HK1084102B - Compounds isolated from gamboge resin having activity in inhibiting the growth of tumor/cancer cells and pharmaceutical compositions comprising the same - Google Patents

Description

Compounds isolated from gamboge resin and having activity of inhibiting tumor/cancer cell growth and pharmaceutical compositions comprising the same

Technical Field

The present invention relates to acetone extraction products from gamboge resin (gamboge resin) and compounds further purified from the acetone extraction products, including novel compounds named formoxathone a, confirming the activity of the acetone extraction products and the further purified compounds in inhibiting tumor/cancer cell growth. The invention also relates to a method for obtaining the further purified compounds, and the use of the acetone extraction product and the further purified compounds for preparing a pharmaceutical composition for inhibiting tumor/cancer cell growth.

Background

Garcinia resin is a colloid resin (the gamboges of Garcinia sp.) secreted by plants of Garcinia of family Fucaceae (Guttiferae), which has been used as a source of vegetable dyes and pigments since ancient times, and is a folk medicine in some areas such as India, Thailand and the like.

Gamboge (latin name: Garcinia morella Desv; TENGHUANG for chinese pinyin; gamcogoe for english name) is an evergreen tree, which grows in tropical regions, mainly Garcinia morella Desv in india and g.harburyi Hook in thailand. Before blooming, the bark of the stem is cut spirally at about 2m from the ground, and the resin flowing out is collected and heated to dry, thus obtaining the solid gamboge resin.

According to the record of Traditional Chinese Medicine (TCM) classics, gamboge has the effects of diminishing swelling, removing toxicity, stopping bleeding and killing parasites. Since 1934 there have been many reports of studies on the components contained in gamboge resins and it is known that many compounds can be isolated from extracts of gamboge resins, including: gambogin (morellin), gambogic acid (morellicacid), gamboge resin acid (gambogic acid), gambogol (morellinol), isogambogin (isomorelin), isogambogic acid (isomorelic acid), isogamboge resin acid (isogambogic acid), isogambogol (isomorenol), neogamboge resin acid (neogambogic acid), desoxygambogic (desoxymedellin), dihydroisogambogin (dihydroisocoryllin), alpha-gambogin (alpha-guttiferin), and beta-gambogin (beta-guttiferin), and the like.

It has been reported in the literature that Garcinia resin contains certain components having cytotoxic activity against human lung cancer cells (HELcell), human cervical cancer cells (HeLa cells), human nasopharyngeal cancer cells (KB cells), etc. (M.Tada et al, (1996), Phytochemistry, 41, 815 & 920; G.A.Cordell et al, (1993), Magnetic research in Chemistry, 31, 340 & 347).

It has also been reported that, a chinese folk medicine, the root of cyperus rotundus (anerone raddeana) is useful for the treatment of rheumatism and neuralgia (neuralgis), and betulin and betulinic acid can be isolated from an ethanol extract of the root of cyperus rotundus, wherein betulin is shown to prevent tyrosyl phosphorylation of proteins in human leukocytes, thereby inhibiting superoxide production (k. yamashita et al, (2002), clinical Chimica Acta, 325, 91-96).

Darrick S.H.L.Kim et al have reported in Bioorganic & Medical chemistry letters (1998), 8, 1707-1712 that simple modifications to the parent structure of betulinic acid can lead to derivatives that can be developed as potent anticancer agents. Betulinic acid has also been reported to trigger apoptosis by acting directly on mitochondria (Simone Fulda et al, (1998), The Journal of Biological Chemistry, 278, 33942-33948) and to be selectively cytotoxic to tumor cells (Valentina Zuco et al, (2002), Cancer Letters, 175, 17-25).

To the best of the applicant's knowledge, no document or patent has so far disclosed the isolation of betulin and betulinic acid from gamboge resins.

US 6462041 discloses gamboge resin acids and analogues and derivatives thereof represented by the following chemical formula I, II or III:

(wherein R is₁To R₅As defined in that case),

the compounds of formula I, II or III are disclosed as activators of cysteamine proteases (caspases) and inducers of apoptosis.

EP 0428815 a1 discloses a process for extracting cytolytic toxins from a resin of Garcinia morella Desv, which involves the use of ethanol with chloroform and Thin Layer Chromatography (TLC) to give the product designated GMD 1630. However, this patent publication does not disclose whether the GMD1630 product comprises a single compound or a mixture of compounds with similar properties.

Based on the above, it would be of great industrial value to prepare biologically active extracts or pure compounds from gamboge resin for the manufacture of drugs such as anticancer drugs.

Disclosure of Invention

The invention provides an acetone extraction product of gamboge resin, and the acetone extraction product can be further purified to obtain the following 9 compounds: betulin, betulinic acid, gambogic acid, isogambogic acid, gambogic resin acid, isogambogic alcohol, deoxygamboge, and a novel compound named formoxanthenone a.

It was confirmed that the acetone-extracted products of gamboge resin according to the present invention and these further purified compounds were effective in inhibiting the growth of tumor/cancer cells. Accordingly, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any one of:

(i) extracting the gamboge resin with acetone to obtain a product;

(ii) formoxanthone a;

(iii) the formoxanthone A and at least one compound selected from betulin, betulinic acid, gambogic acid, isogambogic alcohol and deoxygamboge.

Drawings

The above and other objects and features of the present invention will become more apparent by referring to the following description, appended claims and accompanying drawings, when taken in conjunction with the accompanying drawings and embodiments, in which:

FIG. 1 shows the HPLC elution profile of the acetone extraction product of Garcinia resin according to the present invention, hereafter designated TSB-14;

FIG. 2 is an HPLC elution profile in which 7 products purified from Garcinia resin obtained according to the present invention (which will be hereinafter designated as TSB-2 to TSB-8, respectively) are used as standards for HPLC analysis, and the numbers on the respective peaks correspond to the numbers of TSB-2 to TSB-8 of these products, respectively;

FIG. 3 is an HPLC elution profile showing the comparison of FIGS. 1 and 2 to show that the 7 products TSB-2 to TSB-8 purified from Garcinia resin obtained according to the present invention correspond to those peaks in the elution profile of TSB-14, wherein the sample concentration of the product TSB-14 is adjusted higher to facilitate the comparison;

FIG. 4 is an HPLC elution profile showing the HPLC results of product TSB-14 according to the present invention (upper) and product TSB-14 plus 500 μ L each of product TSB-4 acetone solution and product TSB-8 acetone solution (lower) to identify the elution peaks corresponding to products TSB-4 and TSB-8; and

FIG. 5 is an HPLC elution profile showing the corresponding positions of the 7 products TSB-2 to TSB-8 purified from Garcinia resin obtained according to the present invention appearing in the elution profile of the product TSB-14 according to the present invention.

Detailed Description

Based on the solubility of the gamboge resin powder in the extraction solvent and the boiling point and safety of the extraction solvent, acetone was likely the best choice, and the applicant then attempted to extract the gamboge resin with acetone, followed by removal of the acetone, to give an acetone extracted product, designated TSB-14.

The acetone extraction product TSB-14 is further fractionated mainly by column chromatography, and the column packing used is preferably silica gel and Sephadex LH-20, which are generally used as column packing for adsorption matrix and size exclusion chromatography.

The acetone extract product TSB-14 was first subjected to adsorption chromatography on silica gel and eluted with an n-hexane/ethyl acetate gradient (100/0 → 4/1), i.e., first eluted with n-hexane and then the elution gradient of ethyl acetate was gradually increased to obtain 3 fractions, i.e., GME1, GME2 and GME3, wherein GME1 was the elution fraction of 100/0 → 10/1, GME2 was the elution fraction of 10/1 → 4/1 and GME3 was the elution fraction of 4/1 → 1/1, as described in example 2 below.

The elution solvent from the GME1, GME2 and GME3 fractions described above were removed by vacuum rotary evaporator.

In the fractionation, detection of each fraction is carried out by Thin Layer Chromatography (TLC) with a color-developing reagent, and separation of components in each fraction is examined with reference to a general procedure.

