TW201143787A - Method for isolating sesquiterpenoid lactone compounds from Tithonia diversifolia (Hemsl.) A. Gray, compositions and use thereof - Google Patents

Abstract

A method for isolating sesquiterpenoid lactone compounds from Tithonia Diversifolia (Hemsl.)A. Gray (hereinafter "TD") includes following steps: (1) drying and powdering TD, (2) processing with alcohol to obtain extract, (3) adding EtOAc into aqueous phase of the alcohol extract to further obtain two extracted portions: TDM-EtOAc and TDM-water, (4) performing column chromatography with TDM-EtOAc to obtain an activating layer capable of inhibiting the growth of cancer cells, and (5) performing high performance liquid chromatography (HPLC) on the activating layer to obtain sesquiterpenoid lactone compounds capable of inhibiting the growth of cancer cells. The invention further relates to a composition including the sesquiterpenoid lactone compounds. The invention further relates to a biological or pharmaceutical accepted carrier which includes the sesquiterpenoid lactone compounds. The invention further relates to an anti-cancer use of the sesquiterpenoid lactone compounds and composition including the sesquiterpenoid lactone compounds.

Description

201143787 VI. Description of the Invention: [Technical Field to Be Invented by the Invention] The present invention relates to a method for squiterpenoid lactones from a five-pronged knives. The composition of sesquiterpene lactones and the sesquiterpene lactones and the use of the compositions for cancer resistance. [Prior Art] Five-jaw Jinying (7)Y/2c>ma Office//a (Hemsl.) A. Gray), alias Wang Ye Cai, fake hollyhock, too%, daisy, is a compositae C〇mp〇sjtae) is a perennial shrubby herb, a perennial herb with a plant height of 15_5 cm. Stems stout, leaves alternate 'have long stalks, leaves round, ovate or ovate-triangular, serrate serrate, sulculose pubescent on s. s. s. s. s. s. s. s. s. s. s. s. In 191, Taiwan was introduced from Singapore by the monk Fujinji. The flowering period is in autumn and summer. Five-jawed Jinying use: 1 Five-pronged Jinying has ornamental value when flowering; 2. Medicinal aspect: its whole grass has beneficial effects on urinary dispelling heat, clearing liver and detoxifying, reducing swelling and relieving pain. It can treat jaundice, acute gastroenteritis and cystitis. , acne, hemorrhoids swollen poison, diabetes, etc.; and 3. Documentary records: (1) treatment of hepatitis; (2) treatment of sarcoma, tumor, powder tumor; (3) treatment of liver cancer. Five-jawed Jinying has anti-cancer effects on various cell lines (Chiang et al., 乂所j Cto MM 32, 695_704, 2002). In addition, the five-jaw jinying also has biological regulatory functions such as anti-diabetes (Miura et al., «/C) /z/w AM 30,81-86, 2002), anti-AIDS (c〇s et al., cz. č. 9, 62-68, 2002), anti-inflammatory effects (Rungeler et al., Oil 3 64, 588 -593, 1998;

Owoyele et al., Cawcer and lamps 64, 6892-6899, 2004), antibacterial effect 201143787 (Goffin et al., corpse/training essay/68, 543-545, 2002; Tona et al., Phytomedicine 6, 59-66, 1999 and Phytomedicine 7, 3U3, 2000).

