CN107602524A - The miscellaneous terpenoid of formyl phloroglucin and its pharmaceutical composition and its preparation method and application - Google Patents

Abstract

The present invention provides the miscellaneous terpenoid of formyl phloroglucin isolated from guava (Psidium guajava) fruit and branches and leaves, pharmaceutical composition using it as active component, and preparation method thereof, its application in tumor is prepared, and its application in functional health product is prepared.The method raw material of the present invention is easy to get, and method is simple, easy to operate, and gained compound proves that the compound of the present invention has by Bioexperiment and preferably suppresses tumor cell growth activity；And compound 1,2,12,14 and 16 has the suppression topology enzymatic activity similar to camptothecine, compound 1 and 2 has the activity of stronger promotion cancer cell-apoptosis.

Description

The miscellaneous terpenoid of formyl phloroglucin and its pharmaceutical composition and its preparation method and Using

Technical field：

The invention belongs to drug field, and in particular to the isolated formyl from guava (Psidium guajava) The miscellaneous terpene of phloroglucin and its pharmaceutical composition, their applications in treating cancer medicine is prepared, and they are preparing function Application in property food.

Background technology：

All the time, cancer is all an important global public health problem.In recent years, the incidence of disease of cancer and death The trend sharply increased is all presented in rate.According to WHO report, by 2008, the whole world is estimated to be to be reached with cancer occurrence numbers 12700000, there are 7,800,000 people to die from cancer.To the year two thousand thirty, it is contemplated that newly-increased 21,000,000 cancer patients, the annual number of cancer deaths in the whole world It is up to 13,200,000 (Wong A.S.T., et al.Nat.Prod.Res.2015,32: 256–272).Currently, chemotherapy is The main policies for the treatment of cancer.However, in view of drug resistance is inevitable, there is cytotoxic drug effectively to treat for some Side effect and complication (such as suffer from diarrhoea, vomit, nauseous, alopecia) can be produced while cancer to normal cell, these medicines are simultaneously Can not be effective for the prevention of cancer.Therefore, it is necessary to find more effective cancer treatment method.There are some researches show people's DNA topoisomerase ferment I can adjust DNA engineering properties in the main activities such as DNA replication dna and genetic transcription of cell (Pommier Y.,et al. Chem.Biol.2010,17:421-433).Although topoisomerase ferment I inhibitor at present and seldom See, but they are proved to prevent the formation of covalent complexes in cancer cell so as to play preferable anticancer effect.Topology is different Structure ferment I inhibitor can regard into a kind of 21 century new antineoplastic as.

Myrtaceae Psidium (Psidium) the plant whole world there are about 150 kinds, originate in South America, China only introduces a fine variety 2 Kind, guava (P.guajava) and strawberry guava (P.littorale) (Chinese Plants will volume 1984,53:122–123).Kind The leaf all parts of the world of pomegranate is mainly used in treating enterogastritis and diarrhoea (Guti é rrez R.M.P., et al.J. Ethnopharmacol.2008,117:1-27), its fruit is edible, and many beverages conscientiously, the raw material of fruit juice and wine.

Have no that they are making including the miscellaneous terpenoid of 16 formyl phloroglucins and its pharmaceutical composition in the prior art Application in the medicine of standby treating cancer and the complication being induced by it, and the report of the application in functional food is prepared Road.

The content of the invention：

It is an object of the invention to provide isolated formyl isophthalic from guava (P.guajava) fruit and branches and leaves Triphenol miscellaneous terpenoid is the pharmaceutical composition of active constituents of medicine, and its preparation method, they are in cancer and are induced by it Application in the medicine of complication.Present invention formyl isophthalic isolated from guava (P.guajava) fruit and branches and leaves The miscellaneous terpenoid of triphenol, show that such compound is topoisomerase I inhibitor, is had by the research of multiple pharmacological testing The significant activity for suppressing cancer cell and increasing；And compound 1 and 2 has the activity of stronger promotion cancer cell-apoptosis.

In order to realize the above-mentioned purpose of the present invention, the invention provides following technical scheme：

The miscellaneous terpene A (1) of the miscellaneous terpenoid guava fruit of formyl phloroglucin in guava shown in following structural formula.

The miscellaneous terpene A (1) of compound guava fruit shown in following structural formula containing therapeutically effective amount, psiguajadial E(2), guadial A(3),guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7), psiguadial D(8), guajadial(9),psidial A(10),4,5-diepipsidial A(11),guajadial B (12), guajadials C-F (13-16) any one or two kinds of mixture and the medicine group of pharmaceutically acceptable carrier Compound,

Invention also provides the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A (3),guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D (8),guajadial(9),psidial A (10),4,5-diepipsidial A(11),guajadial B(12), Applications of the guajadials C-F (13-16) in the medicine for the complication for preparing the medicine of anticancer and being induced by it.

Apply as mentioned, wherein described cancer be colon cancer, it is acute lymphatic leukaemia, prostate cancer, liver cancer, non-small Cell lung cancer.

Provide the miscellaneous terpene A (1) of compound guava fruit simultaneously, psiguajadial E (2), guadial A (3), guadial B(4), guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D(8), guajadial(9),psidial A(10), 4,5-diepipsidial A(11),guajadial B(12),guajadials C-F (13-16) are preparing the application in suppressing growth of tumour cell agent.

With, the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2) are in preparing and promoting cancer cell-apoptosis agent Application.

And the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guajadial B (12), Guajadial D, guajadial F are preparing the application in suppressing topological enzymatic activity agent.

Present invention further provides the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A(3), guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D (8),guajadial(9), psidial A(10),4,5-diepipsidial A(11),guajadial B(12), Applications of the guajadials C-F (13-16) in functional food is prepared.

The present invention still further provides the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A(3), guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D (8),guajadial(9), psidial A(10),4,5-diepipsidial A(11),guajadial B(12), Guajadials C-F (13-16) preparation method, it is characterised in that this method using petroleum ether including being extracted, silica gel separates, Gel removal of impurities, reverse phase silica gel Rp-18 separation and half prepare HPLC after purification the compound.

More specifically method is：Guava (Psidium guajava) fruit and branches and leaves are taken, it is cold with petroleum ether after crushing Extraction takes 3 times, 48 hours every time, merges extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract point is mixed with the mesh of silica gel 80~100 Sample, through silica gel (200~300 mesh) column chromatography, eluant, eluent petroleum ether:Ethyl acetate (1:0→5:1, v/v) gradient elution, TLC Combining data detection obtains 6 fraction Fr.1-6；Fr.2a (fruit) and Fr.2b (branches and leaves) passes through normal-phase silica gel column chromatography petroleum ether-second Acetoacetic ester (100:1→30:1, v/v), Rp-18 reversed phase column chromatographies MeCN-H₂O-TFA (90:10:0.1→100:0:0.1, v/ V), Saphadex LH-20,16 miscellaneous terpenes of phloroglucin are obtained：The miscellaneous terpene A of pomegranate fruit, psiguajadial E, guadial A, guadial B, guadial C, psiguadial B, psiguadial C, psiguadial D, guajadial, Psidial A, 4,5-diepipsidial A, guajadial B and guajadials C-F.