This fraction GME2 was then subjected to molecular sieve chromatography on a column packed with Sephadex LH-20. After elution with n-hexane/ethyl acetate/methanol (2: 1), according to TLC detection, the 23 rd vial portion was subjected to a repetition of molecular sieve chromatography (n-hexane/ethyl acetate/methanol, 2: 1) followed by silica gel column chromatography (n-hexane/chloroform/methanol, gradient elution 1: 0 → 0: 10: 1), whereas two colorless needle-like crystal products were obtained in succession during silica gel column chromatography, designated TSB-0 and TSB-1, respectively. In addition, a 46 th vial was subjected to Thin Layer Chromatography (TLC), and a portion corresponding to a pink color point indicated by a coloring reagent (see example 2 and Table 1 below) on the TLC plate was removed to repeat molecular sieve chromatography, silica gel column chromatography, and finally preparative TLC was performed to obtain two products, which were designated TSB-6 and TSB-7, respectively. Then, the 47 th flask was subjected to TLC, and a portion corresponding to a pink color point (see example 2 and Table 1 below) indicated by the coloring agent on the TLC plate was removed to repeat the molecular sieve chromatography and the silica gel column chromatography, and finally, the molecular sieve chromatography and the recrystallization were further performed to obtain orange-yellow plate crystals designated as TSB-2. The crude product was subjected to silica gel column chromatography in flask 63, eluted with dichloromethane/methanol (30: 1), and subjected to preparative thin layer chromatography (n-hexane/ethyl acetate, 2: 1) and recrystallization to give a yellow needle-like crystal product designated TSB-4.

Subjecting the part of GME1 to silica gel column chromatography, eluting with n-hexane/acetone (5: 1), subjecting the 26 th bottle to molecular sieve column chromatography (n-hexane/ethyl acetate/methanol, 2: 1), and subjecting to preparative thin layer chromatography (n-hexane/ethyl acetate, 4: 1) to obtain two products, named as TSB-5 and TSB-3. Further, each of the 23 rd and 51 th bottles was subjected to silica gel column chromatography eluting with n-hexane/dichloromethane (1: 1) and gradient elution with n-hexane and n-hexane/ethyl acetate (7: 1), followed by TLC and taking out and combining the portions showing the same Rf value on the TLC plates and showing pink color points, followed by silica gel column chromatography eluting with dichloromethane, followed by TLC and collecting the portions showing pink color points on the TLC plates, followed by chromatography (including silica gel and molecular sieve column chromatography), and finally preparative thin layer chromatography and recrystallization to give an orange needle-like crystal product designated TSB-8.

Purifying the 9 kinds of the raw materials to obtainThe product obtained is subjected to spectral analysis including infrared spectroscopy, nuclear magnetic resonance spectroscopy (f)¹H-and¹³C-NMR), mass spectrum, etc.

The products TSB-0 and TSB-1 are identified by chemical structure identification as two known betulinic acid derivatives, namely betulin and betulinic acid, which respectively have the following chemical formulas:

TSB-0 (betulin) TSB-1 (betulinic acid)

The products TSB-7 and TSB-2 were identified by chemical structure as two known stereoisomers of gambogic acid, i.e., gambogic acid and isogambogic acid, which have the following chemical formulas:

TSB-7 (gambogic acid) TSB-2 (Isogambogic acid)

The products TSB-5 and TSB-3 were identified by chemical structure identification as two known stereoisomers of gambogic acid, i.e. gambogic acid and isogambogic acid, having the following respective chemical formulae:

TSB-5 (gamboge resin acid) TSB-3 (isogamboge resin acid)

The product TSB-6 was identified by chemical structure as isomannide, which has the following formula:

TSB-6 (Isogamboge)

The product TSB-8 is confirmed to be deoxyluteolin after chemical structure identification, and has the following chemical formula:

TSB-8 (deoxyluteolin)

The chemical structure identification and the comparison result with the spectral data of the known compound confirm that TSB-4 is a novel compound which is not reported in the literature, is named as formoxanthone A and has the following chemical formula:

the product TSB-4 (formoxanthone a) obtained from a gamboge resin according to the present invention has, on the xanthone skeleton: (1)3 hydroxyl groups are respectively positioned at the C-1, C-3 and C-5 positions; (2)1 gamma, gamma-dimethylallyl chain position at the C-4 position; (3) the 12, 2-dimethyl pyran ring is positioned at the C-6 and C-7 positions; (4)1 disubstituted double bond between the C-11 and C-12 positions and 1 trisubstituted double bond between the C-17 and C-18 positions.

According to the above chemical structure, a formoxanthone A derivative having the following general formula can be prepared through a chemical reaction:

wherein each R independently represents: hydrogen; is optionally substitutedLower alkyl of (a), preferably having 1 to 6 carbon atoms; aryl optionally substituted and having 3 to 20 carbon atoms, preferably C₅-C₁₂An aryl group; aralkyl optionally substituted and having 3 to 30 carbon atoms, preferably (C)₅-C₁₂) Aryl radical (C)₁-C₆) Alkyl, more preferably (C)₆) Aryl radical (C)₁-C₃) An alkyl group; or an acyl group of the formula Ra-CO, wherein Ra is: optionally substituted lower alkyl, preferably having 1 to 6 carbon atoms; aryl optionally substituted and having 3 to 20 carbon atoms, preferably C₅-C₁₂An aryl group; aralkyl optionally substituted and having 3 to 30 carbon atoms, preferably (C)₅-C₁₂) Aryl radical (C)₁-C₆) Alkyl, more preferably (C)₆) Aryl radical (C)₁-C₃) An alkyl group.

For example, since the above-mentioned hydroxyl groups are all located on an aromatic ring, a methoxy derivative may be obtained by methylation, or an acetyl derivative may be obtained by acetylation. Alternatively, the double bond located outside the xanthone ring may be treated by hydrogenation to form a hydrogenated derivative, or by other addition reactions, such as hydration to form an alcohol-based derivative, and peroxidation to form an epoxide derivative, such as those of the formula:

the applicant further investigated the biological activities of the acetone-extracted products of the gamboge resin obtained in the present invention, the 9 products (TSB-0 to TSB-8) obtained by further purifying the acetone-extracted products, and the mixture composed of these purified products, and found that the acetone-extracted products, the products TSB-2 to TSB-8, and the mixture composed of these products are effective in inhibiting the growth of tumor/cancer cells (e.g., liver cancer cells, lung cancer cells, breast cancer cells, colorectal cancer cells, leukemia cells, lymphoma cells, etc.). The products TSB-0 and TSB-1 may not be as effective as the products TSB-2 to TSB-8 in inhibiting the growth of tumor/cancer cells, but the applicants have found that when they are used in combination with other products, the products TSB-0 and TSB-1 appear to exhibit utility in activating the other products, and thus it is expected that the products TSB-0 and TSB-1 may be used as a drug for introduction within a pharmaceutical composition containing at least one of the products TSB-2 to TSB-8.

According to the literature, betulin and betulinic acid, which are triterpenes, have diverse pharmacological activities, including being effective in human melanoma cancer cells in vitro and in vivo, and they are excellent in safety. Thus, when used in combination with the products TSB-2 to TSB-8 belonging to the xanthone group, the two triterpene compounds TSB-0 and TSB-1 should have auxiliary or synergistic effects.

Accordingly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of any one of:

(i) extracting the gamboge resin with acetone to obtain a product;

(ii) formoxanthone a;

(iii) the formoxanthone A and at least one compound selected from betulin, betulinic acid, gambogic acid, isogambogic alcohol and deoxygamboge.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising the product of extracting gamboge resin with acetone.

In another preferred embodiment, the present invention provides a pharmaceutical composition comprising formoxanthenone a and at least one compound selected from betulin, betulinic acid, gambogic acid, isogambogic acid, gambogic resin acid, isogambogic alcohol and deoxygamboge.

In a more preferred embodiment, the present invention provides a pharmaceutical composition comprising formoxanthenone a, gambogic acid, isogambogic alcohol and deoxygamboge, betulin and betulinic acid. In a more preferred embodiment, the present invention provides a pharmaceutical composition comprising 5% formoxanthone a, 8% gambogic acid, 8% isogambogic acid, 18% gambogic acid, 15% isogambogic acid, 2% isogambogic alcohol and 4% deoxygambogic acid, 8% betulin and 8% betulinic acid as active ingredients, by weight of the composition.

The 9 products separated and purified from gamboge resin of the present invention comprise gamboge, deoxygamboge, two betulinic acid derivatives, namely betulin and betulinic acid, and two sets of stereoisomers, namely gambogic acid and isogambogic acid and gamboge resin acid and isogamboge resin acid. Furthermore, according to the experimental results of extraction and separation of compounds, the highest content of two stereoisomers, gambogic acid and isogambogic acid, followed by two stereoisomers, gambogic acid and isogambogic acid.