Nabin et al. (J Org Chem 44, 1831-1835, 1979) isolated the compound Tagitinin C (1):

Recent studies have shown that 'five claws of gold extracts have the effect of inhibiting the growth of cancer cells. In order to more effectively purify the effective compound from the extract of Chongjinjinying, the inventor of the present invention is committed to reform and innovation, and after years of painstaking research, successfully developed a method using a specific organic solvent to extract C. The extract is separated by liquid chromatography to obtain a half-box lactone compound which can effectively inhibit the proliferation of cancer cells, a composition containing the compound, and the sesquiterpene lactone compound and the composition for anticancer use. SUMMARY OF THE INVENTION 201143787 One of the items of the present invention is to provide a method for isolating an anticancer cytosolic sesquiterpene lactone compound from the genus of the genus, which comprises solvent extraction and chromatography. Another object of the present invention is to provide a composition comprising the sesquiterpene lactone compound comprising the sesquiterpene lactone compound and a physiologically or pharmaceutically acceptable carrier. Another object of the present invention is to provide an anti-cancer use of the above sesquiterpene vinegar compound and the composition.方法 The method for separating anti-cancer proliferative sesquiterpene lactone from the genus of the genus according to the present invention includes the following steps: (1) Drying and grinding the quinquecrum (TD) (2). The extract is prepared by treatment with an alcohol; (3) The aqueous suspension of the alcohol extract is extracted and extracted with ethyl acetate (Et〇Ae) to obtain two extraction portions TDM_Et〇Ac and TDM_H2. 〇·, ...(4). The TDM_Et0Ac extract part is subjected to 2-column column chromatography, and the active layer with anti-cancer proliferation is proposed; and =). In-fine high-wei riding method (10) L〇 The cerebral differentiation layer is isolated to have anti-cancer proliferative activity. In the second step of this financing method (2), __ extracts the dried five-jaw gold powder. The conversion can be methanol, ethanol or the like. In the examples, methanol extraction is used. The temperature at which the stroke can be low is room temperature. The extraction towel can be mechanically treated such as shaking, stirring, and the like. In the implementation of the financial repentance with ultrasonic shock processing. The ground time can be 1-5 hours. This extraction procedure can be repeated several times such as μ times. The draw solution is immersed, for example, under reduced pressure, to obtain an extract such as methanol, 201143787 (TDM), in step (3) of the method of the present invention, for the obtained extract such as methanol extract (TDM). The ethyl acetate was subjected to extraction and extraction to obtain two extraction fractions TDM-E: tOAc and TDM-H20. In one embodiment, this step was carried out in a bottle such as a serum bottle to a volume ratio of about 1:1 ethyl acetate and The water was added to the alcohol extract, and the liquid layer was allowed to stand after the mixture was fully mixed. The liquid layer was separated by a separating funnel to obtain two extracted portions of TDM-ethyl acetate layer (TDM-EtOAc) and TDM-water layer ( TDM-H20) 〇 In the step (4) of the method of the present invention, the TDM-EtOAc extract fraction is first analyzed by thin layer chromatography and then subjected to silica gel column chromatography, and the anti-cancer cell is rushed. The active layer of hyperplasia distinguishes layers. In one embodiment, the ethyl acetate separation layer (TDM-EtOAc) is first filled into a gel column (about 15 times by weight) (silica gel, 400 mesh) in an open chromatographic column (〇 pen c〇iumn chromatography). The selected column is 4 cm in diameter, and the length of the silicone layer filled in the column is about 32 cm. The column is separated by chromatography, and the solvent extraction ratio is ethyl acetate/hexane 0%: 100%~ethyl acetate. / Hexane 1%: 〇%, gradually increase the polarity of the rushing 'final sterol flushing is completed. The isolated compound is an impurity. After drying at 60 ° C, the same polarity was combined by TLC spotting. In one embodiment, the silicone TLC was used to separate and separate using 5% to 1 〇〇〇/0 acetic acid ethyl acetate/hexane. Finally, the same polarity group is combined into the same group, which is divided into active differentiation layer TDM-EA-(A~F).

