 mg/kg to about 400 mg/kg.

The present invention also provides a pharmaceutical or dietary supplement comprising the extract or pharmaceutical formulation of the present invention, at least one macronutrient selected from the group consisting of proteins, carbohydrates and fats, which is compounded for use in ameliorating cancer Related diseases. Both dietary supplements and pharmaceutical meals of the present invention are preferably used in powder form which is soluble in a liquid suitable for human consumption, such as water or fruit juice. However, the dietary supplements and pharmaceutical meals of the present invention may be used in any suitable form, such as a solid stick, paste, gel, lozenge, capsule or liquid.

Instance

Example 1 Extraction of agarwood shell

1.5 Kg of agarwood husks were dried and pressed and then extracted with 40 L of methanol. The resulting solution was concentrated under reduced pressure and then separated with ethyl acetate and water. The ethyl acetate fraction was mixed with diatomaceous earth 545 and separated by a solvent gradient (10.5 cm (inner diameter) × 95 cm) using a dissolution gradient of ethyl acetate/n-hexane in the case of low-polarity dissolution to high-polarity dissolution. . The sample was dissolved in methanol. Thin layer chromatography (TLC) was performed to identify the components in each division.

The agarwood leaves and seeds were extracted with methanol. The agarwood stem was extracted with ethyl acetate. The resulting solution was concentrated under reduced pressure and dissolved in methanol for HPLC analysis.

Example 2 Separation of Compounds of the Invention

The elution gradient of n-hexane/ethyl acetate was used in the case of low-polarity dissolution to high-polarity dissolution (hexane: EtOAc 1:0→10:1→10:2→10:3→10:4→10:5→10: 6→10:8→1:1→0:1, v/v) The ethyl acetate fraction was separated by oxime column chromatography (10.5 cm (inner diameter) × 22 cm), and then dissolved in methanol. Using hexane: EtOAc (10: 1) as the mobile phase by silica gel column VersaPak ^TM filter cartridge (40 × 150 mm) was purified dividing section 3 (10: 2) to give about 0.3g Compound 1 (β- plant sterol ). The partitioning portion 7 (10:5) was filtered to obtain 210 mg of Compound 2 (sucrose) (yellow powder). Using chloroform as the mobile phase by silica gel column VersaPak ^TM filter cartridge (40 × 150 mm) was further purified with hexane -EtOAc (10: 5) eluting the divided portions 8 and 9, to obtain 2.4 g of compound 3 in the divided portion 8 (cucurbitacin E) (crystal or white precipitate) and 0.5 g of compound 4 (cucurbitacin I) (white crystal) and 5.1 g of compound 5 (cucurbitacin E glucoside) were obtained in the section 9. Using chloroform:methanol (5:1) as the mobile phase by gel column chromatography (2.5 cm (inside diameter) × 65 cm) or by Sephadex LH-20 column (1.5 cm (inner diameter) × 36 cm) The fraction 11 obtained by using hexane: EtOAc 1:1 eluent was purified, and 0.14 g of Compound 6 (cucurbitacin I glucoside) was obtained. Purification of fraction 12 obtained with EtOAc eluent by hydrazine gel column chromatography (2.5 cm (inside diameter) x 65 cm) using chloroform:methanol (20:1) as the mobile phase afforded 19 mg of compound 7 5-O-glucoside). The insoluble matter from the partitioning portion 12 was filtered to obtain 3.79 g of Compound 8 (mangiferin).

Figure 1 shows the flow of separating active ingredients (compounds 1 to 8) from agarwood.

Example 3 Identification of Chemical Structure of Compounds Contained in Agarwood Extracts of the Invention

The methanol extract of the agarwood shell prepared in Example 1 (the ethyl acetate fraction and the water fraction thereof), the methanol extract of the agarwood leaf, the ethyl acetate extract of the agarwood stem and the methanol extract of the agarwood seed were dissolved in methanol. (5 mg/ml or 1 mg/ml) for HPLC analysis.

HPLC analysis conditions:

Column: LiChrospher 100 RP-18e (4 mm (inside diameter) × 250 mm, 5 μm)

Column temperature: 40 ° C

Flow rate: 1.0 ml/min

Wavelength: 236 nm

Mobile phase: 0.05% TFA/MeOH (42:58, fixed ratio) for cucurbitacin E (residence time 19.85 minutes), cucurbitacin I (residence time 10.33 minutes), cucurbitacin E glucoside (residence time 8.76 minutes) Cucurbitacin I glucose (residence time 5.15 minutes) and saponin 5-O-glucoside (residence time 4.06 minutes); 0.05% TFA-MeOH (75:25, fixed ratio) for mangiferin (residence time 13.40) minute). The results of HPLC chromatography are shown in Figure 2 (2-1 to 2-6).

