TWI393725B - Furost-5-ene-3,22,26-triol glucoside compounds for cancer prevention and treatment - Google Patents

Description

Glycerol glycoside compound which can prevent and treat cancer

The present invention relates to a compound for preventing and treating cancer of a steroidal glycoside, and more particularly to a succinol (furost-5-ene-3) for preventing and treating cancer by promoting apoptosis. , 22, 26-triol) glycoside compound.

According to the death statistics of the Health Department of the Executive Yuan over the years, cancer is the top ten cause of death in Taiwan, indicating that cancer is still an area for medical treatment.

There are many methods for treating cancer at present, which are roughly classified into non-surgical and surgical treatment. In non-surgical treatment, chemical drugs are often used as treatments, but for transferable and late-stage cancers, regular treatment effects are limited, and with the occurrence of serious side effects, the effect of chemotherapy is disappointing, even if it is currently up to date. The anti-angiogenic therapy developed has a limited therapeutic effect on cancer.

In view of the above, the present invention has been directed to the above-mentioned problems, and proposes a steroidal glycoside compound containing a furan structure for preventing and treating cancer by a mechanism for promoting apoptosis, thereby effectively overcoming the conventional deficiency. The following is a brief description of the case.

A first concept of the present invention is to provide a pharmaceutical composition comprising:

An effective amount of a furano-5-ene-3,22,26-triol glycoside of the formula I, wherein the R ₁ is selected from the group consisting of monohydrogen, monoglucose, rhamnose, and galactose , a xylose, an arabinose, a group consisting of glucose, rhamnose, galactose, xylose, arabinose, and the like, one of a group consisting of sucrose, a sugar, and a lanose; The steric configuration of the 25-position carbon atom may be R, S or racemization; R ₂ is selected from monohydrogen or monomethyl; and R ₃ is selected from monohydrogen, monoglucose, rhamnose, and half lactose. Any one of the group consisting of xylose and arabinose; and a pharmaceutically acceptable carrier or excipient.

According to the above concept, the pharmaceutical composition has an effect of promoting apoptosis.

According to the above concept, the pharmaceutical composition has the effect of preventing and treating cancer.

According to the above concept, the pharmaceutical composition has an action of preventing and treating liver cancer, lung cancer, and colorectal cancer.

According to the above concept, wherein the pharmaceutical composition is dichotomin.

According to the above concept, wherein the pharmaceutical composition is 26- O- β-D-glucopyranosyl-22α-methoxy-(25 S )-furazan-5-ene-3β, 26-diol 3- O -α-L-pyranosyl-(1→4)-β-D-glucopyranoside (26- O- β-D-glucopyranosyl-22α-methoxy-(25 S )-furost-5 -ene-3β,26-diol 3- O -α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside).

According to the above concept, wherein the pharmaceutical composition is 26- O- β-D-glucopyranosyl-22α-methoxy-(25 R )-furazan-5-ene-3β, 26-diol 3- O -α-L-pyranosyl-(1→4)-β-D-glucopyranoside (26- O- β-D-glucopyranosyl-22α-methoxy-(25 R )-furost-5 -ene-3β,26-diol 3- O -α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside).

According to the above concept, wherein the pharmaceutical composition is 26- O- β-D-glucopyranosyl-22α-hydroxy-(25 R )-furan steroid-3,26-diol 3- O -α-L -pyranosyl-(1→2)-[α-L-pyran rhamido-(1→4)]-β-D-glucopyranoside (26- O- β-D- Glucpyranosyl-22α-hydroxy-(25 R )-furostane-3,26-diol 3- O -α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β- D-glucopyranoside).

According to the above concept, the s-triol glycoside system of the formula I of the pharmaceutical composition is derived from a water extract or a partial purification of one of the genus.

According to the above concept, the sulphate glycoside system of formula I of the pharmaceutical composition is derived from one of the water extracts or a portion of the purified extract of the Asparagus plant.

A second concept of the present invention is to provide a method of preparing a composition comprising a furost-5-ene-3,22,26-triol glycoside of formula I, which comprises: providing a genus Plant or asparagus plant; and extracting the plant with an aqueous solution to obtain the composition comprising the sulphate glycoside of formula I.

