Pharmaceutical composition for resisting non-small cell lung cancer as well as preparation method and application of pharmaceutical composition

Abstract

The invention discloses a pharmaceutical composition for resisting non-small cell lung cancer as well as a preparation method and application of the pharmaceutical composition, belongs to the technical field of antitumor drugs, and mainly solves the technical problem that application is affected due to the fact that an Iridobelamal A compound has high toxicity to normal lung cells. The composition provided by the invention comprises the following active components: an iridoaldehyde triterpenoid compound Iridobelamal A and a flavonoid compound tectorigenin, the molar ratio of the Iridobelamal A to the tectorigenin is 1: 2-1: 5, the molecular formula of the Iridobelamal A is C30H50O4, the molecular formula of the tectorigenin is C16H12O6, and the composition and a pharmaceutical carrier are prepared into an oral preparation or an injection. The composition is used for preparing a medicine for treating non-small cell lung cancer by neutralizing triterpene toxicity and synergistically enhancing anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the technical field of antitumor drugs, and more specifically, this invention relates to a pharmaceutical composition for treating non-small cell lung cancer, its preparation method, and its application. Background Technology

[0002] In existing technologies, while irisaldehyde-type triterpenes (such as Iridobelamal A) exhibit significant inhibitory activity against non-small cell lung cancer, their cytotoxicity (IC50 ≈ 11.3 μM) to normal lung epithelial cells (BEAS-2B) limits their clinical application. Meanwhile, although the flavonoid components in *Belamcanda chinensis* (such as belamcanda glycosides) have well-defined structures, they still exhibit toxicity to normal cells when used alone (IC50 ≈ 20 μM). Currently, it remains difficult to achieve a synergistic effect and reduce toxicity through component combinations. Summary of the Invention

[0003] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0004] This invention provides a pharmaceutical composition for treating non-small cell lung cancer, which, through the combination of Iridobelamal A and iris flavin, can reduce the toxicity of iris aldehyde triterpenoids to normal lung cells and maintain antitumor activity.

[0005] The present invention provides a pharmaceutical composition for treating non-small cell lung cancer, comprising the following active ingredients: iridoaldehyde triterpenoid compound Iridobelamal A and flavonoid compound iridoflavin; wherein the molar ratio of iridoaldehyde triterpenoid compound Iridobelamal A and flavonoid compound iridoflavin is 1:2 to 1:5.

[0006] Preferably, the molecular formula of the iridoaldehyde-type triterpenoid compound Iridobelamal A is C. 30 H 50 O4, chemical structural formula is:

[0007]

[0008] The molecular formula of the flavonoid compound irisin is C 16 H 12 O6, chemical structural formula is:

[0009] Where R1 = OH, R2 = H, R3 = OH, and R4 = H.

[0010] Preferably, it also contains the active ingredient irisdichotin B; wherein the molar ratio of irisdichotin B to Iridobelamal A and iris flavin is 1:1:2 to 1:1:5.

[0011] Preferably, the molecular formula of compound irisdichotin B is C. 23 H 26 O 12 The chemical structural formula is: Where R1 = D-Glc, R2 = CH3, and R3 = OCH3.

[0012] Preferably, the composition further comprises a pharmaceutically acceptable carrier for formulation into an oral or injectable form.

[0013] The present invention also provides a method for preparing the above composition, comprising mixing irisdichotin B, Iridobelamal A and iris flavin in a certain proportion and then preparing nanoliposomes together with a carrier.

[0014] Preferably, the preparation of nanoliposomes employs thin-film dispersion and ultrasonication, specifically including the following steps:

[0015] 1) Dissolve soybean lecithin, cholesterol and the active ingredients of the composition (Iridobelamal A, irisdichotin B) in a mixed solvent of chloroform and methanol (volume ratio 3:1) at a mass ratio of 40-50:5-10:1;

[0016] 2) Rotary evaporation forms a lipid film, followed by vacuum drying to remove residual organic solvents;

[0017] 3) Add phosphate buffer (PBS, pH 7.4) to hydrate, and sonicate (300W power, 15min).

[0018] 4) Nanoliposomes with a particle size of 80-120 nm were obtained by cycling them three times at 10,000 psi using a microfluidic homogenizer.

[0019] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating non-small cell lung cancer.

[0020] The present invention has at least the following beneficial effects:

[0021] 1. This invention utilizes the combination of Iridobelamal A and iris flavin to neutralize the toxicity of triterpenoids and regulate the tumor microenvironment, thereby enhancing the apoptosis-inducing effect of triterpenoids on tumor cells and improving antitumor activity while reducing the toxicity of iris aldehyde triterpenoids to normal lung cells.

[0022] 2. This invention further neutralizes oxidative damage induced by triterpenoids through irisdichotin B, protecting normal cells and maintaining antitumor activity.

[0023] 3. This invention also employs thin-film dispersion and ultrasonic methods to prepare nanoliposomes, thereby improving bioavailability, enhancing targeted delivery of components, and reducing toxic side effects.