Present invention selection guava (Psidium guajava) fruit and its branches and leaves are material, carry out extracting and developing, knot Structure is identified and the systematic development work such as screening active ingredients, therefrom obtains 16 miscellaneous terpenes of formyl phloroglucin, wherein there is 1 new such Compound：psiguajavadial A.

The present invention select all compounds carried out in vitro to five plants of human cancer cells (HCT116, CCRF-CEM, DU145, Huh7 and A549) cytotoxic activity test experiments, it is found that all compounds have to all cell lines and preferably suppress to grow Activity, the IC of individual compound₅₀Value reaches nM, such as compound 11 and the IC of 12 pairs of A549 cell lines₅₀Value respectively reaches 160 and 150nM, IC of the compound 14 to HCT116 cell lines₅₀It is worth for 610nM, IC of the compound 9 to CCRF-CEM cell lines₅₀ It is worth for 570nM.Further active testing experiment shows that compound 1,2,12,14 and 16 has the suppression similar to camptothecine Topological enzymatic activity, compound 1 and 2 have the activity of stronger promotion cancer cell-apoptosis.

The compound of the present invention and available for preparing treating cancer and the complication that is induced by it, and preparing feature Applied in food.

It when the compounds of this invention is used as medicine, can directly use, or be used in the form of pharmaceutical composition.The medicine Composition contains 0.1-99%, preferably 0.5-90% the compounds of this invention, remaining to be pharmaceutically acceptable, to people and The nontoxic and inert pharmaceutical acceptable carrier of animal and/or excipient.

Described pharmaceutical carrier or excipient is one or more solids, semisolid and liquid diluent, filler and medicine Tetramune assistant agent.The pharmaceutical composition of the present invention is used in the form of per weight dose.All compounds for effectively into The pharmaceutical composition being grouped is prepared into various formulations, such as liquid preparation (injection using the generally acknowledged method of pharmacy and field of food Agent, supensoid agent, emulsion, solution, syrup etc.), solid pharmaceutical preparation (tablet, capsule, granule, electuary etc.), spray, aerosol Agent etc..The medicine of the present invention can be through injecting (intravenous injection, drip-feed, intramuscular injection, intraperitoneal injection, hypodermic injection) and mouth The methods of administration such as clothes, sublingual administration, mucous membrane dialysis carry out the treatment of cancer and the complication being induced by it.

Brief description of the drawings：

Fig. 1 is the structural representation of the compounds of this invention；

Fig. 2 is relaxing assay (concentration gradient) experimental result of Top1 mediations.Swimming lane 1 and 2 is respectively single PBR322 DNA and pBR322DNA and Top1；Swimming lane 3-7, pBR322 DNA, Top1 and various concentrations from guava fruit Isolated compound 1,2,12,14 and 16 (0.2,1,5,25,125 μM).R：Loose DNA；Sc：Super coiled DNA；

Fig. 3 .A:For the cleavage assay of Top1 mediations.Swimming lane 3-8, pBR322 DNA, Top1 and various concentrations Reactive compound is incubated simultaneously.B：Tested for EMSA.Swimming lane 3-8, pBR322DNA, Top1 and CPT, (25,50 μM) of compound are altogether With incubation.C:For the unwinding assay of Top1 mediations.Substrate be supercoil type DNA (on)；Substrate is loose type DNA (under).Swimming lane 3-11, pBR322 DNA, excessive Top1 and EB, the reactive compound of various concentrations of supercoil.R：Loosely DNA；Sc：Super coiled DNA；N：Breach DNA；C：Top1-DNA compounds；

Fig. 4 A:For the influence of compound 1 and 2 pairs of HCT116 Apoptosis, the compound 1 and 2 of various concentrations is handled The influence of 24 hours of HCT116 cells,

Fig. 4 B:For the influence of compound 1 and 2 pairs of HCT116 Apoptosis, compound 1 and 2 acts on the quantitative post of 24 hours Shape figure；

Fig. 5 is the formation that compound 1 and 2 induces γ H2AX.

Embodiment：

Below in conjunction with the accompanying drawings, the substantive content of the present invention is further illustrated with the embodiment of the present invention, but the present invention's is interior Appearance is not limited thereto.

In following experiments, EI-MS and HRESI-MS are determined by the UPLC/6540Q-TOF mass spectrographs of Agilent 1290, its Middle EI-MS is determined under 70eV；¹H,¹³C NMR and 2D H NMR spectroscopy are surveyed on Bruker Avance III-600 NMRs Fixed (TMS is internal standard)；Column chromatography is Qingdao Haiyang Chinese workers' factory products with silica G (200-300 mesh) and thin-layer chromatography.Thin layer Chromatography observes its spot by 10% ferric trichloride-ethanol solution.Saphadex LH-20 are Pharmcia Products.It is anti-phase Material RP-18 and RP-18 lamellae are Merck Products.

Embodiment 1：

The miscellaneous terpene A of guava fruit and two compounds of guadial A preparation：

After guava (Psidium guajava) fruit 6.0kg is crushed, 3 times are taken with petroleum ether extraction, 48 hours every time, Merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract (320g) silica gel (80-100 mesh) mixes sample, through silica gel (200-300 mesh) Column chromatography (eluent petroleum ether-ethyl acetate, 1:0→5:1v/v) gradient elution, TLC combining data detections obtain 6 parts (Fr.1- 6).Fr.2a (32g) is through sephadex Sephadex LH-20 (CHCl₃-MeOH,3:2v/v) remove aliphatic acid after through RP-18 Reversed phase column chromatography (MeCN-H₂O, 85:15→100:0v/v) obtain 4 part (Fr.2a₁–2a₄)。Fr.2a₁(35 mg) is passed through HPLC anti-phase half prepare (MeCN -0.01%TFA, 80:20→90:10v/v) it is further purified to obtain psiguajavadial A (1,3.2mg) and guadial A (3,2.8mg).

Embodiment 2：

The preparation of Guadials B and guadial two compounds of C：

After guava (Psidium guajava) branches and leaves 10.0kg is crushed, 3 times are taken with petroleum ether extraction, 48 hours every time, Merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract (740g) silica gel (80-100 mesh) mixes sample, through silica gel (200-300 mesh) Column chromatography (eluent petroleum ether-ethyl acetate, 1:0→5:1v/v) gradient elution, TLC combining data detections obtain 6 parts (Fr.1- 6).Fr.2b (85g) is through sephadex Sephadex LH-20 (CHCl₃-MeOH,3:2v/v) except anti-through RP-18 after aliphatic acid Phase column chromatography (MeCN-H₂O, 85:15→100:0v/v) obtain 4 parts (Fr.2b1-2b4). Fr.2b₁(50mg) is through HPLC Anti-phase half prepare (MeCN -0.01%TFA, 80:20→90:10v/v) be further purified to obtain guadial B (4,5.2mg) and Guadial C (5,6.5mg).