According to the above concept, it further comprises: extracting the aqueous solution with an organic solvent.

According to the above concept, the organic solvent is n-butanol or monoethyl acetate.

The present invention will be fully understood from the following description of the embodiments of the present invention, and the invention may be practiced otherwise.

(1) Separation of the extract of the palmetto of the present invention: 200 g of the seed of the shelled sunflower was ground and boiled for 8 hours with 0.8 liter of water, and the supernatant was removed by centrifugation and concentrated to a volume of 200 ml under reduced pressure. Ethyl acetate (200 ml × 3), n-butanol (200 ml × 3) was subjected to polar division, and concentrated under reduced pressure to obtain n-butanol soluble (PK-1-1, 2.34 g) and aqueous layer (PK-1). -2, 13.78 grams). A part of n-butanol solubles (PK-1-1, 1.55 g) was treated with n-butanol:methanol:water=4:1:4 as the separation solvent system, first with the organic layer as the mobile phase, then reversed the water layer. Centrifugal partition chromatography (CPC) separation was carried out for the mobile phase, followed by dextran LH-20 chromatography (Sephadex LH-20, 140 ml; 50% methanol/water) to give 18 mg of dichotomin (1, PK-22-1), a partially purified product containing dichotomin PK-22-2 (21 mg) and PK-22-3 (17 mg) without dichotomin.

(2) Preparation of a partially purified compound containing the compound dichotomin of the present invention PK-17-1, PK-22-4: a portion of n-butanol soluble (PK-1-1, 1.03 g) was used as a glucan LH-20 chromatography (Sephadex LH-20, 140 ml; 50% methanol/water) afforded a fraction of purified product of P.sub. Chromatography with -20, further processing to obtain another purified product (PK-22-4, 36 mg) containing dichotomin.