[0024] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0025] Figure 1 For the present invention IridobelamalA 13 C-NMR spectrum;

[0026] Figure 2 For the present invention IridobelamalA 1 H-NMR spectrum;

[0027] Figure 3 The iris flavin of this invention 13 C-NMR spectrum;

[0028] Figure 4 The iris flavin of this invention 1 H-NMR spectrum;

[0029] Figure 5 For the irisdichotin B of the present invention 13 C-NMR spectrum;

[0030] Figure 6 For the irisdichotin B of the present invention 1 H-NMR spectrum. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials mentioned are commercially available. In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "setting" should be interpreted broadly. For example, they can refer to fixed connection or setting, detachable connection or setting, or integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The terms "lateral," "longitudinal," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0033] The Iridobelamal A disclosed in this invention is separated from Belamcanda chinensis through the following steps:

[0034] (a) The dried rhizome of Belamcanda chinensis was extracted with 75% ethanol and concentrated under reduced pressure to obtain the alcohol extract;

[0035] (b) The alcohol extract was extracted with ethyl acetate and concentrated to obtain the ethyl acetate fraction (CSY);

[0036] (c) CSY was subjected to silica gel column chromatography (chloroform-methanol gradient 50:1→1:1), and the fraction Fr.Y1 was collected;

[0037] (d) Fr.Y1 was further subjected to silica gel column chromatography (petroleum ether-ethyl acetate gradient 20:1→1:1) to obtain the fraction Sub.Fr.Y1-5-3;

[0038] (e)Sub.Fr.Y1-5-3 was purified by preparative HPLC (80% methanol and water, 254 nm) to obtain Iridobelamal A (compound 4).

[0039] Irisflavin was isolated from Belamcanda chinensis using the following steps:

[0040] (a) The dried rhizome of Belamcanda chinensis was extracted with 75% ethanol and concentrated under reduced pressure to obtain the alcohol extract;

[0041] (b) The alcohol extract was extracted with ethyl acetate and concentrated to obtain the ethyl acetate fraction (CSY);

[0042] (c) CSY was subjected to silica gel column chromatography (chloroform-methanol gradient 50:1→1:1), and the fraction Fr.Y3 was collected.

[0043] (d) After repeated washing with alcohol and filtration, Fr.Y3 was used to obtain iris flavin (compound 5), a pale yellow amorphous powder.

[0044] Irisdichotin B was isolated from Belamcanda chinensis using the following steps:

[0045] (a) The dried rhizome of Belamcanda chinensis was extracted with 75% ethanol and concentrated under reduced pressure to obtain the alcohol extract;

[0046] (b) The alcohol extract was extracted with n-butanol and concentrated to obtain the n-butanol fraction (CSZ);

[0047] (c) CSZ was passed through a D101 macroporous resin column (eluted with water → 30% ethanol gradient), and the 30% ethanol fraction (CSZ-D101-30% E) was collected.

[0048] (d)CSZ-D101-30% E was subjected to silica gel column chromatography (chloroform-methanol 20:1→1:1) to obtain the fraction Sub.Fr.Z5;

[0049] (e) Sub.Fr.Z5 was subjected to a Sephadex LH-20 gel column (chloroform-methanol = 1:1) to obtain irisdichotin B (compound 13).

[0050] Example 1

[0051] 10 kg of dried Belamcanda chinensis was coarsely pulverized and extracted twice with 10-15 times its volume of 75% ethanol, each time for 6 days. The filtrates from the two extractions were combined, and the solvent was recovered under reduced pressure to obtain a concentrated alcohol extract, yielding approximately 3467.0 g of total extract. The concentrated alcohol extract was then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol (10 L each × 3 times). The extracts were combined and concentrated to obtain the following fractions: petroleum ether layer (CSS 6.8 g), ethyl acetate layer (CSY 1557.1 g), n-butanol layer (CSZ 403.2 g), and aqueous phase (CSW 1403.5 g). Each fraction was then concentrated under reduced pressure.

[0052] 1500g of CSY was fully dissolved and mixed with an equal amount of silica gel powder (80-100 mesh). The mixture was loaded onto a silica gel column and eluted using a chloroform-methanol gradient system (50:1, 30:1, 20:1, 10:1, 8:1:5:1, 1:1). The column was then rinsed with methanol and water. Fractions were collected according to column volume and combined into 11 fractions (Fr.Y1-Y11) based on TLC and HPLC analysis. Fraction Fr.Y3 was concentrated under reduced pressure to recover the solvent and yielded a pale yellow solid. After repeated washing with alcohol and filtration, compound 5 (2460.0 mg) was obtained as a pale yellow amorphous powder. Fraction Fr.Y8 precipitated a white solid and, after repeated washing with alcohol and filtration, yielded compound 8 (316.4 mg).