Embodiment 3：

Psiguajadial E, guajadial, psidial A, 4,5-diepipsidial A and guajadial B five The preparation of individual compound：

After guava (Psidium guajava) fruit 6.0kg is crushed, 3 times are taken with petroleum ether extraction, 48 hours every time, Merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract (320g) silica gel (80-100 mesh) mixes sample, through silica gel (200-300 mesh) Column chromatography (eluent petroleum ether-ethyl acetate, 1:0→5:1v/v) gradient elution, TLC combining data detections obtain 6 parts (Fr.1- 6).Fr.2a (32g) is through sephadex Sephadex LH-20 (CHCl₃-MeOH,3:2v/v) remove aliphatic acid after through RP-18 Reversed phase column chromatography (MeCN-H₂O, 85:15→100:0v/v) obtain 4 part (Fr.2a₁–2a₄).Guajadial (9, 320mg) and psidial A (10,110mg) are respectively from Fr.2a₂(1.2g) and 2a₃(0.5g) is obtained by the method for recrystallization. Fr.2a₃Mother liquor prepare through HPLC anti-phase half (MeCN -0.01%TFA, 90:10→95:5v/v) it is further purified to obtain Psiguajadial E (2,6.2mg), 4,5-diepipsidial A (11,8.3mg) and guajadial B (12,9.4mg).

Embodiment 4：

Psiguadials B-D, guajadial, psidial A and six compounds of 4,5-diepipsidial A preparation：

After guava (Psidium guajava) branches and leaves 10.0kg is crushed, 3 times are taken with petroleum ether extraction, 48 hours every time, Merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract (740g) silica gel (80-100 mesh) mixes sample, through silica gel (200-300 mesh) Column chromatography (eluent petroleum ether-ethyl acetate, 1:0→5:1v/v) gradient elution, TLC combining data detections obtain 6 parts (Fr.1- 6).Fr.2b (85g) is through sephadex Sephadex LH-20 (CHCl₃-MeOH,3:2v/v) except anti-through RP-18 after aliphatic acid Phase column chromatography (MeCN-H₂O, 85:15→100:0v/v) obtain 4 part (Fr.2b₁–2b₄).Guajadial (9,800mg) With psidial A (10,2.5g) respectively from Fr.2b₂(2.4g) and 2b₃(5.5g) is obtained by the method for recrystallization.Fr.2b₃'s Mother liquor prepare through HPLC anti-phase half (MeCN -0.01%TFA, 90:10→95:5v/v) it is further purified to obtain psiguadial B (6,25.8mg), psiguadial C (7,5.1mg), psiguadial D (8,32.6 mg) and 4,5-diepipsidial A (11,35.2mg) and guajadial B (12,9.4mg).

Embodiment 5：

The preparation of Guajadials C four compounds of-F：

After guava (Psidium guajava) fruit 6.0kg is crushed, 3 times are taken with petroleum ether extraction, 48 hours every time, Merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract (320g) silica gel (80-100 mesh) mixes sample, through silica gel (200-300 mesh) Column chromatography (eluent petroleum ether-ethyl acetate, 1:0→5:1v/v) gradient elution, TLC combining data detections obtain 6 parts (Fr.1- 6).Fr.2a (32g) is through sephadex Sephadex LH-20 (CHCl₃-MeOH,3:2v/v) remove aliphatic acid after through Rp-18 Reversed phase column chromatography (MeCN-H₂O, 85:15→100:0v/v) obtain 4 part (Fr.2a₁–2a₄)。Fr.2a₄(1.1 g) is passed through HPLC anti-phase half prepare (MeCN -0.01%TFA, 95:5→100:0v/v) be further purified to obtain guajadial C (13, 28.7mg), guajadial D (14,25.4mg), guajadial E (15,32.1mg) and guajadial F (16,29.6 mg).

Note：Compound Guajadials C-F (13-16) are present in guava fruit and its branches and leaves, but the former Content is slightly higher in total miscellaneous terpene；In addition, Guajadial (9) and psidial A (10) are main in guava fruit and its branches and leaves Composition, but its ratio difference.

The physical constant and spectral data of the miscellaneous terpene A (1) of guava fruit：Colorless gum；(c 0.1, MeOH)；UV(MeOH)λ_max(logε)205(4.47),285(4.54)nm；ECD(MeOH)250(Δε–1.8),278(Δε + 9.6)nm；IR(KBr)ν_max 3441,2957,2929,1633,1442,1384,1301,1221,1179cm^–1；¹H(600 MHz, CDCl₃)and ¹³C(150MHz,CDCl₃) NMR data is shown in Table 1；HRESIMS m/z 429.1680[M+Na]⁺ (calcd for C₂₅H₂₆O₅Na,429.1672)。

The miscellaneous terpene A's of table 1, guava fruit¹H and¹³C NMR datas are (in CDCl₃Middle measure)

Psiguajadial E (2) physical constant and spectral data：Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI- MS m/z 475.2477[M+H]⁺；¹H NMR(CDCl₃, 600MHz)：δ 4.90 (1H, br d, J=9.2Hz, H-1), 1.54 (1H, br d, J=14.0Hz, H-2a), 2.03 (1H, dd, J=15.1,4.9Hz, H-3a), 1.80 (1H, t, J=13.9Hz, ), H-3b 4.45 (1H, d, J=7.1Hz, H-5), 0.93 (1H, t, J=7.8Hz, H-6), 0.57 (1H, br t, J=9.9Hz, H-7), 0.99 (1H, q, J=12.8Hz, H-8b), 1.95 (1H, t, J=13.9Hz, H-9a), 1.87 (1H, t, J= 12.5Hz,H-9b),1.17(3H,s, Me-12),1.23(3H,s,Me-13),1.08(3H,s,Me-14),1.13(3H,s, Me-15),3.79(1H,s,H-1'),6.91 (1H,br s,H-9'),7.49(1H,br s,H-12'),7.33(1H,br s, H-13'),10.18(1H,s,H-14'),10.07(1H,s, H-15'),13.74(1H,s,OH-5'),13.05(1H,s,OH- 7')；¹³C NMR(CDCl₃,150MHz):δ127.1(d,C-1), 24.3(t,C-2),40.0(t,C-3),40.9(s,C-4), 80.0(d,C-5),28.5(d,C-6),32.7(d,C-7),23.3(t,C-8), 37.6(t,C-9),131.3(s,C-10), 19.4(s,C-11),30.5(q,Me-12),19.9(q,Me-13),18.0(q,Me-14), 26.0(q,Me-15),48.6(d, C-1'),105.7(s,C-2'),163.0(s,C-3'),103.6(s,C-4'),168.6(s,C-5'), 104.0(s,C-6'), 168.8(s,C-7'),142.8(s,C-8'),128.9(d,C-9'),128.2(d,C-10'),126.9(d,C-11'), 132.1(d,C-12'),128.7(d,C-13'),192.7(d,C-14'),191.4(d,C-15')。