The applicant has found that the 9 products can be combined according to the structural characteristics of the compounds, and that each combination of the 9 products can show excellent effect of inhibiting the growth of cancer cells. Therefore, referring to the preliminary pharmacological test results and considering the industrial application of the above 9 products, the above 9 products can be formulated into pharmaceutical compositions having different active ingredients and composition ratios with a desired effect.

Thus, in a preferred embodiment, the present invention provides a pharmaceutical composition comprising formoxanthenone a, betulinic acid and any one of the following combinations of compounds:

(1) isoresinic acid, resinic acid and deoxygambogic acid;

(2) isogambogic acid, gambogic acid and isogambogic alcohol;

(3) gambogic acid, gambogic acid and deoxygambogic acid;

(4) isogambogic acid, isogambogic acid and isogambogic alcohol.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising 5% of formoxanthone a, 10% of betulinic acid, 20% of isogambogic acid, 50% of gambogic acid and 5% of deoxygamboge as active ingredients, based on the weight of the composition.

In another preferred embodiment, the present invention provides a pharmaceutical composition comprising 5% of formoxanthone a, 10% of betulinic acid, 20% of isogambogic acid, 50% of gambogic acid and 5% of isogambogol as active ingredients, by weight of the composition.

In yet another preferred embodiment, the present invention provides a pharmaceutical composition comprising 5% of formoxanthone a, 10% of betulinic acid, 40% of gamboge resin acid, 30% of gambogic acid and 5% of deoxygamboge as active ingredients, based on the weight of the composition.

In another preferred embodiment, the present invention provides a pharmaceutical composition comprising 5% of formoxanthone a, 10% of betulinic acid, 40% of isogambogic acid, 30% of isogambogic acid and 5% of isogambogol as active ingredients, based on the weight of the composition.

The pharmaceutical compositions according to the present invention may be formulated into suitable pharmaceutical forms for parenteral, topical or oral administration using techniques well known to those skilled in the art, including, but not limited to, injections (e.g., sterile aqueous solutions or dispersions), sterile powders, lozenges, tablets, pills, capsules, and the like. In addition, the bioactive components according to the present invention may be incorporated into sustained release preparations and formulations.

Optionally, the pharmaceutical composition of the present invention may additionally comprise pharmaceutically acceptable carriers widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more of the following agents: solvents (e.g., water, physiological saline, buffer solutions, glycerin, organic solvents), emulsifiers, suspending agents, disintegrating agents, binders, excipients, stabilizers, preservatives, lubricants, absorption delaying agents, liposomes, and the like.

For the production of oral solid preparations, excipients and, if desired, binders, disintegrants, lubricants, colorants, flavourings and/or the like can be mixed with the gamboge resin extract according to the invention. The resulting mixture may then be formed into tablets, coated tablets, granules, powders, capsules or the like by methods known per se in the art. These additives may be those generally used in the art today, including excipients: sugars [ such as glucose, lactose, sucrose, brown sugar, sorbitol, mannitol, starch ], sodium chloride, calcium carbonate, kaolin, microcrystalline cellulose and silicic acid; adhesive: water, ethanol, propanol, sucrose solution, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate and polyvinylpyrrolidone; disintegrating agent: dry starch, sodium alginate, powdered agar, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, glycerol monostearate and lactose; lubricant: purified talc, stearate, borax and polyethylene glycol; and a flavoring agent: sucrose, bitter orange peel, citric acid and tartaric acid.

In a preferred embodiment according to the present invention, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable excipient, and the excipient comprises at least one saccharide selected from the group consisting of sucrose, brown sugar, lactose, sorbitol, mannitol, corn starch, and crystalline cellulose. When sugars are used as excipients, they have the effect of assisting dissolution in addition to enhancing the properties of the powdered drug compound.

To manufacture an oral liquid product, flavors, buffers, stabilizers and the like may be mixed with the gamboge resin extract according to the present invention. The resulting mixture may then be formed into a solution, syrup, elixir or the like for oral use by methods known per se in the art. In this case, the flavouring agent may be the same as that previously mentioned. An exemplary buffering agent is sodium citrate, and exemplary stabilizing agents are gum tragacanth, gum acacia, and gelatin.

For the manufacture of injectables, pH regulators, buffers, stabilizers, isotonicity agents (isotonicity) and the like can be mixed with the gamboge resin extract according to the present invention. The resulting mixture may then be formed into a subcutaneous, intramuscular or intravenous injection by methods known per se in the art. Examples of the pH control agent and buffer include sodium citrate, sodium acetate, and sodium sulfate. Exemplary of such stabilizers include sodium metabisulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid and thioglycolic acid. Examples of these isotonicity agents include sodium chloride and glucose.

As used herein, the term "therapeutically effective amount" refers to an amount sufficient to provide the desired therapeutic efficacy without causing undesirable serious harm to non-target tissues or organs when a pharmaceutical composition according to the present invention is administered to an individual in need of treatment with the composition. The therapeutically effective amount will vary depending on various factors including, for example, the type of disorder, the weight, age, physical condition and response of the individual to be treated, the route of administration of the drug, and the like. Such therapeutically effective amounts can be determined by one skilled in the art.

The dosage and frequency of administration of the pharmaceutical composition according to the present invention may vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the individual to be treated. For example, the pharmaceutical composition according to the present invention may be administered in a daily dose of 2.1 to 3.0mg/Kg body weight, and administered in a single dose or in divided doses, for example, in three doses each of 0.7 to 1.0mg/Kg body weight.

The pharmaceutical compositions according to the invention may be administered alone or in combination with other therapeutic methods or agents for the treatment of tumors or cancers. These treatments include chemotherapy and external beam radiation therapy (external beam radiation therapy), and these include, but are not limited to, paclitaxel (paclitaxel), cisplatin, carboplatin, cyclophosphamide, and doxorubicin.

The invention will be further described with respect to the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limiting the practice of the invention.

Examples

General operating procedures:

the infrared spectrum was measured using a JASCO FT-IR 5300 spectrometer.

The UV spectrum was measured using a HITACHI U-3000 spectrometer.

Mass Spectra (EIMS) were determined using a JEOL HX110 or JEOL SX-102A mass spectrometer.

Proton and carbon-13 nuclear magnetic resonance (¹H NMR & ¹³C NMR) was determined using BRUKERAM type 400, VARIAN GEMINI-400 or BRUKER ADVANCEDMX-600.

The column chromatography includes molecular sieve column chromatography using Sephadex LH-20(Pharmacia Fine Chemicals) and silica gel column chromatography using Merck Kiesegel 60(70-230 mesh ASTM).

Thin Layer Chromatography (TLC) plates using Merck Silica gel 60F₂₅₄Glass sheets (20X 20cm) with a layer thickness of 0.25mm can be cut to the appropriate size for use.

Preparation of thin layer chromatography (PLC) slides Using Merck Silica gel 60F₂₅₄Glass sheets (20X 20cM) with a thickness of 0.5mm or 1 mm.

The color development by Thin Layer Chromatography (TLC) used the following color development reagent formula: (a) ce (SO)₄)₂·4H₂O(0.8g)，(b)(NH₄)₆Mo₇O₂₄·4H₂O (20.0g), (c) concentrated sulfuric acid (23 mL); (d) distilled water (377 mL).

The color reaction was detected at short wavelength (254nm) and long wavelength (365nm) by irradiation with UV light.

The High Performance Liquid Chromatography (HPLC) instrument used was Rainin, type SD-200; the UV detector detects at a fixed wavelength of 360 nm; the analytical column is LiChrospher100RP-18e 5 μm (Merck); mobile phase: 90% acetonitrile and 10% deionized water with 0.05% trifluoroacetic acid (v/v); the flow rate is 1.0 ml/min; analysis conditions were as follows: the sample was dissolved in acetone to form the appropriate dilution and injected into the HPLC instrument.

Example 1 preparation of acetone extracted product derived from Garcinia resin

And (3) extraction procedures:

garcinia resin purchased from local shop as Chinese medicinal material was pulverized, and the powder (100g) was immersed in acetone solvent at room temperature for extraction (6X 500mL) for 24 hours each time. The combined extracts were filtered through filter paper and the filtrate was then freed of acetone solvent on a vacuum rotary evaporator to give a tan gummy solid (65g) (S.A. Ahmad et al, (1966), Journal Chemical Society (C), 772-.