Compound 1 was detected by GC-MS to determine its molecular weight and Compound 1 was detected by 126 MHz ¹³ C-NMR to determine its structure. ^{The 13} C NMR spectrum data is shown in Table 1. Compound 1 is a beta-phytosterol.

Compound 3 was detected by 500 MHz ¹ H-NMR and 126 MHz ¹³ C-NMR to determine its structure. The results shown in Table 2 indicate that Compound 3 is cucurbitacin E.

The framework of Compound 4 is very similar to Compound 3 according to the 500 MHz ¹ H-NNR and 126 MHz ¹³ C-NMR spectra (see Table 3 below). According to the HMBC and HMQC spectra, Compound 4 was found to have one ethyl fluorenyl group on the side chain of cucurbitacin E. Compound 4 is cucurbitacin I.

According to the NMR spectrum (see Table 4 below), Compound 5 is a cucurbitacin E compound. The OH on carbon 2 is replaced by O-glycoside, so compound 5 is cucurbitacin E glucoside.

According to the NMR spectrum (see Table 5 below), Compound 6 was similar in structure to cucurbitacin I. Compound 6 has one more glucose than cucurbitacin I. Compound 6 is also similar to cucurbitacin E glucoside and one less ethyl. Compound 6 is cucurbitacin I glucoside.

According to the NMR spectrum (see Table 6 below), Compound 2 is a flavonoid compound, lycopene, which is especially present in the genus Rutaceae. The framework of Compound 2 is similar to the framework of the flavonoid compound, but has OCH ₃ on C7 (δC 165.0) and chemical shift at 56.0 ppm.

According to the NMR spectrum (see Table 7 below), Compound 7 has a framework of the flavonoid compound saponin which further has a substituent O-glucoside on C5 (δ 102.7). Compound 7 is saponin 5-O-glucoside.

According to the NMR spectrum (see Table 8 below), compound 8 is a xanthone structure in which a C-glycosid is attached to carbon 2. According to HMBC and ¹ H- ¹ H COSY, it can be inferred that compound 8 is mangiferin.

Example 5 Effect of Cucurbitacin E and Cucurbitacin I on the Cytotoxicity of Agarwood Husk Extract

The effects of cucurbitacin E and cucurbitacin I on the cytotoxicity of HT29 cells in agarwood husk extract were tested. HT cells were treated with diluted cucurbitacin E and cucurbitacin I (100-1.56 μg/ml). After treating the cells for 24 hours, the cell viability was determined by quantitatively measuring the MTT assay of the metabolic activity of the living cells. The results showed that the concentrations of cucurbitacin E and cucurbitacin I above 14.1 μM and 15.8 μM, respectively, significantly reduced the cell viability of HT29 cells. See Table 9. The results clearly indicate that both cucurbitacin E and cucurbitacin I have effective toxicity against HT29 cancer cells. Further, when the HT29 cells were incubated with the ethyl acetate fraction and the water fraction of 100 μg/ml of the methanol extract of the agarwood husk of the present invention for 24 hours, the ethyl acetate fraction exhibited better toxicity than the water fraction. (See Figure 3).

In addition to HT29 cells, P-388 leukemia cells were also assayed by MTT assay. The results showed that cucurbitacin E and cucurbitacin I concentrations higher than 6.5 μM and 7.4 μM, respectively, inhibited cell proliferation of P-388 cells. See Table 9.

Example 6 Effect of Cucurbitacin E and Cucurbitacin I on the Cell Morphology of HT29 Cells in Agarwood Husk Extract

The effects of cucurbitacin E and cucurbitacin I on the cell morphology of HT29 cells were measured. HT cells were treated with different concentrations of cucurbitacin E or cucurbitacin for 24 hours and stained with 10% Giemsa. The change in HT cells was measured under a light microscope and shown in Fig. 4 (4-1 to 4-3), and the results showed that as the concentrations of cucurbitacin E and cucurbitacin I were increased, the nuclei were squeezed and chromatin was concentrated.

Example 7 Effect of Cucurbitacin E and Cucurbitacin I on the Cell Cycle of HT29 Cells in Agarwood Husk Extract

The cell cycle of the treated cells was studied by flow cytometry. The effects of cucurbitacin E and cucurbitacin I on the cell cycle distribution of HT29 cells in the extract of Agarwood husk were shown in Figure 5 (5-1 to 5-2). Changes in the cell cycle profile of HT29 cells after treatment with cucurbitacin E and cucurbitacin I were dose-dependent. In HT29 cells, with increasing concentrations of cucurbitacin E and cucurbitacin I, the sub-G1 population increased, indicating that treatment with cucurbitacin E and cucurbitacin I increased cell death.