Guadial A (3) physical constant and spectral data：Colorless gum；Molecular formula is C₂₅H₂₆O₅；ESI-MS m/z 429 [M+Na]⁺；¹H NMR(CDCl₃,600MHz):δ 1.35 (1H, dd, J=7.5,2.8Hz, H-2), 0.82 (1H, ddd, J= 5.1,3.7Hz, H-3a), 2.34 (1H, dd, J=14.1,7.2Hz, H-7a), 2.01 (1H, dd, J=14.1,10.5Hz, H- 7b), 0.97 (3H, d, J=6.8Hz, Me-9), 0.91 (3H, d, J=6.8Hz, Me-10), 4.21 (1H, dd, J=10.3, 7.3Hz, H-1'), 7.15 × 2 (2H, d, J=7.3Hz, H-9', 13'), 7.29 (2H, t, J=7.5Hz, H-10', 12'), 7.21 (1H, t, J=7.4 Hz, H-11'), 10.10 (1H, s, H-14'), 10.13 (1H, s, H-15'), 13.53 (1H, s, OH- 5'),13.17(1H,s,OH-7')；¹³C NMR(CDCl₃,150MHz):δ88.9(s,C-1),28.3(d,C-2),12.3(t,C- 3),34.7(s,C-4),24.5(t,C-5), 33.6(t,C-6),42.4(t,C-7),32.7(d,C-8),19.9(q,Me-9), 19.8(q,Me-10),35.2(d,C-1'),103.7(s, C-2'),165.9(s,C-3'),104.6(s,C-4'),168.7 (s,C-5'),104.4(s,C-6'),170.0(s,C-7'),144.8(s,C-8'), 126.9×2(d,C-9',13'), 128.8×2(d,C-10',12'),126.6(d,C-11'),192.6(d,C-14'),191.9(d, C-15')。

Guadial B (4) physical constant and spectral data：Colorless gum；Molecular formula is C₂₅H₂₆O₅；ESI-MS m/z 407 [M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 3.00 (1H, br t, J=9.2Hz, H-3), 0.83 (1H, d, J= 9.6Hz, H-7b), 1.01 (3H, s, Me-8), 1.28 (3H, s, Me-9), 1.06 (3H, s, Me-10), 4.24 (1H, br s, H- 1'), 7.09 × 2 (2H, d, J=7.6Hz, H-9', 13'), 7.28 (2H, t, J=7.6Hz, H-10', 12'), 7.20 (1H, t, J=7.3Hz, H-11'), 10.03 (1H, s, H-14'), 10.19 (1H, s, H-15'), 13.52 (1H, s, OH-5'), 13.21 (1H,s,OH-7')；¹³C NMR (CDCl₃,150MHz):δ56.2(d,C-1),88.9(s,C-2),41.3(d,C-3),36.5 (t,C-4),40.7(d,C-5),40.4(s, C-6),27.5(t,C-7),29.1(q,Me-8),27.9(q,Me-9),23.8 (q,Me-10),39.1(d,C-1'),101.5(s,C-2'), 166.4(s,C-3'),103.7(s,C-4'),168.8(s,C- 5'),104.0(s,C-6'),169.3(s,C-7'),142.5(s,C-8'), 127.4×2(d,C-9',13'),128.6×2 (d,C-10',12'),126.7(d,C-11'),192.2(d,C-14'),191.6(d, C-15')。

Guadial C (5) physical constant and spectral data：Colorless gum；Molecular formula is C₂₅H₂₆O₅；ESI-MS m/z 407 [M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 2.29 (1H, t, J=5.3Hz, H-1), 1.49 (1H, d, J=10.3Hz, H-7b), 2.43 (1H, dd, J=14.5,7.0Hz, H-8a), 2.11 (1H, dd, J=14.5,10.2Hz, H-8b), 1.30 (3H, S, Me-9), 0.99 (3H, s, Me-10), 3.99 (1H, dd, J=10.2,6.9Hz, H-1'), 7.13 × 2 (2H, d, J= 7.2Hz, H-9', 13'), 7.28 (2H, t, J=7.5Hz, H-10', 12'), 7.21 (1H, t, J=7.4Hz, H-11'), 10.11×2(2H,s,H-14', 15'),13.51(1H,s,OH-5'),13.13(1H,s,OH-7')；¹³C NMR(CDCl₃, 150MHz):δ47.5(d,C-1),86.2 (s,C-2),29.9(t,C-3),34.5(t,C-4),40.5(d,C-5),38.3(s, C-6),26.3(t,C-7),43.6(t,C-8),27.5(q, Me-9),23.2(q,Me-10),35.1(d,C-1'),102.9 (s,C-2'),164.6(s,C-3'),104.4(s,C-4'),168.6(s, C-5'),104.2(s,C-6'),169.5(s,C- 7'),144.4(s,C-8'),126.7×2(d,C-9',13'),128.6×2(d,C-10', 12'),126.3(d,C-11'), 191.6(d,C-14'),192.2(d,C-15')。

Psiguadial B (6) physical constant and spectral data：Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 497[M+Na]⁺；¹H NMR(CDCl₃,600MHz):δ1.01(3H,s,Me-13),1.01(3H,s,Me-14),0.86 (3H, s,Me-15),10.07×2(2H,s,H-14',15'),13.51(1H,s,OH-5'),13.05(1H,s,OH-7')；¹³C NMR (CDCl₃,150MHz):δ33.4(s,C-1),36.9(t,C-2),35.4(t,C-3),35.1(s,C-4),44.0(d,C- 5),20.1(t, C-6),29.3(t,C-7),84.1(s,C-8),50.0(d,C-9),23.9(t,C-10),37.6(t,C- 11),47.4(t,C-12),30.6(q, Me-13),20.8(q,Me-14),26.1(q,Me-15),40.4(d,C-1'), 105.7(s,C-2'),163.5(s,C-3'),104.6(s, C-4'),168.5(s,C-5'),104.1(s,C-6'),169.6 (s,C-7'),143.4(s,C-8'),128.2×3(d,C-9',11',13'), 126.2×2(d,C-10',12'),191.5 (d,C-14'),192.4(d,C-15')。

Psiguadial C (7) physical constant and spectral data：Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ1.19×2(6H,s,Me-12,13),1.33(3H,s,Me-14), 0.76 (3H, s, Me-15), 4.23 (1H, s, H-1'), 6.78 (1H, d, J=7.6Hz, H-9), 10.12 × 2 (2H, s, H-14', 15'), 13.60(1H,s,OH-5'),13.15(1H,s,OH-7')；¹³C NMR(CDCl₃,150MHz):δ64.0(d,C-1), 22.5(t, C-2),31.6(t,C-3),40.6(s,C-4),84.0(s,C-5),26.5(d,C-6),31.6(d,C-7),21.3 (t,C-8),39.3(t, C-9),60.5(s,C-10),21.6(s,C-11),19.3(q,Me-12),30.3(q,Me-13), 17.2(q,Me-14),18.5(q, Me-15),43.9(d,C-1'),104.6(s,C-2'),165.8(s,C-3'),104.2 (s,C-4'),168.3(s,C-5'),104.8(s, C-6'),170.6(s,C-7'),140.4(s,C-8'),127.6(d,C- 9'),127.4(d,C-10'),126.5(d,C-11'),130.3(d, C-12'),127.7(d,C-13'),192.0(d,C- 14'),191.6(d,C-15')。

Psiguadial D (8) physical constant and spectral data：Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 5.27 (1H, br d, J=8.5Hz, H-1), 3.71 (1H, d, J= 7.1 Hz,H-5),1.18(3H,s,Me-12),1.12(3H,s,Me-13),1.71(3H,s,Me-14),0.71(3H,s,Me- 15), 4.37 (1H, s, H-1'), 6.78 (1H, d, J=7.8Hz, H-9), 10.11 (1H, s, H-14'), 10.09 (1H, s, H- 15'),13.60 (1H,s,OH-5'),13.14(1H,s,OH-7')；¹³C NMR(CDCl₃,150MHz):δ127.5(d,C-1), 23.4(t,C-2), 35.4(t,C-3),41.4(s,C-4),85.3(d,C-5),26.9(d,C-6),31.5(d,C-7),22.4 (t,C-8),38.1(t,C-9), 130.7(s,C-10),19.8(s,C-11),30.4(q,Me-12),19.3(q,Me-13), 17.5(q,Me-14),18.6(q, Me-15),43.9(d,C-1'),105.2(s,C-2'),166.4(s,C-3'),104.4 (s,C-4'),168.3(s,C-5'),104.2(s, C-6'),170.6(s,C-7'),140.4(s,C-8'),127.5×2(d, C-9',10'),126.3(d,C-11'),130.2(d,C-12'), 127.8(d,C-13'),192.2(d,C-14'),191.6 (d,C-15')。