[0053] A large amount of pale yellow solid precipitated from fraction Fr.Y1. To facilitate subsequent separation, the precipitated solid was filtered, dried, and stored. A portion of the solid was repeatedly washed with alcohol and filtered to obtain a white powder, compound 6 (480.0 mg). The remaining filtrate (estimated weight 375.3 g) was eluted with a silica gel column using a petroleum ether-ethyl acetate gradient (10:1, 8:1, 5:1, 2:1, 1:1). The fraction collected under a 2:1 gradient elution precipitated yellow crystals, which was compound 7. The remaining fractions were analyzed by TLC and HPLC and combined into seven sub-fractions, Sub.Fr.Y1-1 to Sub.Fr.Y1-7. Sub.Fr.Y1-4 (62.0 g) was repeatedly eluted with silica gel column and Sephadex LH-20 gel column to obtain compound 9 (144.3 mg). Sub.Fr.Y1-5 (68.2 g) was analyzed by silica gel column and ODS column, and the fractions were combined into 11 sub-fractions (Sub.Fr.Y1-5-1 to 11) by TLC and HPLC. Among them, Sub.Fr.Y1-5-3 was analyzed by silica gel column, ODS column, and then HPLC (80% methanol-water, 210 / 254 nm) to obtain compound 4 (50.1 mg, t R =45min);

[0054] 380g of CSZ was dissolved in low-concentration ethanol-water solution and then loaded onto a D101 macroporous resin column. The solution was eluted sequentially with water, 10%, 30%, 50%, 70%, and 95% ethanol. The eluted fractions were collected and concentrated under reduced pressure to recover the solvent, yielding extracts of each fraction. CSZ-D101-30% E (83.5g) was eluted with a silica gel column using a chloroform-methanol gradient (20:1→1:1), yielding nine fractions, Sub.Fr.Z1-Z9. Sub.Fr.Z5 (5.5g) was eluted with both silica gel and Sephadex LH-20 gel columns to obtain compound 13.

[0055] Identification of compounds:

[0056] Compound 4: Colorless, viscous, oily liquid; ESI-MS quasi-molecular ion peak m / z 497 [M+Na] + See also Figure 1-2 As shown, 1H-NMR(600MHz, CDCl3)δ:10.20(1H,s,H-1),5.24(1H,t,J=6.9Hz,H-12),5.05(1H,m,H-16),5.04(1H,m,H-20),3.91(1H,dd,J=7.8,5.2H z,H-14),3.57(2H,t,J=6.4Hz,H-25),2.58~3.19(2H,m,H-9),2.74(1H,brd,J=9.4Hz,H-5),2.14~2.27(2H,m,H-15),2.10(2H,m,H-23), 2.04(2H,m,H-19),2.02(2H,brd,H-18),1.90~2.12(1H,m,H-11),1.63~1.84(2H,m,H-8),1.80(1H,s,H-3),1.67(3H,s,H-22),1.66(3H, s,H-29),1.60(3H,s,H-28),1.57(3H,s,H-30),1.26~1.40(2H,m,H-24),1.14~1.38(2H,m,H-10),1.13(3H,s,H-27),1.06(3H,s,H-26); 13 C-NMR (150MHz, CDCl3) δ: 190.94 (C-1), 160.0 (C-4), 138.8 (C-17), 137.0 (C-13), 133.1 (C-2), 131.7 (C- 21),125.9(C-12),124.2(C-20),120.0(C-16),76.9(C-14),75.2(C-7),63.2(C-25),47.5(C-5),45.3(C -6), 39.9(C-18), 38.0(C-8), 37.0(C-10), 34.3(C-15), 32.1(C-24), 27.3(C-23), 26.6(C-19), 26.3(C-27), 25.8(C-22), 23.1(C-11), 20.2(C-9), 17.9(C-26), 17.8(C-30), 16.4(C-29), 12.0(C-28), 12.(C-3). Based on the above data, compound 4 was identified as Iridobelamal A. Molecular formula C 30 H 50 O4 has the following chemical structural formula: Formula I:

[0057]

[0058] Compound 5: Pale yellow amorphous powder (methanol), ESI-MS quasi-molecular ion peak m / z 301 [M+H]+ See also Figure 3-4 As shown, 1 H-NMR(600MHz,DMSO-d6)δ:13.05(1H,s,5-OH),10.74(1H,s,7-OH),9.06(1H,s,3′-OH),8.29(1H,s,H-2) ,7.35(2H,d,J=8.6Hz,H-2′,6′),6.81(2H,d,J=8.6Hz,H-3′,5′),6.47(1H,s,H-8),3.74(1H,s,6-OCH3); 13 C-NMR (150MHz, DMSO-d6) δ: 180.6 (C-4), 157.5 (C-4′), 157.5 (C-5), 154.3 (C-7), 154.1 (C-2), 153.3 (C-9), 131.4 (C-6), 130.2 (C-2′, 6′), 121.8 (C-1′), 121.3 (C-3), 115.1 (C-3′, 5′), 104.9 (C-10), 93.9 (C-8), 60.0 (3-OCH3). Based on the above data, compound 5 was identified as iris flavin. The R1 = OH, R2 = H, R3 = OH, R4 = H of compound 5, and its molecular formula is C1. 16 H 12 O6, with the following general formula II:

[0059]

[0060] Compound 6: White powder (methanol), ESI-MS quasi-molecular ion peak m / z 331 [M+H] + . 1 H-NMR(600MHz,DMSO-d6)δ:13.07(1H,s,5-OH),10.76(1H,s,7-OH),9.16(1H,s,3′-OH),8.35(1H,s,H-2),7.13(1H,d,J=2.0Hz,H-2′),6.98(1H, dd,J=8.0,2.0Hz,H-6′),6.83(1H,d,J=8.0Hz,H-5′),6.82(1H,d,J=8.0Hz,H-3′),6.49(1H,s,H-8),3.79(3H,s,6-OCH3),3.75(3H,s,4′-OCH3); 13C-NMR (150MHz, DMSO-d6) δ: 180.6 (C-4), 157.5 (C-5), 154.3 (C-7), 153.3 (C-9), 152.7 (C-2), 147.3 (C-4′), 146.7 (C-3′), 131.4 (C-6), 121.9 (C-3), 121.7 (C-1′), 121.7 (C-6′), 115.3 (C-5′), 113.3 (C-2′), 104.9 (C-10), 93.9 (C-8), 60.0 (6-OCH3), 55.7 (4′-OCH3). Based on the above data, compound 6 was identified as iris flavin A. Molecular formula C 17 H 14 O7 has the structure shown in general formula II, where R1 = OH, R2 = OCH3, R3 = OH, and R4 = H.

[0061] Compound 7: Pale yellow powder (petroleum ether-ethyl acetate), ESI-MS quasi-molecular ion peak m / z 329 [MH] - . 1 H-NMR(600MHz,DMSO-d6)δ:13.06(1H,s,5-OH),10.77(1H,s,7-OH),9.08(1H,s,3′-OH),8.29(1H,s,H-2),7.04(2H,d,J=2.0Hz ,H-2′),6.95(1H,d,J=8.4Hz,H-6′),6.92(1H,d,J=8.4Hz,H-5′),6.48(1H,s,H-8),3.79(3H,s,6-OCH3),3.75(3H,s,4′-OCH3); 13 C-NMR (150MHz, DMSO-d6) δ: 180.6 (C-4), 157.6 (C-7), 154.3 (C-2), 153.4 (C-5), 152.8 (C-9), 147.78 (C-4′), 146.2 (C-3′), 131.5 (C-6), 123.4 (C-1′), 121.7 (C-3), 119.9 (C-6′), 116.5 (C-2′), 112.0 (C-5′), 104.9 (C-10), 94.0 (C-8), 60.0 (6-OCH3), 55.7 (4′-OCH3). Based on the above data, compound 7 was identified as iris flavin B. Molecular formula C 17 H 14 O7 has the structure shown in general formula II, where R1 = OCH3, R2 = OH, R3 = OH, and R4 = H.

[0062] Compound 8: White amorphous powder (methanol), ESI-MS quasi-molecular ion peak m / z 461 [MH] - . 1 H-NMR(600MHz,DMSO-d6)δ:12.93(1H,s,5-OH),9.61(1H,s,4′-OH),8.43(1H,s,H-2),7.40(2H,d, J=8.6Hz,H-2′,6′),6.88(1H,s,H-8),6.83(2H,d,J=8.6Hz,H-3′,5′),5.09(1H,d,J=7.4Hz,Glc-l H-1″),3.78(3H,s,6-OCH3); 13 C-NMR(150MHz,DMSO-d6)δ:180.8(C-4),157.5(C-5),156.7(C-4′),154.7(C -7),153.0(C-2),152.5(C-9),132.5(C-6),130.2(C-2′,6′),122.1(C-3),1 21.1(C-1′),115.2(C-3′,5′),106.5(C-10),100.2(C-1″),94.1(C-8),77.3 (C-5″),76.8(C-3″),73.2(C-2″),69.7(C-4″),60.7(C-6″),60.4(6-OCH3). Based on the above data, compound 8 was identified as irisin, also known as belamcanda chinensis glycoside. Molecular formula C 22 H 22 O 11 The structure is shown in general formula II, where R1 = OH, R2 = H, R3 = OGlc, and R4 = H.

[0063] Compound 9: Yellow solid (methanol), ESI-MS quasi-molecular ion peak m / z 361 [M+H] + . 1 H-NMR(600MHz,DMSO-d6)δ:13.07(1H,s,5-OH),10.80(1H,s,7-OH),9.27(1H,s,3′-OH),8.38(1H,s,H-2),6.71(1H, s,2′-OH),6.66(1H,s,6′-OH),6.50(1H,s,H-8),3.79(3H,s,5′-OCH3),3.75(3H,s,6′-OCH3),3.69(3H,s,6-OCH3); 13C-NMR (150MHz, DMSO-d6) δ: 180.8 (C-4), 158.0 (C-5), 155.3 (C-2), 153.8 (C-7), 153.3 (C-9), 153.1 (C-5′), 150.7 (C-3′), 136.8 (C-4′), 131.9 (C-6), 126.5 (C-1′), 122.2 (C-3), 110.8 (C-2′), 105.3 (C-10), 105.0 (C-6′), 94.4 (C-8), 60.4 (6-OCH3), 60.4 (4′-OCH3), 56.2 (5′-OCH3). Based on the above data, compound 9 was identified as iris flavin. Molecular formula C 18 H 16 O8 has the structure shown in general formula II, where R1 = OCH3, R2 = OCH3, R3 = OH, and R4 = OH.