Example 8 Effect of Administration of Agarwood Husk Extract on CDF1 Mice

The potential of the agarwood husk extract to inhibit cancer progression in vivo was tested in CDF1 mice with P-388D1. Male DBA mice and female BALB/c mice were purchased from the National Science Council (Taipei, Taiwan) and were raised by the present inventors. After mating, the first generation of male CDF ₁ (eight weeks old, body weight 25 ± 2 g) was obtained for the study. To induce cancer, each mouse was subcutaneously inoculated with 0.2 ml of 1 x 10 ⁶ P-388 cells/ml. One hour after the inoculation, the mice were assigned to three groups and subjected to subcutaneous administration to receive a solvent control (Daunorubicin 1 mg/kg/day) or different doses (15 and 30 mg/kg/ The agarwood shell extract of the day) lasted for 9 days. Body weight was measured and shown in Figure 6. The number of days of survival of the tested mice is shown in Table 10.

Student's - t test (Student's-t test): * p <0.05

Life extension percentage

ILS%=[(T-C)/C]×100%

Control: DMSO (0.1%)

T: average survival days of the treatment group

C: average survival days of the control group

The results indicate that administration of the agarwood husk extract of the present invention significantly increases the number of days of survival in mice. The effect of the extract of the invention on lifespan is dose dependent. As shown in Figure 6, the extract of the present invention at a dose of 30/mg/kg significantly inhibited the progression of ascites.

Example 9 Effect of extract of agarwood shell on cytotoxicity

Test Agarwood Husk Extract for T24 cells (human bladder cancer cells), HT29 cells (human colon adenocarcinoma cells), HeLa cells (cervical cancer cells), AGS cells (stomach epithelial cancer cells), and Hep G2 The cytotoxic effects of cells (human hepatocellular carcinoma cells). The cells were treated with agarwood husk extract. The cytotoxicity test procedure was the same as that mentioned in Example 5. Sinensis extract husk for the IC ₅₀ values of the cells shown in Table 11 below.

Example 10 Separation of Cucurbitacin by Centrifugal Partition Chromatography (CPC) from the Ethyl Acetate Layer of the Agarwater Shell Extract

The ethyl acetate layer of the methanol extract of the agarwood husk was obtained according to the materials and methods mentioned in Example 1. A solvent system containing chloroform-methanol-water (6:7:4, v/v/v) was used in CPC. CPC is a method based on countercurrent chromatography (CCC). Separation is based on the difference in the distribution characteristics of the components between the two immiscible liquids. The phase remaining in the column is called the stationary phase and the other phase is called the mobile phase. The ethyl acetate extract of 0.75 g of agarwood husk was dissolved in 4 ml of chloroform-methanol-water. After 60 minutes of CPC, the organic phase was used as the stationary phase and water was used as the mobile phase at a flow rate of 10.0 ml/min. After 60 minutes, the organic phase was used as the mobile phase. The centrifugation rate was 700 rpm. The ethyl acetate sample was subjected to CPC for distribution and then detected by a UV detector (236 nm) (see Figure 7). Nine kinds of divisions (see Fig. 8) were obtained, in which the division portion 1 was obtained in 0 to 14 minutes, the division portion 2 containing 82.49% mangiferin was obtained in 15-18 minutes, and the division portion 3 was obtained in 19 to 22 minutes. Obtaining part 4 containing 73.54% cucurbitacin I glycoside in 23-26 minutes, obtaining part 5 containing 63.24% cucurbitacin E glycoside in 27-38 minutes, and obtaining 80.09% cucurbit in 39-63 minutes The dividing portion 6 of the prime I obtains the partitioning portion 7 containing β-phytosterol in 64-65 minutes, and obtains the dividing portion 8 containing 76.87% of cucurbitacin E in 66-75 minutes and in the last 76-80 minutes. The division section 9 is obtained.

The recovery was 0.749 g and the recovery was 99.87%. Molecular sieve chromatography can be further used to increase the purity of the above compounds. Sephadex LH-20 gel (1.5 cm (inside diameter) × 36 cm) was used in molecular sieve chromatography and methanol was used as the mobile phase. The purity of cucurbitin I glucoside in fraction 4 increased from 73.54% to 92.57%. The purity of cucurbitin E glucoside in fraction 5 increased from 63.24% to 90.24% and the purity of cucurbitin I increased from 80.09% to 95.36%. . Since mangiferin is difficult to dissolve in methanol, the mangiferin in the fraction 2 is partitioned and then centrifuged to obtain mangiferin whose purity is increased from 82.49% to 96.25%. Cucurbitacin E is difficult to dissolve in water and methanol, and cucurbitacin E in the fraction 8 is distributed and then centrifuged to obtain mangiferin having a purity of 96.87%.