Guajadial (9) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ1.03(3H,s,Me-12),1.00(3H,s,Me-13),1.29(3H,s, ), Me-14 5.56 (1H, s, H-15a), 4.06 (1H, s, H-15b), 3.40 (1H, d, J=10.3Hz, H-1'), 10.12 (1H, s, H-14'),10.08(1H,s,H-15'),13.47(1H,s,OH-5'),13.08(1H,s,OH-7')；¹³C NMR(CDCl₃, 150 MHz):δ53.2(d,C-1),22.1(t,C-2),37.0(t,C-3),84.3(s,C-4),43.4(d,C-5),30.3(t, C-6),35.4(t, C-7),150.6(s,C-8),41.3(d,C-9),36.6(t,C-10),33.7(s,C-11),30.4(q, Me-12),21.9(q,Me-13), 21.1(q,Me-14),110.1(t,C-15),43.3(d,C-1'),105.4(s,C-2'), 163.3(s,C-3'),104.1(s,C-4'), 168.3(s,C-5'),104.3(s,C-6'),169.4(s,C-7'),143.7 (s,C-8'),128.7×2(d,C-9',13'),127.8×2(d, C-10',12'),126.1(d,C-11'),192.0(d, C-14'),191.4(d,C-15')。

Psidial A (10) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/ z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 2.12 (1H, dd, J=13.8,9.4Hz, H-3b), 2.06 (1H, br t, J=7.7Hz, H-5), 2.59 (1H, td, J=13.4,2.9Hz, H-7a), 2.27 (1H, br d, J=14.0Hz, H-7b), 0.97(3H,s, Me-12),0.99(3H,s,Me-13),1.18(3H,s,Me-14),5.04(1H,s,H-15a),5.02(1H, S, H-15b), 4.19 (1H, d, J=5.6Hz, H-1'), 10.15 (1H, s, H-14'), 10.11 (1H, s, H-15'), 13.45 (1H,s,OH-5'),13.10 (1H,s,OH-7')；¹³C NMR(CDCl₃,150MHz):δ59.3(d,C-1),23.9(t,C- 2),47.9(t,C-3),88.1(s, C-4),35.0(d,C-5),36.0(t,C-6),35.5(t,C-7),150.7(s,C-8), 42.2(d,C-9),24.4(t,C-10),34.7(s, C-11),29.7(q,Me-12),21.7(q,Me-13),22.1(q,Me- 14),111.2(t,C-15),35.0(d,C-1'),107.5(s, C-2'),164.1(s,C-3'),104.6(s,C-4'), 167.5(s,C-5'),104.1(s,C-6'),168.4(s,C-7'),138.6(s,C-8'), 129.5×2(d,C-9', 13'),128.0×2(d,C-10',12'),126.9(d,C-11'),192.0(d,C-14'),191.6(d, C-15')。

4,5-diepipsidial A (11) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅； ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ0.99(6H,s,Me-12,13),1.20(3H,s, Me- 14), 4.60 (1H, s, H-15a), 4.59 (1H, s, H-15b), 3.42 (1H, d, J=10.3Hz, H-1'), 10.08 (1H, s, H-14'),10.07(1H,s,H-15'),13.48(1H,s,OH-5'),13.08(1H,s,OH-7')；¹³C NMR(CDCl₃,150 MHz):δ57.4(d,C-1),22.4(t,C-2),38.2(t,C-3),84.7(s,C-4),43.8(d,C-5),33.5(t,C- 6),37.2(t, C-7),154.9(s,C-8),42.1(d,C-9),38.7(t,C-10),33.2(s,C-11),29.5(q,Me- 12),22.4(q,Me-13), 20.1(q,Me-14),109.6(t,C-15),44.3(d,C-1'),105.0(s,C-2'), 163.6(s,C-3'),104.2(s,C-4'), 168.4(s,C-5'),104.1(s,C-6'),169.4(s,C-7'),144.1 (s,C-8'),128.3×2(d,C-9',13'),128.0×2(d, C-10',12'),126.4(d,C-11'),192.2(d, C-14'),191.5(d,C-15')。

(±) Guajadial B (12) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅； ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 4.52 (1H, dd, J=10.9,3.6Hz, H-2), 1.44 (3H, s, Me-12), 1.23 (3H, s, Me-13), 1.05 × 2 (6H, s, Me-14,15), 3.60 (1H, d, J=10.0Hz, H- 1'),10.11 (1H,s,H-14'),10.07(1H,s,H-15'),13.46(1H,s,OH-5'),13.01(1H,s,OH-7') ；¹³C NMR(CDCl₃, 150MHz):δ41.3(t,C-1),123.0(d,C-2),136.3(s,C-3),37.4(t,C-4), 30.7(t,C-5),43.4(d,C-6), 85.2(s,C-7),42.4(t,C-8),119.3(d,C-9),143.2(d,C-10), 38.6(s,C-11),16.7(q,Me-12),19.9 (q,Me-13),23.9(q,Me-14),29.8(q,C-15),44.7(d, C-1'),105.8(s,C-2'),163.3(s,C-3'),104.3 (s,C-4'),168.4(s,C-5'),104.2(s,C-6'), 169.3(s,C-7'),144.7(s,C-8'),128.3×4(d,C-9',10',12', 13'),126.5(d,C-11'), 192.2(d,C-14'),191.6(d,C-15')。

Guajadial C (13) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):¹H NMR(CDCl₃,600MHz):δ5.33(1H,s,H-5),0.57 (3H, d, J=6.6Hz, Me-12), 0.88 (3H, d, J=6.5Hz, Me-13), 0.90 (3H, d, J=7.0Hz, Me-14), 2.16 (1H, dd, J=14.3,7.1Hz, H-15a), 2.04 (1H, dd, J=14.3,10.4Hz, H-15b), 4.06 (1H, dd, J=10.3,7.2Hz, H-1'), 7.13 × 2 (2H, d, J=7.3Hz, H-9', 13'), 7.27 × 2 (2H, t, J=7.5Hz, H- 10', 12'), 7.20 (1H, t, J=7.3Hz, H-11'), 10.10 (1H, s, H-14'), 10.12 (1H, s, H-15'), 13.56 (1H,s,OH-5'),13.15 (1H,s,OH-5')。