Dichotomin( 1 ): amorphous powder; [α] D ^{2 5} = -84° ( c 1.0, MeOH); IR v _{m a x} cm ^{- 1} (KBr): 3437 (br s, OH), 2933 (m) , 1633 (m), 1454 (m), 1388 (m), 1262 (m), 1128 (s), 1049 (s), 910 (w), 804 (w); ¹ H-NMR (CD ₃ OD, 400 MHz): δ 1.87 (br d, J = 14.2 Hz, H-1β), 1.07 (m, H-1α), 1.61 (m, H-2β), 1.91 (m, H-2α), 3.59 (m) , H-3α), 2.28 (br t, J = 13.0 Hz, H-4β), 2.44 (dd, J = 2.8, 13.0 Hz, H-4α), 5.38 (br d, J = 2.9 Hz, H-6 ), 1.58 (m, H-7α), 2.01 (m, H-7β), 1.66 (m, H-8), 0.95 (m, H-9), 1.53 (m, H-11β), 1.56 (m) , H-11α), 1.80 (m, H-12β), 1.18 (m, H-12α), 1.13 (m, H-14), 1.27 (m, H-15β), 1.97 (m, H-15α) , 4.36 (dd, J = 7.4, 14.4 Hz, H-16), 1.73 (m, H-17), 0.83 (s, 3H, H-18), 1.04 (s, 3H, H-19), 2.17 ( dq, J = 6.2,7.7 Hz, H -20), 1.00 (d, J = 6.2 Hz, 3H, H-21), 1.58 (m, H-23), 1.81 (m, H-23), 1.15 ( m, H-24), 1.59 (m, H-24), 1.74 (m, H-25), 3.72 (m, H-26), 3.39 (m, H-26), 0.94 (d, J = 6.4 Hz, 3H, H-27), 3- O - glu : 4.49 (d, J = 7.7 Hz, H-1 '), 3.25 (m, H-2'), 3.56 (m, H-3'), 3.52 (m, H-4'), 3.30 (m, H-5'), 3.79 (dd, J = 1.6, 1 2.4 Hz, H-6'), 3.64 (m, H-6'), rha - (1→2): 5.18 (br s, H-1"), 3.93 (m, H-2"), 3.61 ( m, H-3"), 3.40 (m, H-4"), 4.11 (dq, J = 6.0, 9.4 Hz, H-5"), 1.23 (d, J = 6.0 Hz, 3H, H-6" ),rha-(1→4)- rha -(1→4):4.83(buried in DOH,H-1"'),3.77(m,H-2"'),3.74(m,H-3"'), 3.53 (m, H-4"'), 4.03 (dq, J = 6.2, 9.4 Hz, H-5"'), 1.28 (d, J = 6.2 Hz, 3H, H-6"'), 26- O - glu : 4.23 (d, J = 7.8 Hz, H-1""), 3.18 (dd, J = 7.8, 8.7 Hz, H-2""), 3.37 (m, H-3"") , 3.34 (m, H-4""), 3.27 (m, H-5""), 3.85 (dd, J = 1.5, 12.4 Hz, H-6""), 3.66 (m, H-6"" ), rha -(1→4)-rha-(1→4): 5.17(br s,H-1""'),3.93(m,H-2""'),3.66(m,H-3 '"'), 3.38 (m, H-4""'), 3.70 (m, H-5""'), 1.24 (d, J = 6.1 Hz, 3H, H-6""'); ^{1 3} C-NMR (CD ₃ OD, 100 MHz): δ 38.56 (t, C-1), 30.75 (t, C-2), 79.28 (d, C-3), 39.50 (t, C-4), 141.90 (s, C-5), 122.62 (d, C-6), 33.17 (t, C-7), 32.77 (d, C-8), 51.71 (d, C-9), 38.03 (s, C- 10), 21.94 (t, C-11), 40.83 (t, C-12), 41.82 (s, C-13), 57.74 (d, C-14), 32.77 (t, C-15), 82.43 ( d, C-16), 65.03 (d, C-17), 16.82 (q, C 18), 19.85 (q, C-19), 41.15 (d, C-20), 16.13 (q, C-21), 113.98 (s, C-22), 31.39 (t, C-23), 28.97 ( t, C-24), 34.99 (d, C-25), 76.01 (t, C-26), 17.29 (q, C-27), 3- O - glu : 100.48 (d, C-1'), 77.88(d, C-2'), 77.95(d, C-3'), 79.49(d, C-4'), 76.67(d, C-5'), 61.92(t, C-6'), Rha - (1 → 2): 102.34 (d, C-1"), 72.39 (d, C-2"), 72.39 (d, C-3"), 73.83 (d, C-4"), 69.78 ( d, C-5"), 17.85 (q, C-6"), rha-(1→4)- rha - (1→4): 102.61 (d, C-1"'), 72.93 (d, C -2"'), 72.91 (d, C-3"'), 80.84 (d, C-4"'), 69.08 (d, C-5"'), 18.56 (q, C-6"'), 26- O - glu : 104.58 (d, C-1""), 75.16 (d, C-2""), 79.49 (d, C-3""), 71.71 (d, C-4""), 78.14(d,C-5""),62.83(t,C-6""), rha -(1→4)-rha-(1→4):103.16(d,C-1""'), 72.13(d,C-2""'),72.39(d,C-3""'),73.92(d,C-4""'),70.44(d,C-5""'),17.99( q, C-6""'); ESI-MS (negative) [M-H] ^- 1193.5 (calcd. for C _{5 7} H _{9 3} O _{2 6} 1193.6).

(3) Preparation and separation of the perylene succinyl alcohol glycoside derivative of the present invention: the n-butanol soluble fraction (PK-1-1) by gel column chromatography and centrifugal partition chromatography One of the active fractions obtained was Fr.I-2-3, and this fraction (61 mg) was placed in a 10 ml round bottom flask, 0.1 ml of acetic anhydride and 0.1 ml of pyridine were added, and the reaction was sealed at 58 ° C. After concentrating under reduced pressure, it was separated by chromatography on a silica gel column (230-400 mesh gelatin, 25-40% ethyl acetate/toluene) to obtain 25 mg of pseudodichotomin peracetate.