Figure 1 shows the flow of separating active ingredients (compounds 1 to 8) from agarwood.

Figure 2 (2-1 to 2-6) shows cucurbitacin E (residence time 19.85 minutes) (Fig. 2-1), cucurbitacin I (residence time 10.33 minutes) (Fig. 2-2), cucurbitacin E glucoside (Fig. 2-2) Retention time 8.76 minutes) (Figure 2-3), cucurbitacin I glucoside (residence time 5.15 minutes) (Figure 2-4), safflower 5-O-glucoside (residence time 4.06 minutes) (Figure 2-5 And HPLC chromatography of mangiferin (residence time 13.40 minutes) (Figures 2-6).

Figure 3 illustrates that the ethyl acetate layer portion of the agarwood exhibits better toxicity than the aqueous portion of the agarwood.

Figure 4 (4-1 to 4-3) shows that as the concentration of cucurbitacin E and cucurbitacin I increases, the nucleus is squeezed and the chromatin is concentrated.

Figure 5 (5-1 to 5-2) shows the effect of agarwood extract (cucurbitacin E and cucurbitacin I) on the cell cycle distribution of HT29 cells.

Figure 6 illustrates that the extract of the present invention at a dose of 30/mg/kg significantly inhibits the progression of ascites.

Figure 7 shows a centrifugal distribution chromatogram using a chloroform-methanol-water solvent system.

Figure 8 shows a centrifugal distribution chromatogram using a chloroform-methanol-water solvent system, wherein numbers 1-8 indicate divisions 1 to 8.

Claims (16)

An aqueous extract of a woody husk of a therapeutically effective amount of agarwood ( Aquilaria agallocha ) for treating cancer, wherein the extract is obtained by extracting agarwood ( Aquilaria agallocha ) by methanol extraction followed by partitioning with ethyl acetate and water. The ethyl acetate layer was further dissolved by a hexane-ethyl acetate mixture and methanol by a silica gel column chromatography. The extract of claim 1, wherein the ratio is (1), 1:0, 10:1, 10:2, 10:3, 10:4, 10:5, 10:6 by gel column chromatography. The hexane-ethyl acetate mixture of 10:8, 1:1 and 0:1 and (ii) the mobile phase of methanol were further dissolved in the ethyl acetate layer to obtain 13 kinds of partitions. The extract of claim 2, wherein the fractions of the 10:5 and 10:6 fractions contain cucurbitacin E and cucurbitacin I. The extract of claim 2, wherein the partitioning portion 3 (10:2) contains β-phytosterol, the dividing portion 7 (10:5) contains ellagdin, and the dividing portion 8 (10:5) contains cucurbitacin E, The dividing portion 9 (10:5) contains cucurbitacin I and cucurbitacin E glucoside, and the dividing portion 11 (1:1) contains cucurbitacin I glucoside and the dividing portion 12 (0:1) contains scutellarin 5-O- Glucosides and mangifersin. The extract of claim 1, wherein the ethyl acetate layer is further partitioned by centrifugal partition chromatography (CPC) to obtain nine kinds of divisions. The extract of claim 4, wherein the dividing portion 2 contains mangiferin, the dividing portion 4 contains cucurbitacin I glucoside, the dividing portion 5 contains cucurbitacin E glucoside, the dividing portion 6 contains cucurbitacin I, and the dividing portion 7 contains β- The phytosterol and the partitioning portion 8 contain cucurbitacin E. An extract according to claim 6 which can be further purified by molecular sieve chromatography These divisions are made. The extract of claim 1, wherein the aqueous extract of the woody husk of the fruit of the Aquilaria agallocha is from Aquilaria agallocha Roxb. A pharmaceutical formulation consisting essentially of the extract of claim 1 and a pharmaceutically acceptable carrier. The pharmaceutical formulation of claim 9 for oral, rectal administration, topical administration, buccal administration, parenteral administration, and transdermal administration. The pharmaceutical formulation of claim 10, which is in a form selected from the group consisting of a capsule, a blister, a lozenge or lozenge, a powder, a granule, a solution, a suspension or an emulsion. A use of the extract of claim 1 for the preparation of a medicament for killing cancer cells. A use of the extract of claim 1 for the preparation of a medicament for the treatment of cancer. Use of the extract of claim 1 for the preparation of a medicament for inducing apoptosis in cancer cells. The use of claim 12, 13 or 14, wherein the cancer is selected from the group consisting of colorectal cancer, gastric cancer, prostate cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer, colon cancer, cervical cancer, and hepatocellular carcinoma. The use of claim 15, wherein the cancer is bladder cancer, colon cancer, cervical cancer, gastric cancer, and hepatocellular carcinoma.