The acetone extracted product obtained here is referred to as TSB-14, and HPLC analysis of the product TSB-14 to resolve the major compositional distribution of the product TSB-14 was carried out according to the general procedure described previously, resulting in an HPLC elution profile as shown in FIG. 1.

Example 2 preparation of purified Compounds derived from Garcinia resin

Experimental procedure:

the column chromatography described below was performed according to the general procedure described previously.

Silica gel column chromatography (column: 10cm diameter. times.80 cm length) was carried out with 35g of the product TSB-14 from example 1 above and eluted with an n-hexane/ethyl acetate gradient (100/0 → 4/1), the solvent was removed by vacuum rotary evaporator and 3 fractions GME1(10g), GME2(14g) and GME3(8g) were taken.

GME1 is a fraction collected by gradient elution to n-hexane/ethyl acetate (100/0 → 100/10), and the fraction containing TSB-5 (see below) as a main component and other components having an Rf value > TSB-5 was detected by TLC (developing solvent: dichloromethane/methanol, 20: 1).

GME2 is a fraction collected by gradient elution to n-hexane/ethyl acetate (10/1 → 4/1), and components including TSB-5 as a main component and TSB-7, TSB-6 and TSB-2 having an Rf value slightly smaller than TSB-5 were detected by TLC (developing solvent: dichloromethane/methanol, 20: 1).

GME3 is the fraction collected by gradient elution to n-hexane/ethyl acetate (4/1 → 1/1), TSB-5 is not detected by TLC (developing solvent: dichloromethane/methanol, 20: 1), and only contains other components with larger polarity and Rf value smaller than TSB-5, and tailing phenomenon exists.

This fraction, GME2(10g), was subjected to Sephadex LH-20 column chromatography (column: 5 cm. times.80 cm), herein referred to as A-column, eluting with n-hexane/ethyl acetate/methanol (2: 1) [ fraction size: 100mL ], the 23 rd vial was subjected to Sephadex LH-20 column chromatography (gradient of eluting solvent: n-hexane/chloroform/methanol, 2: 1) and silica gel column chromatography (gradient of eluting solvent: n-hexane/chloroform/methanol, 1: 0 → 0: 10: 1) to obtain two colorless needle crystals, TSB-0(320mg) and TSB-1(450mg), respectively, upon elution of silica gel column chromatography.

Further, the portion of the 46 th bottle obtained after the elution from the A column was subjected to thin layer chromatography (developing solvent: dichloromethane/methanol, 20: 1), and a portion corresponding to the pink spot exhibited by the coloring reagent on the TLC plate was taken out to repeat molecular sieve column chromatography (n-hexane/ethyl acetate/methanol, 2: 1) and silica gel column chromatography (eluting solvent: n-hexane/ethyl acetate, 4: 1), and finally preparative thin layer chromatography (developing solvent: dichloromethane/methanol, 100: 1) was carried out to obtain TSB-6(44mg) and TSB-7(220mg) as orange powders.

The 47 th vial fraction obtained after elution from the A column was subjected to thin layer chromatography (developing solvent: dichloromethane/methanol, 20: 1), and the fraction corresponding to the pink spot exhibited by the chromogenic reagent was taken out from the TLC plate and subjected to Sephadex LH-20 column chromatography (eluting solvent: n-hexane/ethyl acetate/methanol, 2: 1). The eluate was removed by vacuum concentration, and the residue was subjected to silica gel column chromatography (eluting solvent: n-hexane/ethyl acetate 4: 1; n-hexane/ethyl acetate 1: 0 → 10: 1 gradient elution; n-hexane/ethyl acetate/methanol 20: 3: 1), and finally to molecular sieve column chromatography (Sephadex LH-20, eluting solvent: n-hexane/ethyl acetate/methanol, 2: 1). The eluting solvent was removed by concentration in vacuo and the residue was recrystallized to give the product TSB-2 as orange yellow plate crystals (815 mg).

The fraction from the 63 rd flask obtained after the elution from the A column was subjected to silica gel column chromatography (elution solvent: dichloromethane/methanol, 30: 1), followed by TLC to extract a fraction corresponding to a yellow spot on the TLC plate (development solvent: dichloromethane/methanol, 20: 1) shown by the coloring reagent (after spraying the coloring reagent and heating, see general procedure and Table 1), the collected fractions were combined, the elution solvent was removed by a vacuum evaporator, and the residue was subjected to preparative thin layer chromatography (development solvent: n-hexane/ethyl acetate, 2: 1) to obtain TSB-4(52mg) as a yellow needle-like crystal.

This fraction of GME1(8g) was additionally subjected to silica gel column chromatography (column: 6cm × 80cm), herein referred to as B column, eluting with n-hexane/acetone (5: 1) [ fraction size: 100mL ], the 26 nd bottle was subjected to molecular sieve column chromatography (elution solvent: n-hexane/ethyl acetate/methanol, 2: 1), the elution solvent was removed by a vacuum evaporator, and the residue was subjected to preparative thin layer chromatography (developing solvent: n-hexane/ethyl acetate, 4: 1) to give TSB-5(1050mg) as an orange powder and TSB-3(850mg) as a dark yellow powder.

Further, the 23 rd bottle and the 51 th bottle obtained after elution of the B column were each subjected to silica gel column chromatography, the former was eluted with n-hexane/dichloromethane (1: 1), the latter was eluted with n-hexane → n-hexane/ethyl acetate (1: 0 → 7: 1) gradient, and the portions corresponding to the same Rf value on TLC plate (developing solvent: n-hexane/acetone, 6: 1) and shown as pink color spot by the color developing reagent were taken out by TLC, the collected portions were combined, and the eluting solvent was removed by a vacuum evaporator, the residue was subjected to silica gel column chromatography (eluting solvent: dichloromethane) elution, and then the portions corresponding to the pink color spot on TLC plate (developing solvent: n-hexane/acetone, 6: 1) were collected and combined by TLC, the eluting solvent was removed by a vacuum evaporator, the residue was subjected to molecular sieve column chromatography (eluting solvent: n-hexane/ethyl acetate/methanol, 2: 1) and the eluting solvent was removed with a vacuum evaporator and the residue was purified by preparative thin layer chromatography (developing solvent: n-hexane/dichloromethane, 2: 1) and recrystallisation gave the product TSB-8(60mg) as orange needle crystals.

Example 3 identification and characterization of compounds purified from gamboge resin:

experimental procedure:

analysis of the physicochemical Properties of the 9 Garcinia resin-derived products obtained in example 2, including Infrared Spectroscopy (IR), Nuclear magnetic resonance Spectroscopy: (IR)¹H-and¹³C-NMR), mass spectrometry (EIMS) and thin layer chromatography.

As a result:

the results of the coloration of the 9 products obtained in example 2 (TSB-0 to TSB-8) on thin-layer chromatography are shown in Table 1, in which the product TSB-4 was observed to appear yellow under the tests carried out.

TABLE 1 color development results of products derived from Garcinia resin on TLC plate

Product numbering	Colour under the naked eye after unfolding	Color under UV Lamp	Color at room temperature after spraying developer	Spraying color developing agent heated color
					TSB-0	Colorless and colorless	Colorless and colorless	Colorless and colorless	Blue color
TSB-1	Colorless and colorless	Colorless and colorless	Colorless and colorless	Blue color
					TSB-2	Yellow pink after standing	Yellow colour	Yellow colour	Pink colour
TSB-3	Yellow colour	Yellow colour	Yellow colour	Green colour
					TSB-4	Yellow colour	Yellow colour	Yellow colour	Yellow colour
TSB-5	Yellow colour	Yellow colour	Yellow colour	Green colour
					TSB-6	Yellow pink after standing	Yellow colour	Yellow colour	Pink colour
TSB-7	Yellow pink after standing	Yellow colour	Yellow colour	Pink colour
					TSB-8	Yellow pink after standing	Yellow colour	Yellow colour	Pink colour

From the observed TLC color behavior and physicochemical properties, the product TSB-4 was initially identified as a novel compound not described in the literature.

These 9 products were then detected by various spectroscopic analyses and the experimental numbers obtained were summarized as follows:

1. product TSB-0:

the TSB-0 properties of the product were determined as follows:

colorless needle-shaped crystals with a melting point of 257-259 ℃.