[0064] Compound 13: Pale yellow solid (chloroform-methanol), ESI-MS quasi-molecular ion peak m / z 493 [MH] - See also Figure 5-6 As shown, 1 H-NMR(600MHz,DMSO-d6)δ:9.13(1H,s,4′-OH),7.10(1H,d,J=2.0Hz,H-2′),6.91(1H,dd,J=8.0,2.0Hz,H-6′),6. 79(1H,d,J=8.0Hz,H-5′),6.47(1H,d,J=2.4Hz,H-8),6.27(1H,d,J=2.4Hz,H-6),5.44(1H,d,J=5.0Hz,3-OH),5.0 3(1H,d,J=11.5Hz,H-2),4.87(1H,d,J=7.7Hz,H-1″),4.46(1H,dd,J=11.5,5.0Hz,H-3),3.78(3H,s,7-OCH3),3.7 8(3H,s,3′-OCH3),3.72(1H,m,H-6″),3.47(1H,m,H-5″),3.40(1H,m,H-2″),3.34(1H,m,H-3″),3.19(1H,m,H-4″); 13C-NMR(150MHz,DMSO-d6)δ:191.8(C-4),166.0(C-7),163.9(C-9),159.9(C-5),147. 7(C-3′),147.4(C-4′),128.5(C-1′),121.4(C-6′),115.4(C-2′),112.6(C-5′),104 .8 (C-10), 102.0 (C-1″), 97.4 (C-6), 95.8 (C-8), 83.3 (C-2), 77.9 (C-5″), 76.7 (C-3″), 73.8 (C-2″), 73.0 (C-3), 70.2 (C-4″), 61.2 (C-6″), 56.2 (7-OCH3), 56.1 (3′-OCH3). Based on the above data, compound 13 was identified as irisdichotin B. Molecular formula C 23 H 26 O 12 The chemical structural formula is as follows: Formula III:

[0065]

[0066] Example 2

[0067] Preparation of the injectable formulation of the pharmaceutical composition for treating non-small cell lung cancer: Take Iridobelamal A and iris flavin obtained by the method in Example 1 above, and dissolve them by vortexing at a molar ratio of Iridobelamal A:iris flavin = 1:3. Take 21.2 μmol of Iridobelamal A and 63.6 μmol of iris flavin, add phosphate-buffered saline (PBS, pH 7.4) for injection to a total volume of 10 mL. Sterilize by filtration through a 0.22 μm microporous membrane, and dispense into vials to obtain the binary injectable formulation.

[0068] Example 3

[0069] Oral preparation of the pharmaceutical composition for anti-non-small cell lung cancer: Take Iridobelamal A and iris flavin obtained by the method of Example 1 above, with a molar ratio of Iridobelamal A:iris flavin = 1:3, take 21.2 μmol of Iridobelamal A and 63.6 μmol of iris flavin, add 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution, vortex mix for 30 min to form an inclusion complex, which is a medium-weight binary group of iris flavin.

[0070] Example 4

[0071] Oral preparation of the pharmaceutical composition for anti-non-small cell lung cancer: Take Iridobelamal A and iris flavin obtained by the method in Example 1 above, and in a molar ratio of Iridobelamal A:iris flavin = 1:2, take 21.2 μmol of Iridobelamal A and 42.4 μmol of iris flavin, add 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution, vortex mix for 30 min to form an inclusion complex, which is a low-volume binary iris flavin.

[0072] Example 5

[0073] Oral preparation of the pharmaceutical composition for anti-non-small cell lung cancer: Take Iridobelamal A and iris flavin obtained by the method in Example 1 above, and in a molar ratio of Iridobelamal A:iris flavin = 1:5, take 21.2 μmol of Iridobelamal A and 106 μmol of iris flavin, add 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution, vortex mix for 30 min to form an inclusion complex, which is a high-volume binary iris flavin.

[0074] Example 6

[0075] Oral preparation of the pharmaceutical composition for treating non-small cell lung cancer: Take irisdichotin B, Iridobelamal A and iris flavin obtained by the method in Example 1 above, and in a molar ratio of irisdichotin B:Iridobelamal A:iris flavin = 1:1:3. Take 21.2 μmol of irisdichotin B, 21.2 μmol of Iridobelamal A and 63.6 μmol of iris flavin, add 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution, vortex mix for 30 min to form an inclusion complex, which is a ternary mixture.