Guajadial D (14) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 497[M+Na]⁺；¹H NMR(CDCl₃,600MHz):δ 5.33 (1H, s, H-5), 0.75 (3H, d, J=6.6Hz, Me-12), 0.93 (3H, d, J=6.5Hz, Me-13), 0.87 (3H, d, J=7.0Hz, Me-14), 2.25 (1H, dd, J=14.2,7.2Hz, ), H-15a 2.03 (1H, dd, J=14.2,9.1Hz, H-15b), 7.13 × 2 (2H, d, J=7.2Hz, H-9', 13'), 7.26 × 2 (2H, t, J=7.5Hz, H-10', 12'), 7.20 (1H, t, J=7.3Hz, H-11'), 10.10 (1H, s, H-14'), 10.13 (1H,s,H-15'), 13.55(1H,s,OH-5'),13.19(1H,s,OH-5')。

Guajadial E (15) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 5.32 (1H, s, H-5), 0.53 (3H, d, J=6.5Hz, Me-12), 0.88 (3H, d, J=6.5Hz, Me-13), 0.92 (3H, d, J=6.9Hz, Me-14), 1.46 (3H, s, Me-15), 3.67 (1H, D, J=6.9Hz, H-1'), 7.15 × 2 (2H, d, J=7.4Hz, H-9', 13'), 7.27 × 2 (2H, t, J=7.4Hz, H- 10', 12'), 7.20 (1H, t, J=7.3Hz, H-11'), 10.15 (1H, s, H-14'), 10.09 (1H, s, H-15'), 13.50 (1H,s,OH-5'), 13.05(1H,s,OH-7')；¹³C NMR(CDCl₃,150MHz):δ34.2(d,C-1),27.1(t,C- 2),44.5(d,C-3), 80.1(s,C-4),126.4(d,C-5),144.4(s,C-6),50.8(d,C-7),22.6(t,C- 8),29.1(t,C-9),33.7(d, C-10),26.5(d,C-11),21.1×2(q,Me-12,13),14.3(q,Me-14), 28.1(q,C-15),38.6(d,C-1'), 104.3(s,C-2'),163.4(s,C-3'),104.0×2(s,C-4',6'), 168.2(s,C-5'),169.7(s,C-7'),144.5(s, C-8'),127.6×2(d,C-9',13'),128.3×2(d, C10',12'),126.2(d,C-11'),192.3(d,C-14'),191.5(d, C-15')。

Guajadial F (16) physical constant and spectral data:Colorless gum；Molecular formula is C₃₀H₃₄O₅；ESI-MS m/z 475[M+H]⁺；¹H NMR(CDCl₃,600MHz):δ 5.59 (1H, s, H-5), 0.78 (3H, d, J=6.5Hz, Me-12), 0.91 (3H, d, J=6.4Hz, Me-13), 0.65 (3H, d, J=7.0Hz, Me-14), 1.50 (3H, s, Me-15), 4.41 (1H, D, J=7.1Hz, H-1'), 10.03 (1H, s, H-14'), 10.14 (1H, s, H-15'), 13.51 (1H, s, OH-5'), 13.30 (1H,s, OH-7')。

Embodiment 6：

Guava fruit four compounds of miscellaneous terpene A and guadials A-C are first made by the method for Examples 1 and 2, by normal Parenteral solution is made in rule plus water for injection, refined filtration, embedding sterilizing.

Embodiment 7：

By implement 3 and 4 method first be made psiguajadial B, psiguadials B-D, guajadial, Eight psidial A, 4,5-diepipsidial A and guajadial B compounds, routinely add water for injection, refined filtration, fill Parenteral solution is made in envelope sterilizing.

Embodiment 8：

Guava fruit four compounds of miscellaneous terpene A and guadials A-C are first made by the method for Examples 1 and 2, by it It is dissolved in sterile water for injection, stirring makes molten, is filtered with sterile suction funnel, then sterile refined filtration is sub-packed in 2 ampoules, low temperature It is sterile after freeze-drying to seal to obtain powder-injection.

Embodiment 9：

By implement 3 and 4 method first be made psiguajavadial B, psiguadials B-D, guajadial, Eight psidial A, 4,5-diepipsidial A and guajadial B compounds, are dissolved in sterile water for injection, Stirring makes molten, is filtered with sterile suction funnel, then sterile refined filtration is sub-packed in 2 ampoules, sterile after frozen drying to seal Powder-injection.

Embodiment 10：

By Examples 1 and 2 it is separated four compounds of guava fruit miscellaneous terpene A and guadials A-C and excipient weight Amount is than being 9:1 ratio adds excipient, and pulvis is made.

Embodiment 11：

By implement 3 and 4 method first be made psiguajavadial B, psiguadials B-D, guajadial, Psidial A, 4,5-diepipsidial A and eight compounds of guajadial B and excipient weight ratio are 9:1 ratio Excipient is added, pulvis is made.

Embodiment 12：

Guava fruit four compounds of miscellaneous terpene A and guadials A-C are first made by the method for Examples 1 and 2, by it It is 1 with excipient weight ratio:5–1:10 ratio adds excipient, pelletizing press sheet.

Embodiment 13：

By implement 3 and 4 method first be made psiguajavadial B, psiguadials B-D, guajadial, Eight psidial A, 4,5-diepipsidial A and guajadial B compounds, it is 1 by itself and excipient weight ratio:5– 1:10 ratio adds excipient, pelletizing press sheet.

Embodiment 14：

Guava fruit four compounds of miscellaneous terpene A and guadials A-C are first made by the method for Examples 1 and 2, by normal Oral liquid is made in rule oral liquid preparation method.

Embodiment 15：

By implement 3 and 4 method first be made psiguajavadial B, psiguadials B-D, guajadial, Eight psidial A, 4,5-diepipsidial A and guajadial B compounds, routinely oral liquid preparation method be made orally Liquid.

Embodiment 16：

By embodiment 1-4 method first be made four compounds of guava fruit miscellaneous terpene A and guadials A-C and Psiguajavadial B, psiguadials B-D, guajadial, psidial A, 4,5-diepipsidial A and Eight compounds of guajadial B, routinely oral liquid preparation method oral liquid is made.

Embodiment 17：

By embodiment 1-4 method first be made four compounds of guava fruit miscellaneous terpene A and guadials A-C and Psiguajavadial B, psiguadials B-D, guajadial, psidial A, 4,5-diepipsidial A and Eight compounds of guajadial B, it is 3 by itself and excipient weight ratio:1 ratio adds excipient, and capsule or particle is made Agent or electuary.

Embodiment 18：

Take by embodiment 3 and 4 method be made psiguajavadial B, psiguadials B-D, guajadial, Psidial A, 4,5-diepipsidial A and guajadial B eight, 12.4 grams of compounds, add 600 grams of starch, lactose 200 grams, 5 grams of menthol, 183 grams of sodium carboxymethyl starch, lozenge is made, as functional food.

Superiority for a better understanding of the present invention, will be below test example with all compound effects results of the present invention Son illustrates, but does not limit the present invention with this.