IR v_maxcm^-1(KBr)：3400，3090，2970，1640，1460，1380，1040，1020，880。

¹H NMR(CDCl₃400 MHz): δ 4.65(1H, d, J ═ 2.0Hz), 4.55(1H, d, J ═ 2.0Hz), 3.77(1H, d, J ═ 10.9Hz), 3.31(1H, br d, J ═ 10.9Hz), 3.16(1H, dd, J ═ 11.2, 5.0Hz), 1.66(3H, s), 1.01, 1.00, 0.95, 0.80, 0.74 (each 3H, s).

¹³C NMR(CDCl₃，100MHz)：δ150.62，109.74，79.14，60.80，55.57，50.67，49.05，47.96，42.92，41.16，38.99，38.93，37.56，37.36，34.48，34.10，30.02，29.42，28.09，27.58，27.27，25.48，21.01，19.19，18.44，16.13，15.37，14.87。

EIMS m/z (relative intensity): 442[ M ]]⁺(76)，427(11)，424(16)，411(77)，399(12)，385(14)，288(15)，273(6)，271(5)，257(9)，247(10)，245(10)，234(56)，220(30)，207(90)，203(80)，189(100)。

Based on the measured spectral data, the product TSB-0 was identified as a known compound having the chemical formula shown below, i.e., betulin:

2. product TSB-1:

the TSB-1 properties of the product were determined as follows:

colorless needle-shaped crystal with the melting point of 290-292 ℃.

IR v _max cm^-1(KBr)：3600-2400，3070，2950，1690，1640，1240，1040，880。

¹H NMR(400MHz，CD₃OD): δ 0.75, 0.86, 0.95, 0.97, 1.00, 1.70 (each 3H, s), 2.23(1H, dt, J ═ 12.4, 2.8Hz), 2.30(1H, dt, J ═ 12.8, 3.6Hz), 3.02(1H, dt, J ═ 10.4, 4.8Hz), 3.12(1H, dd, J ═ 11.2, 5.2Hz), 4.59, 4.71 (each 1H, brs).

¹³C NMR(CD₃OD-CDCl₃，100MHz)：δ178.93，150.51，109.16，78.53，55.98，55.14，50.31，46.75，42.17，40.41，38.53，38.06，36.88，34.08，32.01，30.32，29.39，27.56，26.66，25.29，20.62，18.94，18.02，15.76，15.56，15.03，14.34。

EIMS m/z (relative intensity): 456[ M ]]⁺(70)，438(35)，423(25)，410(20)，395(15)，316(15)，302(18)，259(30)，248(80)，234(55)，220(60)，207(80)，203(60)，189(100)。

Based on the measured spectral data, the product TSB-1 was identified as a known compound having the chemical formula shown below, i.e., betulinic acid:

3. product TSB-2:

the TSB-2 properties of the product were determined as follows:

yellow flaky crystal, melting point 204-209 ℃.

IR v _max cm^-1(KBr)：3500-2400，2960，1735，1680，1650，1590，1430，1380，1325，1130，1040，960，870。

¹H NMR(CDCl₃，400MHz)：δ7.56(1H，d，J＝6.9Hz)，6.63(1H，d，J＝9.6Hz)，5.51(1H，d，J＝9.6Hz)，5.12(1H，dd，J＝13.6，6.8Hz)，3.52(1H，dd，J＝6.2，4.7Hz)，3.26(2H，d，J＝7.4Hz)，2.66(1H，m)，2.55(2H，m)，2.35(1H，dd，J＝13.4，4.6Hz)，1.75(3H，s)，1.72(3H，s)，1.65(3H，s)，1.44(3H，s)，1.43(3H，s)，1.35(3H，s)，1.30(3H，s)。

¹³C NMR(CDCl₃，100MHz)：δ200.93，178.91，171.86，161.10，157.64，157.28，136.97，135.39，133.35，131.70，128.63，126.18，122.12，115.44，108.27，103.18，100.49，90.71，83.71，83.64，78.65，49.06，46.88，29.95，28.93，28.33，25.71，25.31，21.62，18.09，11.34。

EIMS m/z (relative intensity): 560[ M ]]⁺(100)，545(47)，532(22)，517(36)，405(44)，389(11)，363(24)，349(17)，307(12)，287(22)，285(16)，245(15)，215(12)，189(5)。

Based on the measured spectral data, the product TSB-2 was identified as a known compound having the chemical formula shown below, i.e., isogambogic acid:

4. product TSB-3:

the TSB-3 properties of the product were determined as follows:

the deep yellow amorphous powder has a melting point of 110-112 ℃.

IR v _max cm^-1(KBr)：3500-2400，2960，2920，1735，1685，1640，1590，1435，1400，1380，1330，1175，1140，1045，960，805，760。

¹H NMR(CDCl₃400 MHz): δ 12.70, 12.69(1H, s), 7.48(1H, d, J ═ 6.8Hz), 6.59(1H, d, J ═ 10.4Hz), 6.47(1H, t, J ═ 7.2Hz), 5.38, 5.36(1H, d, J ═ 10.0Hz), 5.03(2H, m), 3.44(1H, m), 3.21(1H, m), 3.17(1H, m), 2.56(1H, m), 2.45(1H, m), 2.27(1H, dd, J ═ 12.0, 4.4Hz), 1.97(2H, m), 1.67, 1.64, 1.57, 1.54, 1.52, 1.47, 1.32, 1.27 (each 3H, s).

¹³C NMR(CDCl₃，100MHz)：δ202.95，202.88，178.76，178.71，172.38，172.24，161.34，161.21，157.52，157.27，137.07，136.82，135.32，133.23，133.18，131.77，131.70，131.64，128.86，128.49，124.67，123.76，123.69，122.09，115.86，107.82，102.81，102.70，100.38，100.27，90.66，90.55，83.68，83.55，83.51，81.23，81.19，48.95，46.87，46.78，41.83，29.94，29.83，28.97，28.86，27.42，27.27，25.61，25.57，25.37，25.22，22.68，21.55，18.07，18.00，17.53，11.26。

EIMS m/z (relative intensity): 628[ M ]]⁺(78)，613(11)，600(12)，546(68)，545(100)，517(26)，473(15)，389(8)，355(11)，271(5)，245(12)，214(19)，189(8)。

Based on the measured spectral data, the product TSB-3 was identified as a known compound having the chemical formula shown below, i.e., isogambogic acid:

5. product TSB-5:

the TSB-5 properties of the product were determined as follows:

orange powder with a melting point of 96-99 ℃.

IR v _max cm^-1(KBr)：3600-2400，2950，2910，1735，1680，1625，1585，1450，1430，1400，1380，1325，1255，1170，1135，1040，950，880，800，755。

¹H NMR(CDCl₃400 MHz): δ 12.72, 12.70(1H, s), 7.51, 7.50(1H, d, J ═ 6.9Hz), 6.52, 6.51(1H, d, J ═ 10.0Hz), 6.17, 6.07(1H, t, J ═ 7.4Hz), 5.34, 5.30(1H, d, J ═ 10.0Hz), 5.02(2H, m), 3.44(1H, m), 3.26(1H, m), 3.10(1H, m), 2.97(1H, m), 2.88(1H, m), 2.47(1H, dd, J ═ 9.2, 2.5Hz), 1.99(2H, m), 1.71, 1.70, 1.69, 1.66, 1.54, 1.50, 1.29, 1.22 (3H, s), respectively.

¹³C NMR(CDCl₃，100MHz)：δ203.26，178.82，178.78，171.89，171.70，161.35，161.20，157.51，157.45，157.24，138.51，135.52，135.26，133.26，133.11，131.77，131.61，131.29，131.25，127.38，127.34，124.62，124.30，123.79，122.21，115.80，107.64，107.46，102.77，102.60，100.41，100.33，90.97，90.87，83.70，83.58，81.15，80.93，48.92，46.72，41.89，41.59，29.84，29.78，29.15，28.77，28.71，27.55，26.81，25.51，25.55，25.11，22.65，21.52，20.60，18.05，17.97，17.51。

EIMS m/z (relative intensity): 628[ M ]]⁺(46)，613(7)，600(12)，546(48)，545(100)，517(24)，474(13)，391(7)，355(7)，287(6)，245(9)，214(13)，189(5)。

Based on the measured spectral data, the product TSB-5 was identified as a known compound having the chemical formula shown below, i.e., gambogic acid:

6. product TSB-6:

the TSB-6 properties of the product were determined as follows:

orange powder with a melting point of 137-139 ℃.