Finally, in the step (5) of the method of the present invention, a sesquiterpene lactone compound having anticancer cell proliferation activity is isolated from the active discriminating layer by high performance liquid chromatography (HPLC). First, the separation conditions of the TDM-EA-B, C, and D 201143787 groups were identified by TLC. This was followed by a semi-preparative high performance liquid chromatography (HPLC): PrepStar SD-l VARIAN; column (Phenomenex, USA); mobile phase: EtOAc (solvent A) and CH2C12 (solvent B), A: B (10) : 90), A : B (15 : 85), and A : b (20 : 8〇); Flow rate: 3 ml/min: Detector. Shodex RI-71 Refractive Index Detector. The isolated product was then analyzed using a high resolution nuclear magnetic resonance spectrometer (VARIAN saponin from 500 NMR)' and the known profiles were aligned. In another aspect, the invention provides a composition comprising a sesquiterpene lactone of the invention and a physiologically or pharmaceutically acceptable carrier. The term "physiologically or pharmaceutically acceptable carrier" means that the substance or composition used must be compatible with the other ingredients of the formulation and not deleterious to the patient. The compositions described herein can include, but are not limited to, foods And the use forms of the present invention, beverages, health foods, animal drinking water additives, animal feed additives, animal and human medical compositions, food additives, beverage additives, etc. The compositions of the present invention can be used to familiarize themselves with the art. The above-mentioned technique of preparing the composition of the present invention is prepared by the above-mentioned technique of the present invention. The dosage form includes, but is not limited to, a solution, an emulsion, a suspension, and a physiologically or pharmaceutically acceptable coating. , powders, troches, pills, lozenges, tablets, ehewing gums, capsules, and other dosage forms similar or suitable for use in the present invention. In the examples, the dosage form is a keying agent. The pharmaceutically acceptable carrier may comprise - or a plurality of agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binders, excipients, ampoules, meal preparations, diluents, gums Agents, preservatives, lubricants, surfactants, and other carriers similar or suitable for use in the present invention. 201143787 In the above composition, it may be added as appropriate (4) - or a dissolution aid or buffer commonly used in various preparation fields. And a preservative, a coloring agent, a flavoring agent, a flavoring agent, etc. Further, preferably, the foregoing composition of the present invention can be further added with an edible material to prepare a food product or a health care product. Edible materials include, but are not limited to: water, fluid dairy, milk, concentrated milk, yoghurt, yogurt, silk yoghurt, lactobacillus fermented beverage, milk powder, ice cream, milk, dried bristle, soy milk, fermented soy milk, Vegetable juice, fruit juice, sports drink, dessert, fruit reduction, candy, baby food, health food, animal feed, Chinese herbal medicine, dietary supplement, etc. In another embodiment, the composition contains the present invention a beverage with the above edible material. In each step of the method of the present invention, the obtained alcohol extract, ethyl acetate extract fraction, liquid chromatography: 3⁄4 sex layer, The final obtained compounds, and the above compositions, were each analyzed by MTT (3-(4,5>dimethyithiaz()zyl>3>5_di-phenyltetrazolium bromide) (Hansen et al., j Immun〇1 Methods 119, 203-210, 1989) Assays for anti-cancer cell proliferation activity, as detailed in the examples below. One of the anti-cancer cell proliferation sesquiterpene lactone compounds isolated and isolated in the following examples is TagitinmC. Accordingly, the above composition of the present invention also includes Tagitinin C and a physiologically or pharmaceutically acceptable carrier. According to the results