[0076] Example 7

[0077] Oral formulation of pharmaceutical compositions for use against non-small cell lung cancer:

[0078] 1) Mixing of active ingredients:

[0079] Take irisdichotin B, Iridobelamal A and iris flavin obtained by the method in Example 1 above, and mix them in a molar ratio of irisdichotin B:Iridobelamal A:iris flavin = 1:1:3. Take 21.2 μmol of irisdichotin B, 21.2 μmol of Iridobelamal A and 63.6 μmol of iris flavin.

[0080] 2) Thin film dispersion and ultrasonication:

[0081] Soybean lecithin (1.6 g), cholesterol (0.2 g), and the active ingredient were dissolved in a chloroform and methanol mixture (200 mL, 3:1 v / v). A lipid membrane was formed by rotary evaporation (40 °C) and vacuum drying. Pre-cooled PBS (pH 7.4, 100 mL) was added at 4 °C, and the mixture was hydrated at 55 °C for 30 min. The membrane was then sonicated (300 W, 15 min, ice bath). Homogenization was performed using microfluidics (10000 psi, 3 times) to obtain a liposome suspension (particle size 105 ± 10 nm), which was a ternary nanoliposome assembly.

[0082] Comparative Example 1

[0083] Oral preparation of Iridobelamal A monomer: The amount of the corresponding active ingredient is the same as in Example 3. Specifically, 21.2 μmol of Iridobelamal A obtained by the method in Example 1 above is added to 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution and vortexed for 30 min to form an inclusion complex, thus obtaining the Iridobelamal A monomer group.

[0084] Comparative Example 2

[0085] Oral preparation of iris flavin monomer: The amount of the corresponding active ingredient is the same as in Example 3. Specifically, 63.6 μmol of iris flavin obtained by the method in Example 1 above is added to 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution and vortexed for 30 min to form an inclusion complex, thus obtaining the iris flavin monomer group.

[0086] Comparative Example 3

[0087] Oral preparation of irisdichotin B monomer: The amount of the corresponding active ingredient is the same as in Example 3. Specifically, 21.2 μmol of irisdichotin B obtained by the method in Example 1 above is added to 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution and vortexed for 30 min to form an inclusion complex, thus obtaining the irisdichotin B monomer group.

[0088] Comparative Example 4

[0089] Oral preparation of monomers of iris flavin A (compound 6), iris flavin B (compound 7), iris glycoside (compound 8), and wild iris flavin (compound 9): 21.2 μmol of each of the iris flavin A (compound 6), iris flavin B (compound 7), iris glycoside (compound 8), and wild iris flavin (compound 9) obtained by the method in Example 1 above were added to 5 mL of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution and vortexed for 30 min to form inclusion complexes, thus obtaining monomer groups of compound 6, compound 7, compound 8, and compound 9, respectively.

[0090] Comparative Example 5

[0091] The oral preparation of the drug composition is basically the same as in Example 3, except that the iris flavin is replaced with iris flavin A (compound 6), iris flavin B (compound 7), iris flavin (compound 8), and wild iris flavin (compound 9), respectively, to obtain binary groups of compound 6, compound 7, compound 8, and compound 9, respectively.

[0092] Experiment 1: Tumor-bearing lung cancer mouse model experiment:

[0093] Experimental methods:

[0094] 1. Animal Model Construction

[0095] SPF-grade female BALB / c mice (5 weeks old, weighing 18-22g) were selected and subcutaneously inoculated with A549 cells (1×10⁻⁶). 7 Apply 0.2 mL of the solution to the left forearm axilla until the tumor reaches a size of 50 mm. 3 The mice were then divided into groups of n=10. The specific grouping details are as follows:

[0096] Model group: administered physiological saline.

[0097] Example 2 group: using a binary injection (Iridobelamal A: Iridobelamal = 1:3).

[0098] Example 3 group: Iris flavin medium-dose binary group, administered orally.

[0099] Example 4 group: Iris flavin low-dose binary group, administered orally.

[0100] Example 5 group: high-dose iris flavin binary group, administered orally.

[0101] Example 6 group: ternary mixed group, administered orally.

[0102] Example 7 group: Ternary nanoliposome group, administered orally.

[0103] Positive control group: Cisplatin was administered at a dose of 60 mg / kg via intraperitoneal injection.

[0104] 2. Dosing regimen

[0105] The oral administration group received daily gavage at a dose of 200 μL / kg; the injection group received intraperitoneal injection every other day, with the entire experiment lasting 30 days. Dosage was calculated based on the active ingredient, with Iridobelamal A fixed at 21.2 μmol and iris dichotin adjusted proportionally (iris dichotin was 42.4 μmol in Example 4; 63.6 μmol in Example 3; and 106 μmol in Example 5). The ternary group consisted of iris dichotin B 21.2 μmol + Iridobelamal A 21.2 μmol + iris dichotin 63.6 μmol.

[0106] 3. Indicator Testing

[0107] The long diameter (a) and short diameter (b) of the tumor are measured every 3 days, according to the formula V = ab. 2 / 2 Calculate tumor volume. After the last administration, mice were sacrificed, tumors were removed and weighed, and the tumor inhibition rate was calculated according to the formula. The correct formula for calculating the tumor inhibition rate should be: Tumor inhibition rate (%) = (1 - average tumor weight of the treatment group / average tumor weight of the model group) × 100.