Test example 1：

Miscellaneous 1-16 pair of influence for suppressing five plants of growth of cancer cells and topoisomerase I of terpene compound：

Reference literature method (Mosmann, T.Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays.J.Immunol.Methods.1983,65: 55–63).The experimental procedure of attached cell mtt assay：It is thin to collect logarithmic phase Born of the same parents, adjustment cell density are 5 × 10⁴Individual/mL；According to 100 μ L/ holes, it is seeded in 96 orifice plates, in 37 DEG C, 5%CO₂Under the conditions of train Support 24 hours；

Old culture medium in 96 orifice plates is removed, adds the drug containing of various concentrations (100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM) Culture medium, 100 μ L/ holes, and in 37 DEG C, 5%CO₂Under the conditions of cultivate 72 hours；The MTT that 20 μ L 2.5mg/mL are added per hole is molten Liquid, and in 37 DEG C, 5%CO₂Under the conditions of cultivate 4 hours；Solution is removed, and 100 μ L DMSO are added in every hole；96 orifice plates are put Light absorption value at ELIASA, detection 570/490；The inhibiting rate of compound on intracellular is calculated, formula is：Inhibiting rate=[1- (A_Experiment–A_Blank)]/(A_Control–A_Blank), and half-inhibition concentration IC is obtained according to inhibiting rate₅₀Value.

The experimental procedure of suspension cell mtt assay：Logarithmic phase cell is collected, adjustment cell density is 1 × 10⁵Individual/mL；According to 50 μ L/ holes, are seeded in 96 orifice plates, add the pastille culture medium of various concentrations (200 μM, 20 μM, 2 μM, 0.2 μM, 0.02 μM), 50 μ L/ holes, and in 37 DEG C, 5%CO₂Under the conditions of cultivate 72 hours；20 μ L 2.5mg/mL of the addition MTT solution per hole, and in 37 DEG C, 5%CO₂Under the conditions of cultivate 4 hours；The lysates of 100 μ L tri- are added per hole, and in 37 DEG C, 5%CO₂Under the conditions of be incubated Overnight；96 orifice plates are placed in ELIASA, detect the light absorption value at 570/490；Calculate the inhibiting rate of compound on intracellular, formula For：Inhibiting rate=[1-(A_Experiment–A_Blank)]/(A_Control–A_Blank), and half-inhibition concentration IC is obtained according to inhibiting rate₅₀Value.

DNA is loosely tested (Top1-mediated Relaxation Assay)：Make 1% Ago-Gel；Top1 (concentration is 20U/ μ L) dilutes 100 times with Top1 buffer solutions, obtains the Top1 solution that concentration is 1U/ μ L, stand-by；Will with DMSO Compound is configured to certain concentration gradient；Following reagent is separately added into 20 μ L reaction system：PBR322 after 1 μ L dilutions DNA solution, the Top1 solution after 1 μ L dilutions, 1 μ L compound solutions, adds Top1 buffer solutions to supply volume；Wherein, first sample Product are pBR322 DNA, are not required to add Top1 and compound, and second sample is pBR322 DNA and Top1, are not required to add compound； After preparing sample, it is put into 37 DEG C of water-baths and is incubated 30min；4 μ 6 × loading of L buffer mixing is added in each sample, Add in the sample well of Ago-Gel, electrophoresis 1.5 hours under 100V voltages；After electrophoresis, in 1 × gel red solution Dye half an hour, photograph.

DNA breach tests (Top1-mediated Cleavage Assay)：Make 1% Ago-Gel；Use DMSO Compound is configured to certain concentration gradient；Following reagent is separately added into 20 μ L reaction system：1μL pBR322 DNA Solution (the μ g/ μ L of concentration 0.5), 1 μ L Top1 solution (concentration 10U/ μ L), 1 μ L compound solutions, adds Top1 buffer solutions to supply body Product；Wherein, first sample is pBR322 DNA, is not required to plus Top1 and compound, second sample be pBR322 DNA and Top1, it is not required to add compound.

After preparing sample, it is put into 37 DEG C of water-baths and is incubated 30min；Added after incubation in each sample a certain amount of 10% SDS solution so that final concentration of the 1% of SDS in sample；Proteolytic enzyme K is added in each sample, makes its concentration For 1mg/mL, 10min is incubated in 50 DEG C of water-baths；5 μ 6 × loading of L buffer mixing is added in each sample, adds agar In the sample well of sugared gel, electrophoresis 0.5 hour under 60V voltages；After electrophoresis, Ago-Gel is put into containing 0.125 μ g/ Half an hour is dyed in 1 × TAE cushioning liquid of mL ethidium bromides, is placed again into electrophoresis half an hour in electrophoresis tank；Electrophoresis finishes Afterwards, take a picture.

Gel retardation assasy (Top1-mediated EMSA)：1% agarose containing 5mg/L ethidium bromides is made to coagulate Glue；Following reagent is separately added into 20 μ L reaction system：PBR322 DNA solutions after 1 μ L dilutions, the Top1 after 1 μ L dilutions Solution, 1 μ L compound solutions, adds Top1 buffer solutions to supply volume；Wherein, first sample is pBR322 DNA, is not required to add Top1 and compound, second sample is pBR322 DNA and Top1, is not required to add compound；After preparing sample, incubation at room temperature 3min；4 μ 6 × loading of L buffer mixing is added in each sample, is added in the sample well of Ago-Gel, 20V voltages Lower electrophoresis 6.5 hours；After electrophoresis, directly take a picture.

DNA insertion experiments (Top1-mediated Unwinding)：Make 1% Ago-Gel；With DMSO by chemical combination Thing is configured to certain concentration gradient (1,5,25 μM)；Prepare reaction terminating liquid：5%SDS, 5mg/mL proteolytic enzyme K.Prepare 37 DEG C of preheatings are put into after good；Following reagent is separately added into 20 μ L reaction system：The 1 μ L pBR322 DNA solutions (μ of concentration 0.2 G/ μ L), 1 μ L Top1 solution (concentration 20U/ μ L), 1 μ L compound solutions, add Top1 buffer solutions to supply volume；Wherein, first Individual sample is pBR322 DNA, is not required to add Top1 and compound, and second sample is pBR322 DNA and Top1, is not required to add chemical combination Thing.0.3,0.6,1.2mg/L ethidium bromide (EB) is positive control；After preparing sample, it is put into 37 DEG C of water-baths and is incubated 30min；5mL reaction terminating liquids are added after incubation in each sample；Each sample adds 56 × loading of μ L Buffer is mixed, and is added in the sample well of Ago-Gel, electrophoresis 1.5 hours under 100 V voltages；After electrophoresis, 1 × Half an hour is dyed in gel red solution, is taken a picture.

As a result show, growth of all compounds to five plants of cancer cells has obvious inhibitory action (table 2), finds institute There is compound to have to all cell lines and preferably suppress growth activity, the IC of individual compound₅₀Value reaches nM, such as chemical combination The IC of thing 11 and 12 pairs of A549 cell lines₅₀Value has respectively reached 160 and 150nM, IC of the compound 14 to HCT116 cell lines₅₀ It is worth for 610nM, IC of the compound 9 to CCRF-CEM cell lines₅₀It is worth for 570nM.In addition, compound 1-5,8,12-14,15 and 16 The significant inhibitory activity of topoisomerase 1 (Fig. 2) is shown, its inhibiting rate reaches more than 90%.Compound 1 and 2 pairs Top1 inhibitory activity has concentration dependent.Found by the research with Top1 interaction characteristics and binding mode, activity Compound is a kind of Top1 catalytic types inhibitor (Fig. 3) of novel mechanism.

Table 2, compound 1-16 suppress growth of cancer cells (μM) and Top1 enzymes (%)

^aRelative to the camptothecine under 50 μM of concentration, the Top1 enzyme inhibition activities of compound are expressed as follows：++++, is more than 90%；+++, between 60% and 89%；++, between 30% and 59%.^bPositive control, unit nM.