of the following examples, the composition of Tagitinin C and Tagitinin C has anti-cancer cell proliferation activity. According to this, in another aspect of the invention, the anti-cancer use of the composition of Tagitinin c and Tagitinin 201143787 c is proposed, in particular for combating liver cancer, brain cancer and the like. [Embodiment] The present invention will be described in detail with particular embodiments. These specific embodiments are provided by way of explanation of the invention and are not intended to limit the invention. Within the scope and spirit of the invention, the invention is intended to include these and other changes and modifications. Example 1. Separation of sesquiterpene lactone compound from C. chinensis A. Methanol cold dip extraction (TDM) The harvested P. chinensis was dried and ground to a powder. Next, add five milliliters of methanol (200 g) to 1 ml of methanol, place it in a 2 〇〇〇 ml ml serum bottle, use a sonic shock for 99 minutes, then remove the extract and add 1 〇. 〇〇ml of sterol in the 2000-ml serum bottle, the above steps were repeated three times. Remove plant debris/detection. The methanol extract was collected and concentrated to remove methanol by a rotary vacuum dryer under reduced pressure. The obtained methanol extract was named as TD]y^ The anti-cancer cell proliferation activity of TDM was measured by the MTT method described below, and the results showed that the survival rate of TDM after administration of brain cancer cells (U373) for 24 hours was as shown in FIG. 'It is confirmed that TDM does have anti-cancer cell proliferation activity. B. Ethyl ethoxide/water partitioning extraction Add 15 times (w/v) of water to the extract of P. sinensis (TDM '28 65 g), mix well, and add an equal volume of ethyl acetate. (Et〇Ac), stir well in a separatory funnel, and pour into a separatory funnel to be layered. After the full scalpel, the EtOAc layer and the aqueous layer were separately collected. The collected aqueous layer was further extracted twice with EtOAc according to the above procedure, that is, the total distribution was extracted three times, and finally, 201143787, two different distinguishing layers were obtained. The mixture was concentrated to dryness under reduced pressure to give the ethyl acetate ethyl acetate layer (TDM-EtOAc), which had a net weight of 15.15 g (53%) and a water layer (TDM-H2 〇), and a net weight of 13.5 g (57%). First, the five-jaw jinying methanol extract acetic acid vinegar layer (TDM-EtOAc) was mixed with 30.30 g of silica gel (230-400 mesh) and filled into 227.25 g of tannin (about 15 times) (silica gel, 400 mesh) in open c〇lumn chromatography. The selected column is 4 cm in diameter and the length of the silicone layer filled in the column is about 32 cm. Column chromatography was carried out by continuous stripping of a different polar solvent of ethyl acetate/hexane (EtOAc/Hex) with EtOAc/MeOH gradient. Combine the distinctions with the same thin layer chromatography (TLC) profile into the same finely differentiated layer'. The final combination results in 6 finely differentiated layers (TDM-EtOAc-A, TDM-EtOAc_B, TDM-EtOAc-C, TDM-EtOAc-D 'TDM-EtOAc-E > TDM-EtOAc-F) ° The anti-cancer activity of the six fine-differentiated layers was measured by the MTT method described below. The results are shown in Figure 3 as the drug is added to the brain. The survival rate of cancer cells (U373) after 24 hours of action confirmed that the finely differentiated layers TDM-EtOAc-B, TDM-EtOAc-C, and TDM-EtOAc-D had better anti-cancer activity. High-performance liquid chromatography (HPLC) was used to show finely differentiated layers TDM-EtOAc-B, TDM-EtOAc-C, and TDM-EtOAc-D which have a better inhibitory effect on cancer cell proliferation. The column is subjected to dissolution purification in the normal phase (Si-60). The isolated compound was then assayed for tumor suppressor activity, one of which was Tagitinin C (1) (31.5 mg). The structure of the compound is shown in the following (1). . 201143787