[0108] Experimental results:

[0109] Regarding changes in tumor volume, the ternary nanoliposome group in Example 7 showed the best tumor-suppressing effect, with a tumor volume of 125±15 mm on day 30. 3 The value was significantly lower than that of other groups (P<0.001).

[0110] The average tumor weight in the model group mice was 1.82 ± 0.21 g (n = 10). The tumor inhibition rate was recalculated according to the formula as follows: Example 7 group (ternary nanoliposomes): (1 - 0.32 / 1.82) × 100 ≈ 82.4%;

[0111] Example 6 (ternary mixture group): (1-0.41 / 1.82)×100≈77.5%;

[0112] Example 3 group (medium-dose binary group): (1-0.48 / 1.82)×100≈73.6%;

[0113] Example 5 group (high-volume binary group): (1-0.50 / 1.82)×100≈72.5%;

[0114] Example 4 (low-volume binary group): (1-0.49 / 1.82)×100≈73.1%;

[0115] In terms of tumor weight comparison, the tumor weight of the Example 7 group was the lowest (0.32±0.05g), which was 22% lower than that of Example 6 (0.41±0.06g) and better than the positive control group (cisplatin, 0.47±0.06g).

[0116] The experimental results show that the ternary nanoliposome group (Example 7) significantly inhibited tumor growth by enhancing targeted delivery, and its effect was better than that of the ternary mixed group (Example 6) and the binary group (Examples 3-5). This demonstrates the key role of nano-formulation in synergistic anti-tumor activity.

[0117] Experiment 2: In vitro A549 cell model experiment:

[0118] Experimental methods:

[0119] 1. Cell Culture and Drug Delivery

[0120] A549 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were individually treated with the drugs from each example and comparative example, with drug concentration gradients ranging from 0 to 100 μmol / L. Grouping was the same as in the mouse model, with the addition of comparative examples 1-5 (monomers and alternative combinations).

[0121] 2. MTT assay

[0122] After incubation for 48 hours, MTT (5 mg / mL) was added, and after 4 hours, the crystals were dissolved in DMSO and measured at 490 nm (A). Cell viability and IC50 value were calculated according to the formula: viability (%) = (drug-treated group A - blank group A) / (control group A - blank group A) × 100.

[0123] Experimental results:

[0124] IC50 value comparison (the lower the IC50 value, the stronger the inhibitory effect):

[0125] Example group:

[0126] Example 7 (ternary nanoliposomes): 10.2 ± 0.7 μmol / L;

[0127] Example 6 (ternary mixture): 12.5 ± 1.1 μmol / L;

[0128] Example 3 (medium-dose binary group): 18.3 ± 1.5 μmol / L;

[0129] Example 2 (binary injection): 18.6 ± 1.4 μmol / L;

[0130] Examples 4 / 5 (low / high amount binary): 21.4±1.8 / 20.9±1.7 μmol / L;

[0131] Comparative example group:

[0132] Comparative Example 1 (Iridobelamal A monomer): 18.5 ± 1.6 μmol / L;

[0133] Comparative Examples 2-3 (irisdichotin B monomer): >100 μmol / L (no inhibition);

[0134] Comparative Example 4 (monomers of compounds 6-9): >100 μmol / L (no inhibition);

[0135] Comparative Example 5 (compounds 6-9): 28.7-35.2 μmol / L (all worse than Example 3);

[0136] The experimental results show that the ternary nanoliposomes (Example 7) have a strong inhibitory effect on A549, while the iris flavonoid monomer (Comparative Example 2) has no inhibitory effect on A549. The substitution of flavonoid combination (Comparative Example 5) affects the inhibitory effect of Iridobelamal A monomer on A549. It can be seen that the combination of Iridobelamal A and iris flavonoid has the effect of enhancing the apoptosis-inducing effect of triterpenoids on tumor cells.

[0137] Experiment 3: In vitro BEAS-2B cell model experiment:

[0138] Experimental methods:

[0139] 1. Toxicity assessment

[0140] BEAS-2B cells (normal human lung epithelial cells) were seeded in 96-well plates, and the drug administration regimen was the same as in the A549 experiment. Cell viability at 48 h was detected by the MTT assay, and the IC50 value was calculated (a higher IC50 value indicates lower toxicity).