IR v_max cm^-1(KBr)：3460，2970，2925，1735，1645，1630，1590，1460，1435，1400，1385，1330，1300，1250，1210，1185，1165，1140，1045，960，880，810。

¹H NMR(CDCl₃400 MHz): δ 12.68(1H, s), 7.41(1H, d, J ═ 7.2Hz), 6.59(1H, d, J ═ 10.0Hz), 5.49(1H, d, J ═ 10.0Hz), 5.19(1H, t, J ═ 7.0Hz), 4.73(1H, t, J ═ 8.0Hz), 3.61(2H, q, J ═ 10.2Hz), 3.49(1H, d, J ═ 4.7Hz), 3.47(1H, d, J ═ 4.7Hz), 3.32(1H, dd, J ═ 14.5, 6.8Hz), 3.24(1H, dd, J ═ 14.3, 7.7Hz), 2.60(1H, d, J ═ 7.7), 2.8 (1H, d, J ═ 7.68, 2.8H, 1H, dd, 1.8 Hz), 1H, dd, J ═ 14.3.3.3.3, 7.7, 7.8 (1H, 13.8 Hz), 1H, 13.8 (1H, 13.8H, 13.68, 1H, 1.8 Hz), 1H, d, J ═ 13.8 (1.8, 1H, 13.8 Hz), 1H, d.

¹³C NMR(CDCl₃，100MHz)：δ203.35，180.33，161.06，157.80，157.44，138.01，134.40，133.63，131.87，126.31，121.90，118.20，115.50，108.44，103.00，100.72，90.48，84.50，83.41，78.66，67.92，49.09，47.00，30.09，28.96，28.28，28.21，25.71，25.29，21.57，18.14，12.49。

EIMS m/z (relative intensity): 546[ M]⁺(100)，531(18)，518(44)，503(40)，485(9)，433(7)，405(33)，391(10)，363(19)，349(13)，307(10)，287(25)，245(8)，231(18)，214(12)，189(5)，105(6)。

Based on the measured spectral data, the product TSB-6 was identified as a known compound having the chemical formula shown below, i.e., isomannide:

7. product TSB-7:

the TSB-7 properties of the product were determined as follows:

orange powder with a melting point of 106-109 ℃.

IR v _max cm^-1(KBr)：3500-2400，2960，2920，1735，1680，1650，1625，1590，1430，1325，1120，1040，870，735。

¹H NMR(CDCl₃400 MHz): δ 12.71(1H, s), 7.51(1H, d, J ═ 6.8Hz), 6.49(1H, d, J ═ 10.0Hz), 6.05(1H, t, J ═ 7.0Hz), 5.39(1H, d, J ═ 10.0Hz), 4.99(1H, d, J ═ 6.0Hz), 3.45(1H, dd, J ═ 6.4, 4.7Hz), 3.27(1H, m), 3.08(1H, m), 2.97(2H, sept, J ═ 8.0Hz), 2.49(1H, d, J ═ 9.3Hz), 2.28(1H, dd, J ═ 13.4, 4.5Hz), 1.70, 1.69, 1.67, 1.60, 1.36, 1.34, 1.26, 26H, 26, s).

¹³C NMR(CDCl₃，100MHz)：δ203.47，179.07，171.74，161.22，157.65，157.34，138.46，135.39，133.42，131.46，127.64，126.00，122.22，115.44，108.04，103.16，100.55，90.93，83.82，78.55，49.01，46.80，29.87，29.26，28.82，28.40，28.20，25.68，25.14，21.57，20.63，18.06。

EIMS m/z (relative intensity): 560[ M ]]⁺(100)，545(56)，532(63)，517(48)，487(12)，433(9)，405(81)，391(22)，363(38)，349(24)，307(18)，287(64)，245(40)，231(21)，215(20)，189(10)。

Based on the measured spectral data, the product TSB-7 was identified as a known compound having the chemical formula shown below, gambogic acid:

8. product TSB-8:

the TSB-8 properties of the product were determined as follows:

orange needle-shaped crystal with the melting point of 109-110 ℃.

¹H NMR(CDCl₃400 MHz): δ 12.89(1H, s), 7.45(1H, d, J ═ 7.2Hz), 7.27(1H, s), 6.65(1H, d, J ═ 10.4Hz), 5.53(1H, d, J ═ 9.6Hz), 5.30(1H, br d, J ═ 6.0Hz), 4.43(1H, br s), 3.49(1H, m), 3.32(2H, m), 2.50(2H, m), 2.34(1H, m), 1.78, 1.71, 1.68, 1.59, 1.33, 1.03 (each 3H, s), 1.45(6H, s).

EIMS m/z (relative intensity): 530[ M ]]⁺(100)，515(22)，502(92)，488(30)，487(83)，459(11)，433(20)，405(49)，391(15)，363(24)，349(16)，307(13)，287(27)，231(13)，215(37)，189(6)。

Based on the measured spectral data, the product TSB-8 was identified as a known compound having the chemical formula shown below, i.e., deoxyluteine:

9. product TSB-4:

the TSB-4 properties of the product were determined as follows: yellow needle crystal with melting point of 115-118 ℃. When reacted with ferric chloride reagent (EeCl)₃test) is positive, which means that it is a phenolic compound.

IR v _max cm^-1(KBr)：3640，3450，3150，2960，2900，1632，1602，1570，1500，1460，1420，1395，1340，1295，1230，1130，1055，870，825，790。

¹H NMR (acetone-d)₆，600MHz)：δ13.09(1H，s，OH-1)，7.40(1H，s，H-8)，6.55(1H，d，J＝10.0Hz，H-11)，6.32(1H，s，H-2)，5.87(1H，d，J＝10.0Hz，H-12)，5.36(H，m，H-17)，3.55(2H，d，J＝7.3Hz，H2-16)，1.84(3H，s，H-20)，1.63(3H，s，H-19)，1.48(6H，s，H3-14，H3-15)。

¹³C NMR (acetone-d)₆，125MHz)：δ181.24(C＝O)，163.41(C-3)，162.26(C-1)，155.56(C-4a)，146.94(C-6)，146.43(C-10a)，134.42(C-18)，132.30(C-12)，131.77(C-5)，123.33(C-17)，122.08(C-11)，119.03(C-7)，115.20(C-8a)，113.15(C-8)，107.54(C-4)，103.26(C-9a)，98.40(C-2)，78.84(C-13)，28.26(C-14，15)，25.93(C-19)，22.16(C-16)，18.00(C-20)。

EIMS m/z (relative intensity): 394[ M ]]⁺(66)，393(23)，379(100)，339(30)，323(16)，311(13)，295(5)，278(10)，203(4)，162(7)。

Mass Spectrometry (EI-MS) data of product TSB-4 showed a molecular peak [ M ]]⁺At m/z394, which corresponds to a molecule of formula C₂₃H₂₂O₆Xanthone of (a).

Of product TSB-4¹The H NMR spectrum showed that it had a chelating hydroxyl group (. delta.13.09), a single peak of 2 aromatic protons (. delta.6.32 (1H, s) and. delta.7.40 (1H, s)]Unimodal of 2 methyl protons [ delta 1.48(6H, s)]And 2 mutually coupled cis-olefinic protons [ delta 5.87(1H, d, J ═ 10Hz) and delta 6.55(1H, d, J ═ 10Hz)]. These signals indicate the presence of a dimethylchromene ring in the molecular structure of the product TSB-4.

Furthermore, from product TSB-4¹The H NMR spectrum data revealed 2 vinylmethyl protons (. delta.11. 63(3H, s) and. delta.1. 84(3H, s)]Methylene proton [ delta 3.55(2H, d, J ═ 7.3Hz)]And olefinic protons [ delta 5.36(1H, m)]This indicates that the molecular structure of the product TSB-4 contains a gamma, gamma-dimethylallyl chain.

From the above data, it is clear that there is substitution of one 2, 2-dimethylpyranyl ring and one γ, γ -dimethylallyl chain in the xanthone structure of the product TSB-4.

In addition to using two-dimensional nuclear magnetic resonance (2D-NMR) homonuclear correlation Spectroscopy (homnuudearcorrelation Spectroscopy,¹H-¹h COSY) to verify the above¹In addition to the coupling situation in H NMR spectra, from heteronuclear multiple quantum correlation experiments: (¹H-Detected heteroconjugate-quaternary coherence, HMQC) (J150 Hz) found: δ 7.40 is related to δ 113.15; δ 06.32 is related to δ 198.40; δ 26.55 is related to δ 3122.08; δ 5.87 is related to δ 132.30. This shows that the chemical shifts of the two disubstituted olefinic carbons in the pyran ring are δ 122.08 and δ 132.30. In addition, δ 5.36 for the trisubstituted olefinic proton is associated with δ 123.33, while the methylene proton coupled to it (δ 3.55) is associated with δ 22.16.