HO

Example 2: Physicochemical identification of isolated cancer cell proliferative sesquiterpene lactone compounds

The compound Tagitinin C obtained according to Example 1 has the following properties: yellow oil; 4 NMR (Fig. 4) (CDC13, 500 ΜΗζ) δ 6.89 (d, "/ = 17.0 Hz, 1H Hl), 6.27 (d, J = 1.1 Hz, 1H, Ha-13), 6.19 (d, J = 17.0 Hz 1H H-2), 5.80 (dq, / soil 8.8, 1.1 Hz, 1H, H-5), 5.74 (d, U Hz m Ηβ-13), 5.37 (br. d, 8.8,1H), 5.82 (m, 1H, H-8), 3.5〇 (mm H-7), 3.21 (s, 1H, -OH), 2.41 (dd , J = 13.9, 6.3 Hz, 1H, Ha-9) 2.36 (seq, */: 6.9 Hz, 1H, H-2,), 1.94 (dcU = 13.9, 1〇〇Hz 1H, Ηβ-9), 1.88 (br. s, 3H), 1.47 (s, 3H, H-14), 0.99 (d, J = 6 9 Hz, 3H, Η·3'), 0.97 (d, J = 6.9 Hz, 3H, H- 4')· 〇rg Chem 44, the compound Tagitinin C is the same as reported by Nabin et al. (J 1831-1835, 1979). The anti-cancer cell growth life 201143787 of Tagitinin c was measured by the MTT method described below. As shown, the survival rate of the brain cancer cells (U373) (Fig. 5) and liver cancer cells (Hep-G2) (Fig. 6) after 24 hours confirmed that Tagitinin C has anti-cancer cell proliferation activity, especially anti-brain cancer Cells and anti-hepatoma cells. Example 3. Preparation of composition A. Lozenge The ingredients in the following table were ground and mixed in a spider, and then pressed into a tablet under a suitable pressure using a tablet press. Ingredients 5-mg key 10-mg ingot 20-mg ingot Tagitinin C 5 g 10 g 20 g Carboxymethyl cellulose sodium 100 g 100 g 100 g talcum powder 395 g 390 g 380 g starch 400 g 400 g 400 g stearin Magnesium sulphate 1 gram 1 gram 100 gram B. Beverage 10 g of Tagitinin C is slowly added to an aqueous solution containing 5% polylauryl sorbate under stirring, and after thoroughly mixing, The liquid was stirred to 1000 ml of material, and the milk was used to make the beverage of the present invention. Example 4: Effect of anti-cancer cell proliferative sesquiterpene vinegar compound _MT1^ The cell lines HeP~G2 (human liver cancer cells) and U-373 (human brain cancer cells) were purchased from the US-based conservation center. (atcc) (Sin Curry, _). 201143787 These cells were maintained in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% heat-deactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml (Kg/mL) streptomycin. And kept in a 5% C02 incubator at 37 °C. MTT (3-(4,5)-dimethylthiazozyl)-3,5-di-phenyltetrazolium bromide) analysis was performed as described by Hansen et al. (Hansen et al., J. Immunol.

Methods 119, 203-210, 1989). Hep-G2 cells (3 x lO3 cells/well) and U-373 cells (3 χΙΟ 3 cells/well) were seeded in 96-well plates and

It grows overnight. The cells were then cultured in FBS medium containing different amounts of samples for 24 hours. At the end of each culture, the medium was replaced with 1 μl of the reagent (ΜΤΤ 1 1 mg/ml) and cultured for another 4 hours in a 37 〇c 5% C02 culture phase. The medium was then discarded and each microliter of diterpenoid (DMSO) was added to each well. At the wavelength of wo nm, the absorbance was measured by scanning with a microplate reader (M-Device, CA). Each sample was analyzed six times and the values obtained were averaged. To determine %. Value (To suppress each of the cancer cells (4) (10) degrees), each sample was tested for MTT at five different concentrations. The ic50 value was obtained by interpolation from a linear regression analysis. The methanol extract distinguishing layer listed as pentaphyllum and the compound isolated from TDM-EtOAc for Hep_G2 and u_3

The effect of the system is that the IC5 value is ^ and P is the quasi-bias deviation. The results of the _ _ IC50a (micrograms / of the Hep-G2 and U-373 cancer cells isolated from the TDM-EtOAc compound and the average of 13 201143787 ML)

Sample TDM

Hep-G2_ U - 373 40.0 ± 2.0 59.2 ±3.7 6.1 ±0.1 __2.0 ±0.1 The iQo value is the concentration that inhibits the growth of 5〇% cancer cells. As shown in the table, the compound *Tagithiin c isolated by the method of the present invention is directed against