Pseudodichotomin peracetate (2) : amorphous powder; [α] _D ^{2 5} : -30° ( c 1.0, CHCl ₃ ); IR v _{m a x} cm ^{- 1} (KBr) 2941(m), 1752(s), 1636 (w), 1435 (w), 1373 (m), 1224 (s), 1139 (m), 1042 (s); ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.06 (dt, J = 3.3, 13.1 Hz, H-1α), 1.83 (m, H-1β), 1.91 (m, H-2α), 1.52 (m, H-2β), 3.55 (m, H-3), 2.41 (dd, J = 3.7,12.8 Hz, H-4α), 2.25 (dd, J = 11.0, 12.8 Hz, H-4β), 5.36 (br d, J = 4.4 Hz, H-6), 1.55 (m, H-7α), 2.01 (m, H-7β), 1.57 (m, H-8), 0.94 (dd, J = 5.0, 10.7 Hz, H-9), 1.53 (m, H-11α), 1.49 (m, H-11β) ), 1.22 (m, H-12α), 1.77 (m, H-12β), 0.96 (m, H-14), 2.13 (m, H-15α), 1.38 (m, H-15β), 4.69 (ddd , J = 5.6, 7.7, 9.8 Hz, H-16), 2.44 (br d, J = 9.8 Hz, H-17), 0.64 (s, 3H, H-18), 0.98 (s, 3H, H-19) ), 1.54 (s, 3H, H-21), 2.o6 (m, H-23), 2.04 (m, H-23), 1.22 (m, H-24), 1.53 (m, H-24) , 1.68 (m, H-25), 3.27 (dd, J = 6.0, 9.5 Hz, H-26), 3.67 (m, H-26), 0.85 (d, J = 6.6 Hz, 3H, H-27) , 3- O - glu : 4.54 (d, J = 7.8 Hz, H-1 '), 3.53 (dd, J = 7.8, 9.4 Hz, H-2'), 5.26 (t, J = 9.4 Hz, H- 3'), 3.69 (t , J = 9.5 Hz, H-4'), 3.58 (m, H-5'), 4.31 (dd, J = 3.6, 12.4 Hz, H-6'), 4.45 (m, H-6'), rha -(1→2): 4.87 (d, J = 1.3 Hz, H-1"), 5.01 (m, H-2"), 5.22 (dd, J = 3.4, 10.1 Hz, H-3"), 5.02 (m, H-4"), 4.34 (m, H-5"), 1.16 (d, J = 6.2 Hz, 3H, H-6"), rha-(1→4) -rha - (1→4 ): 4.73 (d, J = 1.7 Hz, H-1"'), 5.08 (m, H-2"'), 5.15 (m, H-3"'), 3.58 (t, J = 9.5 Hz, H -4"'), 3.79 (dq, J = 6.1, 9.5 Hz, H-5"'), 1.26 (d, J = 6.1 Hz, 3H, H-6"'), 26- O - glu : 4.43 ( d, J = 7.9 Hz, H-1""), 4.96 (dd, J = 7.9, 9.5 Hz, H-2""), 5.16 (t, J = 9.5 Hz, H-3""), 5.05 ( m, H-4""), 3.65 (m, H-5""), 4.10 (dd, J = 2.2, 12.2 Hz, H-6""), 4.23 (dd, J = 4.6, 12.2 Hz, H -6""), rha -(1→4)-rha-(1→4): 4.89(d, J =1.6 Hz, H-1""'), 5.03(m,H-2""') , 5.04 (m, H-3""'), 5.03 (m, H-4""'), 3.93 (dq, J = 6.2, 9.5 Hz, H-5""'), 1.17 (d, J = 6.2 Hz, 3H, H-6""'), 1.94~2.11 (OAc groups); ^{1 3} C-NMR (CDCl ₃ , 100MHz.): δ 37.13(t, C-1), 29.54(t, C- 2), 79.17 (d, C-3), 38.26 (t, C-4), 140.07 (s, C-5), 122.00 (d, C-6), 32.15 (t, C-7), 31.20 ( d, C-8), 50.01 (d, C-9), 36.79 (s, C-10), 20.95 (t, C-11), 39.45 (t, C-12), 43.20 (s, C-13) ), 54.93 (d, C-14), 34.07 (t, C-15), 84.23 (d, C-16), 64.14 (d, C-17), 13.93 (q, C-18), 19.24 (q , C-19), 103.62 (s, C-20), 11.60 (q, C-21), 151.60 (s, C-22), 23.22 (t, C-23), 30.79 (t, C-24) , 32.74 (d, C-25), 75.14 (t, C-26), 16.51 (q, C-27), 3- O - glu : 99.62 (d, C-1 '), 76.30 (d, C- 2'), 75.44 (d, C-3'), 77.78 (d, C-4'), 72.25 (d, C-5'), 62.14 (t, C-6'), rha - (1 → 2 ): 97.27 (d, C-1"), 70.01 (d, C-2"), 68.59 (d, C-3"), 71.70 (d, C-4"), 66.38 (d, C-5" ), 17.28(q,C-6"),rha-(1→4)- rha -(1→4):99.47(d,C-1"'),70.96(d,C-2"'), 68.68(d,C-3"'),79.07(d,C-4"'),68.50(d,C-5"'),17.72(q,C-6"'),26- O - glu : 100.96(d,C-1""), 71.30(d,C-2""),72.86(d,C-3""),68.50(d,C-4""),71.70(d,C- 5""), 61.98(t,C-6""), rha -(1→4)-rha-(1→4):99.41(d,C-1""'),70.08(d,C- 2""'), 70.24(d,C-3""'), 70.85(d,C-4""'),67.23(d,C-5""'),17.17(q,C-6""'), 20.50~20.90 and ^. 169.20~170.70 (OAc group); FAB-MS (posi ^{tive) [M + Na] +} 1787.7 (calcd.for C 8 5 H 1 2 0 O 3 9 + Na 1787.7).