Test example 2：

Cell apoptosis assay：Logarithmic phase cell is collected, adjustment cell density is 1.5 × 10⁵Individual/mL, plant in 6 orifice plates, Per hole 2mL, in 37 DEG C, 5%CO₂Under the conditions of cultivate 24 hours；Old culture medium is discarded, and adds being trained containing compound for various concentrations Support base (10 μM, 20 μM, 40 μM), in 37 DEG C, 5%CO₂Under the conditions of cultivate respectively 24 hours；Old culture medium is collected, pancreatin digests, Cell under digesting is mixed with old culture medium, and cell is washed with cold PBS solution；Add 500 μ L1 × Binding Buffer is into cell and mixes.Under light protected environment, 10 μ L PI and 5 μ L Annexin-V-FITC, room temperature lucifuge are added Reaction 15 minutes, sample was placed in flow cytometer test rapidly in 1 hour.

Cell apoptosis assay is using HCT116 as test cell, experimental result such as Fig. 4.When adding various concentrations (10 μM, 20 μ M, 40 μM) compound 1 and 2 act on 24 hours after, relative to the 0.82% of blank group, the early apoptosis ratio of compound 1 is distinguished Increase to 1.40%, 7.37% and 21.25%, the early apoptosis ratio of compound 2 increases respectively to 1.17%, 6.53% and 36.23%.Apoptosis ratio increases with being incremented by for concentration, and certain concentration dependent is presented.As a result compound 1 and 2 is shown Ability with inducing cell apoptosis and into concentration dependent.

Test example 3：

Immunofluorescence experiment：Logarithmic phase cell is collected, adjustment cell density is 3 × 10⁴Individual/mL, plant in 24 orifice plates, often Hole 1mL, in 37 DEG C, 5%CO₂Under the conditions of cultivate 24 hours；Discard old culture medium, and add 0.5 μM of CPT in 37 DEG C, 5%CO₂Bar The compound of 3 hours and 14 μM is cultivated under part in 37 DEG C, 5%CO₂Under the conditions of cultivate 15 hours；Old culture medium is discarded, more than 4% Polyformaldehyde room temperature fixes cell 15 minutes；Fixer is removed, cell is washed 2 times with PBS, 5 minutes every time, adds and contains 0.5% The Triton-X 100 μ L of permeabilization liquid 500, in 37 DEG C of permeabilizations 30 minutes；Permeabilization liquid is washed away with 5% lowlenthal serum confining liquid 3 times, 10 minutes every time, add 1mL confining liquids and closed 30 minutes in 37 DEG C；Primary antibody is added, 100 μ L are per hole, in 4 DEG C of overnight incubations； Cell is washed with confining liquid 6 times, 5 minutes every time, adds DAPI, fluorescence secondary antibody, and 100 μ L are small in 37 DEG C of lucifuges incubations 2 per hole When；Cell is washed with confining liquid 6 times, 10 minutes every time, and sample is placed in laser confocal microscope imaging.

Immunofluorescence experiment is carried out using HCT116 as test cell, detects the mark γ H of DNA damage₂AX formation.Knot Fruit has gone out in the nucleus of most cells as shown in figure 5, positive control CPT is acted on 3 hours under 0.5 μM of concentration Existing γ H₂AX signals, show now to have occurred and that different degrees of DNA damage into the cell.And compound 1 and 2 is dense at 14 μM The lower effect of degree is up to the γ H in 15 hour cell cores₂AX signals are very weak.Show that compound 1 and 2 can not produce under the concentration The DNA damage of Top1 mediations, this is consistent with the conclusion that Top1 enzymes of the present invention are tested.

Claims (10)

1. the miscellaneous terpene A (1) of the miscellaneous terpenoid guava fruit of formyl phloroglucin in the guava shown in following structural formula.

2. the miscellaneous terpene A (1) of compound guava fruit shown in the following structural formula containing therapeutically effective amount, psiguajadial E (2),guadial A(3),guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7), psiguadial D(8),guajadial(9),psidial A(10),4,5-diepipsidial A(11),guajadial B (12), the medicine group of guajadials C-F (13-16) any one or two kinds of mixture and pharmaceutically acceptable carrier Compound.

3. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A (3), guadial B (4), guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D(8),guajadial(9), Psidial A (10), 4,5-diepipsidial A (11), guajadial B (12), guajadials C-F (13-16) exist Prepare the medicine of anticancer and the medicine of complication that is induced by it in application.

4. application as claimed in claim 3, it is characterised in that described cancer is colon cancer, acute lymphatic leukaemia, forefront Gland cancer, liver cancer, non-small cell lung cancer.

5. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A (3), guadial B (4), guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D(8),guajadial(9), Psidial A (10), 4,5-diepipsidial A (11), guajadial B (12), guajadials C-F (13-16) exist Prepare the application suppressed in growth of tumour cell agent.

6. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2) answering in promotion cancer cell-apoptosis agent is prepared With.

7. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guajadial B (12), guajadials D, guajadials F are preparing the application in suppressing topological enzymatic activity agent.

8. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A (3), guadial B (4), guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D(8),guajadial(9), Psidial A (10), 4,5-diepipsidial A (11), guajadial B (12), guajadials C-F (13-16) exist Prepare the application in functional food.

9. the miscellaneous terpene A (1) of compound guava fruit, psiguajadial E (2), guadial A (3), guadial B (4), guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D(8),guajadial(9), Psidial A's (10), 4,5-diepipsidial A (11), guajadial B (12), guajadials C-F (13-16) Preparation method, it is characterised in that this method is included using petroleum ether extraction, silica gel separation, gel removal of impurities, Rp-18 points of reverse phase silica gel From and half prepare HPLC after purification the compound.

10. the miscellaneous terpene A (1) of compound guava fruit as claimed in claim 9, psiguajadial E (2), guadial A (3),guadial B(4),guadial C(5),psiguadial B(6),psiguadial C(7),psiguadial D (8),guajadial(9),psidial A(10),4,5-diepipsidial A(11),guajadial B(12), Guajadials C-F (13-16) preparation method, it is characterised in that this method includes：Guava fruit and branches and leaves are taken, is crushed Afterwards, extracted 3 times with petroleum ether cold soaking, 48 hours every time, merge extract solution, solvent is recovered under reduced pressure and obtains medicinal extract.Medicinal extract point uses silica gel 80~100 mesh mix sample, and through the mesh column chromatography of silica gel 200~300, eluant, eluent is with 1:0→5:1, v/v petroleum ether:Ethyl acetate ladder Degree elution, TLC combining data detections obtain 6 fraction Fr.1-6；Fr.2a and Fr.2b passes through normal-phase silica gel column chromatography 100:1→30: 1, v/v petroleum ether-ethyl acetate, RP-18 reversed phase column chromatographies MeCN-H₂O-TFA, 90:10:0.1→100:0:0.1, v/v, Saphadex LH-20, obtain 16 miscellaneous terpenes of phloroglucin：The miscellaneous terpene A of pomegranate fruit, psiguajadial E, guadial A, Guadial B, guadial C, psiguadial B, psiguadial C, psiguadial D, guajadial, psidial A, 4,5-diepipsidial A, guajadial B and guajadials C-F.