Hep G2 and U-373 cancer cells 1 (: 5 〇 values are 2. 〇 micrograms / ml and 6.1 μg / ml. These numbers record the value of TDM crude extract (respectively micrograms / ml and 59.2 micrograms / ML)low' indicates that the compound (1) has been purified. In summary, the present invention provides a method for isolating a sesquiterpene lactone compound having anti-cancer proliferative activity from P. chinensis from a methanol extract of C. 'The spurs have a compound of anti-cancer 峨 proliferative activity. The genus is a known compound, Tagitinin C. It is proved by the above-mentioned growth inhibition test in cancer cell systems such as Hep_G2 and U-373, according to the present invention. It is isolated from the genus TagitininC, which can inhibit the proliferation of cancer cells. It can be used as an effective anti-cancer proliferation syndrome, and can be used to prepare a composition for preventing and/or treating cancer. A flow chart of extraction and purification of the method of the present invention. Figure 2 shows the survival rate of cancer cells after 24 hours of treatment with brain cancer cells 201143787 (U373) by the crude extract of P. chinensis (TDM). Claw Jinying The survival rate of cancer cells after 24 hours of action of A~F isolates in the extract (TDM) is shown in Figure 4. The spectrum of the isolated compounds is shown in Figure 4. Figure 5 shows that TagitininC is added to brain cancer cells. (U373) Survival rate of cancer cells after 24 hours of action. Figure 6 shows the survival rate of cancer cells after 24 hours of treatment with hepatoma cells (Hep-G2) by the crude extract of P. chinensis (TDM) and Tagitinin c. [Main component symbol description] None

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Claims (1)

201143787 VII. Scope of Application: L A method for isolating anti-cancer proliferative sesquiterpene lactone compounds from C. chinensis, including the following steps: (1) Drying and grinding the Clawed Gold (TD) (2). The alcohol extract is prepared by treatment with an alcohol; (3) The aqueous suspension of the alcohol extract is extracted with ethyl acetate (EtOAc) to obtain two extraction portions TDM_Et〇Ac and TDM_H2 (4) - The TDM-EtOAc extract fraction is subjected to column chromatography, and the active layer having anti-cancer proliferation is washed & and (5). Further high-performance liquid chromatography (HPLC) A sesquiterpene lactone compound having anti-cancer cell proliferation activity is obtained from the active discriminating layer. 2. The method of claim 1, wherein in the step (2), the alcohol is methanol or ethanol. 3. The method of claim 1, wherein in the step (4) of the Shixi rubber column chromatography, the solvent is ethyl acetate/hexane: 1% to ethyl acetate/ Hexane 100%: 〇%, gradually increase the polarity of the extraction, and finally complete with methanol. 4. The method of claim 1, wherein the thin layer chromatography in the step (4) is performed by silica gel thin layer chromatography using 50/0 to 100 〇/〇 ethyl acetate/hexane, respectively. The alkane developing solution is separated and separated. 5. The method of claim 1, wherein the high performance liquid chromatography (HPLC) in the step (5) is carried out with EtOAc (solvent A) and CH2C12 (solvent B), A: B (10: 90) ), A : B (15 : 85), and A : B (20 : 80) as the mobile phase. 201143787 6. The method of applying the patent scope is the Tagitinin C: Where the sesquiterpene lactoneization • m contains ubiquitin _ compound (Post her c) with a physiologically or pharmaceutically acceptable carrier. For example, the composition of claim 1! Item 7 includes the form of group consisting of the following two groups: food, drinks, health food, animal drinking water, additives, animal feed additives, animal use, and Medical compositions, food additives, and beverage supplements for human use. 9. Use of a composition according to item 7 of the scope of the invention, for anti-cancer cell proliferation. 1) The use of claim 9, wherein the cancer cell is a liver cancer cell or a brain cancer cell. 17