In addition, multinuclear multiquantum correlation experiments from product TSB-4: (¹The data from the H-Detectedmulple-bond heterolepte multiple-quaternary consensus HMBC (J. about.8 Hz) spectrum also show that the chelating hydroxyl group (delta. 13.09) is associated with two quaternary carbons (delta. 103.26 and delta. 0162.26) and with one unsubstituted aromatic ring carbon (delta. 98.40). This demonstrates that the C-2 position of the xanthone backbone is not substituted with a hydroxyl group. The C-2 proton signal, delta 6.32, is associated with two quaternary carbons (delta 103.26, 107.54) and two hydroxyl-linked quaternary carbons (delta 163.41, 162.26). Methylene protons (δ 3.55) are associated with quaternary carbons (δ 163.41, 155.56, 134.42, and δ 107.54); and to the tertiary carbon (. delta. 123.33). This demonstrates that the gamma, gamma-dimethylallyl chain is attached at the C-4 position. Therefore, the ring A of the xanthone skeleton is substituted by 1, 3-dihydroxy-4-gamma, gamma-dimethylallyl.

From the HMBC spectrum, it was found that the other aromatic proton (δ 7.40) was associated with the carbonyl carbon (δ 181.24) and the two oxygenated carbons (δ 146.43, δ 146.94), as well as with one of the olefinic carbons in the pyran ring (δ 122.08). It is thus understood that the proton (. delta.7.40) is located at C-8, the peri-position of the carbonyl group, so that the attached pyran ring and xanthone skeleton must be linearly bonded. The last remaining hydroxyl group has only the possibility of being attached to the C-5 position, and the chemical shift of the carbon spectrum is δ 131.77.

By combining the above data, it was identified that TSB-4 is a novel compound having the chemical formula shown below

The product TSB-4 was named Fumuxanthenone A { IUPAC name: 1, 3, 5-Trihydroxyl-6 ', 6' -dimethyl-2H-pyrano (2 ', 3': 6, 7) -4- (3-methylbut-2-enyl) xanthone or 7, 9, 12-Trihydroxyl-2, 2-dimethyl-10- (3-methyl-but-2-enyl) -2H-pyrano [3, 2-b ] xanthen-6-one }.

Component identification of gamboge resin acetone extraction product TSB-14

To further understand the composition distribution of acetone-extracted product TSB-14 of Garcinia resin, HPLC analysis of TSB-14 was performed according to the general procedure above, and the results are shown in FIG. 1.

Since TSB-0 and TSB-1 have no ultraviolet absorption property, 7 kinds of purified compounds such as TSB-2 to TSB-8 are used as standards for HPLC analysis, and HPLC elution curves as shown in FIG. 2 are obtained, in which the numbers on the respective peaks correspond to the numbers of TSB-2 to TSB-8 of these products.

To find those peaks in the elution profiles of the products TSB-2 to TSB-8, respectively, corresponding to the acetone-extracted product TSB-14 shown in FIG. 1, the sample concentration of the acetone-extracted product TSB-14 was increased for HPLC to obtain the HPLC elution profile shown in FIG. 3.

Comparing the results of FIGS. 1 to 3, it was found that TSB-4 and TSB-8 did not appear to be present in TSB-14 in such high amounts that the corresponding peaks shown in the HPLC elution profile were very low. In order to confirm the peak positions of TSB-4 and TSB-8, HPLC was performed using TSB-14 (upper) containing only acetone extract and TSB-14 (lower) containing TSB-4 (500. mu.l) and TSB-8 (500. mu.l) added thereto, to obtain the HPLC elution profiles shown in FIG. 4.

To show that the 7 products TSB-2 to TSB-8 purified from Garcinia resin obtained according to the present invention appeared in the corresponding positions in the elution profile of the product TSB-14 according to the present invention, HPLC elution profiles as shown in FIG. 5 were obtained.

Accordingly, it was confirmed from the HPLC results that the gamboge resin acetone extraction product TSB-14 according to the present invention contained the following 9 compounds: betulin (TSB-0), betulinic acid (TSB-1), isogambogic acid (TSB-2), isogambogic acid (TSB-3), formosanxanthone A (TSB-4), gamboge acid (TSB-5), isogamboge alcohol (TSB-6), gambogic acid (TSB-7) and deoxygamboge (TSB-8). In addition to the 9 compounds purified above, the acetone extract product TSB-14 also contained approximately 25% of minor components including triterpenes and xanthones, as well as other unknown compounds.

Example 4 pharmacological experiments with acetone extracted products from Garcinia resin and purified Compounds

In order to determine the biological activity of the acetone-extracted product TSB-14 according to the present invention and the products TSB-1 to TSB-8 further purified from the extract, the following pharmacological activity analysis was performed.

Pharmacological test 1. in vitro anticancer test

The in vitro anti-cancer tests of the acetone-extracted product TSB-14 according to the invention and the products TSB-1 to TSB-8 which have been further purified from this extract were carried out by the International famous MDSPanlabs pharmacological Services.

The cancer cell growth inhibition assay is mainly used for detecting the proliferation effect of a drug candidate on cancer cells, and the action principle involved in the growth inhibition assay is that living cells can convert alamarBlue reagent (Biosource, USA) from an original non-fluorescent oxidation state (non-fluorescent, blue) into a fluorescent reduction form (fluorescent, red) through metabolic reaction, and the proliferation condition and the cell activity of the living cells can be quantitatively detected according to the measured fluorescence data result generated by the alamarBlue reagent.

In this experiment, the test concentrations of drug candidates were set at 0.01, 0.1, 1, 10 and 100 μ g/ml and were performed using the following cell lines: 6 human cancer cells MCF-7 (breast cancer), HT-29 (colorectal cancer), HL-60 (leukemia), HepG2 (liver cancer), A549 (lung cancer) and U937 (lymph cancer), and 1 normal Human Umbilical Vein Endothelial Cell (HUVEC). In addition, Dimethylsulfoxide (DMSO) (40%) was used as a negative control and mitomycin was used as a positive control.

According to the experimental report of MDS Panlabs pharmacological Services, the acetone-extracted product TSB-14 and the products TSB-1 to TSB-8 further purified from the extract according to the present invention showed 50% cell growth Inhibitory Concentrations (IC) against the above 6 kinds of human cancer cells and 1 kind of normal human cells₅₀) And 50% cytotoxic concentration (LC)₅₀) Are shown in the tables below, respectively.

TABLE 1 acetone extraction of Garcinia resin product TSB-14 50% growth Inhibitory Concentration (IC) for cancer cells₅₀[μg/ml]) And half Lethal Concentration (LC)₅₀[μg/ml])

TABLE 2 50% growth Inhibitory Concentration (IC) of TSB-1 to TSB-8 products purified from Garcinia resin on cancer cells₅₀[μg/ml])

"-" indicates not measured.

TABLE 3 median Lethal Concentrations (LC) of TSB-1 to TSB-8 purified from Garcinia resin on cancer cells₅₀[μg/ml])

"-" indicates not measured.

As is apparent from the results shown in tables 1 to 3, neither the acetone-extracted product TSB-14 according to the present invention nor the products TSB-2 to TSB-8 further purified from the extract have significant effects of inhibiting the growth of tumor cells, and thus have high potential for development into anticancer drugs.

Pharmacological test 2 in vitro anticancer Effect of mixture formulations containing products TSB-0 to TSB-8

To further understand the biological activity of the products TSB-0 to TSB-8 according to the invention when used in admixture, the mixture formulations TSB-9, TSB-10, TSB-11, TSB-12 and TSB-13 were formulated according to Table 4 below and also submitted to MDS Panlabs PharmacologyServices for in vitro anti-cancer tests, the results of which are shown in tables 5 to 9, respectively.

TABLE 4

TABLE 5 growth inhibitory Effect of formulation TSB-9 on cancer cells

TABLE 6 growth inhibitory Effect of formulation TSB-10 on cancer cells

TABLE 7 growth inhibitory Effect of formulation TSB-11 on cancer cells

TABLE 8 growth inhibitory Effect of formulation TSB-12 on cancer cells

TABLE 9 growth inhibitory Effect of formulation TSB-13 on cancer cells

As is apparent from the results shown in tables 5 to 9, the products TSB-1 to TSB-8 purified from Garcinia cambogia resin according to the present invention, in various combinations, clearly show the effect of inhibiting the growth of tumor cells, and thus have high potential for developing anticancer drugs.