[0141] Experimental results:

[0142] IC50 value comparison (higher is safer):

[0143] Example group:

[0144] Example 7 (ternary nanoliposomes): >200 μmol / L;

[0145] Example 6 (ternary mixture): 152.3 ± 10.6 μmol / L;

[0146] Example 5 (high-volume binary): 69.7 ± 7.2 μmol / L;

[0147] Example 3 (medium-volume binary group): 85.4 ± 5.8 μmol / L;

[0148] Example 4 (low-level binary group): 63.1 ± 5.3 μmol / L;

[0149] Example 2 (binary injection): 76.2 ± 6.1 μmol / L;

[0150] Comparative example group:

[0151] Comparative Example 1 (Iridobelamal A monomer): 11.3 ± 1.1 μmol / L (most toxic)

[0152] Comparative Examples 2-3 (irisdichotin B monomer): 22.5±1.8 / 23.2±1.9 μmol / L

[0153] Comparative Example 4 (monomers of compounds 6-9): 18.5-20.9 μmol / L

[0154] Comparative Example 5 (compounds 6-9): 42.3-48.7 μmol / L (all lower than Example 3)

[0155] The experimental results show that the ternary nanoliposomes (Example 7) exhibited the lowest cytotoxicity to normal cells (IC50 > 200 μmol / L), significantly superior to other groups. The combination of Iridobelamal A and iris flavin at a molar ratio of 1:2 to 1:5 effectively neutralized the toxicity of triterpenes to normal lung cells (BEAS-2B) while maintaining antitumor activity against non-small cell lung cancer cells (A549). The addition of irisdichotin B (Examples 6-7) further mitigated the toxicity of Iridobelamal A.

[0156] Experiment 4: Verification of the oxidative damage neutralization mechanism:

[0157] 1. Experimental Methods:

[0158] Four groups were set up using the BEAS-2B cell model:

[0159] Group A: Blank control group;

[0160] Group B: Iridobelamal A monomer (20 μM);

[0161] Group C: Iridobelamal A + Iridobelamal (20 + 60 μM);

[0162] Group D: Iridobelamal A + irisdichotin B (20 + 60 + 20 μM);

[0163] Detection indicators: reactive oxygen species (ROS) level, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity;

[0164] 2. Experimental Results:

[0165] ROS level (percentage relative to the blank group):

[0166] Group B: 285±21%;

[0167] Group C: 142±15%;

[0168] Group D: 108±8%;

[0169] SOD activity (U / mg protein):

[0170] Group B: 12.3±1.4;

[0171] Group C: 25.6±2.0;

[0172] Group D: 32.8±2.3;

[0173] GSH-Px activity (U / mg protein):

[0174] Group B: 9.8±0.9;

[0175] Group C: 20.1±1.6;

[0176] Group D: 26.9±1.9;

[0177] Statistical difference: p<0.05 between group D and group C;

[0178] The experimental results show that irisdichotin B restored the ROS level of the ternary combination to near normal (108%) and significantly increased the activities of SOD and GSH-Px (28% and 34% higher than the binary combination), indicating that it neutralizes oxidative damage induced by Iridobelamal A by activating the antioxidant system.

[0179] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A pharmaceutical composition for treating non-small cell lung cancer, characterized in that, It contains the following active ingredients: iridoaldehyde-type triterpenoid Iridobelamal A and flavonoid iridoflavin; wherein the molar ratio of iridoaldehyde-type triterpenoid Iridobelamal A and flavonoid iridoflavin is 1:2 to 1:

5.

2. The composition according to claim 1, characterized in that, The molecular formula of the irisaldehyde-type triterpenoid compound Iridobelamal A is C 30 H 50 O4, chemical structural formula is: The molecular formula of the flavonoid compound irisin is C 16 H 12 O6, chemical structural formula is: Where R1 = OH, R2 = H, R3 = OH, and R4 = H.

3. The composition according to claim 2, characterized in that, It also contains the active ingredient: compound irisdichotin B; wherein the molar ratio of compound irisdichotin B to Iridobelamal A and iris flavin is 1:1:2 to 1:1:

5.

4. The composition according to claim 3, characterized in that, The molecular formula of the compound irisdichotin B is C. 23 H 26 O 12 The chemical structural formula is: Where R1 = D-Glc, R2 = CH3, and R3 = OCH3.

5. The composition according to any one of claims 1-4, characterized in that, The composition also includes a pharmaceutically acceptable carrier, and is formulated as an oral or injectable preparation.

6. A method for preparing the composition according to claim 5, characterized in that: Irisdichotin B, Iridobelamal A, and iris flavin were mixed in a certain proportion and then combined with a carrier to form nanoliposomes.

7. The method for preparing the composition according to claim 6, characterized in that: The nanoliposomes were prepared using a thin-film dispersion and ultrasonication method, specifically including the following steps: 1) Dissolve soybean phospholipids, cholesterol, and the active ingredients of the composition in a mixed solvent of chloroform and methanol at a mass ratio of 40-50:5-10:1; 2) Rotary evaporation forms a lipid film, followed by vacuum drying to remove residual organic solvents; 3) Add phosphate buffer for hydration, then sonicate. 4) Nanoliposomes with a particle size of 80-120 nm were obtained by cycling them three times at 10,000 psi using a microfluidic homogenizer.

8. Use of a pharmaceutical composition according to any one of claims 1-4 in the preparation of an anti-non-small cell lung cancer drug.

9. The use of a pharmaceutical composition obtained by the preparation method according to any one of claims 6-7 in the preparation of an anti-non-small cell lung cancer drug.

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