Pharmacological test 3. in vivo biological Activity of mixture formulation TSB-9

The mixture formulation TSB-9 was further subjected to animal testing using Severe combined immunodeficiency Syndrome (SCID) female mice (6-8 weeks old, 20-22 grams in weight) derived from a C.B-17/Icr background. The animal experiments were also conducted by MDS Panlabs pharmacological Services.

Human breast cancer cell MCF-7(ATCC HTB-22) (1X 10)⁷Individual cells/0.2 ml) were implanted subcutaneously into the dorsal side of the mice and estradiol benzoate (Sigma, USA) was injected subcutaneously into the mice at a dose of 50 μ g/mouse for 4 weeks per week as a supplement. When the tumor grew to a diameter of 5mm or more (this day is expressed as day 1), the tumor-bearing nude mice were randomly divided into 4 groups (each group had 6 mice))。

Mixture formulation TSB-9 was dissolved in 10% DMSO and administered orally to mice separately at two doses of 40mg/kg and 80mg/kg daily for a total of 21 consecutive days. Mitomycin was administered intraperitoneally to mice at a dose of 2mg/kg and at 4 day intervals for a total of 5 doses, and this treatment group served as a positive control.

After administration of the test substance, these mice were observed for the following: significant signs of toxicity, body weight and tumor volume. Measurements were taken and recorded every 4 days during the experiment. The results of the experiment are shown in tables 10 and 11.

TABLE 10 in vivo tumor growth inhibition efficacy of mixture formulation TSB-9

*: when the% T/C is less than or equal to 42%, the anti-tumor activity is obvious.

TABLE 11 weight effects of mixture formulation TSB-9 on the experimental animals

*: the body weight values of the experimental groups when analyzed by unpaired Stirling test (unpaired student test) showed significant changes (p < 0.05 ═ relative to the body weight values of the vehicle control group.

Over a 29 day period, an 80mg/kg dose of the mixture formulation TSB-9 caused significant tumor weight inhibition, while a 40mg/kg dose of the mixture formulation TSB-9 caused moderate tumor weight inhibition relative to vehicle controls.

All documents and patents cited in this specification are herein incorporated by reference in their entirety. In conflict, the present detailed description, including definitions, will control.

While the invention has been described with reference to the specific embodiments described above, it will be apparent that numerous modifications and variations can be made without departing from the scope and spirit of the invention. It is the intention, therefore, to be limited only as indicated by the claims appended hereto.

Claims (29)

1. A compound characterized by:

the compound has the following chemical formula:

2. a pharmaceutical composition characterized by:

the pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1.

3. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition comprises a therapeutically effective amount of a product obtained by extracting gamboge resin with acetone, said product comprising a compound of claim 1.

4. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition further comprises at least one compound selected from betulin, betulinic acid, gambogic acid, isogambogic acid, isogamboge alcohol and desoxygamboge.

5. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition comprising a compound of claim 1, gambogic acid, isogambogic acid, gambogic resin acid, isogambogic resin acid, isogamboge alcohol and deoxygamboge, betulin and betulinic acid.

6. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition comprises a compound of claim 1, betulinic acid and any one of the following combinations of compounds:

(1) isoresinic acid, resinic acid and deoxygambogic acid;

(2) isogambogic acid, gambogic acid and isogambogic alcohol;

(3) gambogic acid, gambogic acid and deoxygambogic acid;

(4) isogambogic acid, isogambogic acid and isogambogic alcohol.

7. The pharmaceutical composition of claim 6, wherein:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 20% isogambogic acid, 50% gambogic acid and 5% deoxygamboge, by weight of the composition.

8. The pharmaceutical composition of claim 6, wherein:

the pharmaceutical composition comprising 5% of the compound of claim 1, 10% betulinic acid, 20% isogambogic acid, 50% gambogic acid and 5% isogambogic alcohol, by weight of the composition.

9. The pharmaceutical composition of claim 6, wherein:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 40% gambogic acid, 30% gambogic acid and 5% deoxygamboge, by weight of the composition.

10. The pharmaceutical composition of claim 6, wherein:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 40% isogambogic acid, 30% isogambogic acid and 5% isogambogic alcohol, by weight of the composition.

11. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, wherein:

the pharmaceutically acceptable carrier comprises one or more of the following agents: excipient, solvent, emulsifier, suspending agent, disintegrant, binder, stabilizer, antiseptic, lubricant, absorption delaying agent and liposome.

13. The pharmaceutical composition of claim 12, wherein:

the pharmaceutically acceptable carrier is an excipient and comprises at least one saccharide selected from the group consisting of sucrose, brown sugar, lactose, D-sorbitol, D-mannitol, corn starch and crystalline cellulose.

14. The pharmaceutical composition of claim 2, wherein:

the pharmaceutical composition is manufactured in a form suitable for oral administration.

15. Use of a pharmaceutical composition according to claim 2 for the preparation of a medicament, characterized in that:

the medicament is useful for the treatment of at least one cancer selected from breast cancer, colorectal cancer, leukemia, liver cancer, lung cancer and lymphoma.

16. Use of a pharmaceutical composition for the preparation of a medicament for inhibiting the growth of a tumor/cancer cell selected from the group consisting of breast cancer cells, colorectal cancer cells, leukemia cells, liver cancer cells, lung cancer cells and lymphoma cells, wherein:

the pharmaceutical composition comprising a compound of claim 1.

17. Use according to claim 16, characterized in that:

the pharmaceutical composition comprises a product obtained by extracting a gamboge resin with acetone, said product comprising a compound of claim 1.

18. Use according to claim 16, characterized in that:

the pharmaceutical composition further comprises at least one compound selected from betulin, betulinic acid, gambogic acid, isogambogic acid, isogamboge alcohol and desoxygamboge.

19. Use according to claim 16, characterized in that:

the pharmaceutical composition comprising a compound of claim 1, gambogic acid, isogambogic acid, gambogic resin acid, isogambogic resin acid, isogamboge alcohol and deoxygamboge, betulin and betulinic acid.

20. Use according to claim 16, characterized in that:

the pharmaceutical composition comprises a compound of claim 1, betulinic acid and any one of the following combinations of compounds:

(1) isoresinic acid, resinic acid and deoxygambogic acid;

(2) isogambogic acid, gambogic acid and isogambogic alcohol;

(3) gambogic acid, gambogic acid and deoxygambogic acid;

(4) isogambogic acid, isogambogic acid and isogambogic alcohol.

21. Use according to claim 20, characterized in that:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 20% isogambogic acid, 50% gambogic acid and 5% deoxygamboge, by weight of the composition.

22. Use according to claim 20, characterized in that:

the pharmaceutical composition comprising 5% of the compound of claim 1, 10% betulinic acid, 20% isogambogic acid, 50% gambogic acid and 5% isogambogic alcohol, by weight of the composition.

23. Use according to claim 20, characterized in that:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 40% gambogic acid, 30% gambogic acid and 5% deoxygamboge, by weight of the composition.

24. Use according to claim 20, characterized in that:

a pharmaceutical composition comprising 5% of a compound according to claim 1, 10% betulinic acid, 40% isogambogic acid, 30% isogambogic acid and 5% isogambogic alcohol, by weight of the composition.

25. Use according to claim 16, characterized in that:

the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

26. The use according to claim 25, characterized in that:

the pharmaceutically acceptable carrier comprises one or more of the following agents: excipient, solvent, emulsifier, suspending agent, disintegrant, binder, stabilizer, antiseptic, lubricant, absorption delaying agent and liposome.

27. The use according to claim 26, characterized in that:

the pharmaceutically acceptable carrier is an excipient and comprises at least one saccharide selected from the group consisting of sucrose, brown sugar, lactose, sorbitol, mannitol, corn starch and crystalline cellulose.

28. Use according to claim 16, characterized in that:

the pharmaceutical composition is manufactured in a form suitable for oral administration.

29. A product obtained by extracting gamboge resin with acetone, characterized in that: the product has a composition distribution of HPLC elution profile as shown in figure 1, and comprises the compound of claim 1, betulin, betulinic acid, gambogic acid, isogambogic alcohol and deoxygamboge.