(4) Separation of the glycoside component of the aspartame containing the furan structure of the present invention: freshly ripened fruit of Asparagus (4.26 Kg), added with water (3 liters), crushed and extracted with a blender, and concentrated to obtain water. Extract (203 grams). The extract was subjected to polar division to give ethyl acetate (EtOAc) soluble, n-butanol ( n- BuOH) soluble (18.2 g) and water-soluble three portions. The n-butanol soluble fraction (17.0 g) was partitioned by large-scale centrifugal partition chromatography (CPC, Sanki LLI type) and combined into four parts according to their similarity (Fr.A, 4.8 g; Fr.B.3.2 g ; Fr. C, 2.1 g; Fr. D, 7.0 g).

Part B (1.70 g) was also subdivided into dextran LH-20 (MeOH) to obtain four blocks, of which the second group weighed 217 mg and was separated by semi-preparative high performance liquid chromatography. The conditions were as follows: : Column, Merck, purospher STAR RP-18e, 5 μm, 10 × 250 mm; extract, methanol/water 70% (18 min), 70-90% (1 min, linear gradient), 90% (8 Min); flow rate, 3 ml/min; column temperature, 40 ° C; evaporative light scattering detector (ELSD) (5% extract), gain 2, temperature: 40 ° C, pressure: 3.3 bar. Compound 5 (10 mg, retention time approximately 15.0 min), 6 (5 mg, retention time approximately 19.3 min), and 7 (30 mg, retention time approximately 12.3 min) were obtained (Figure 6).

Compound 5 : 26- O- β-D-glucopyranosyl-22α-methoxy-(25 S )-furost-5-ene-3β,26-diol 3- O -α-L-rhamnopyranosyl-(1→4)-β -D-glucopyranoside

White solid; [α] _D ^{2 7} -43.0 ^o ( c 1.0, MeOH); IR v _{m a x} cm ^{- 1} (KBr): 3406, 2934, 1639, 1378, 1037; ¹ H and ^{1 3} C-NMR: Tables 1 and 2; HMBC (CD ₃ OD, 400 MHz): H-4 to C-3, C-5; H-6 to C-4, C-8; H-15 to C-13, C- 16; H-18 to C-12, C-13, C-14, C-17; H-19 to C-1, C-5, C-9, C-10; H-20 to C-13, C-17, C-21; H-21 to C-17, C-20, C-22; 22-OMe to C-22; H-27 to C-24, C-25, C-26; H- 1' to C-3;H-1''' to C-26.ESI-MS[M+H] ⁺ m/z 917(C _{4 6} H _{7 6} O _{1 8} +H)

Compound 6 : 26- O- β-D-glucopyranosyl-22α-methoxy-(25 R )-furost-5-ene-3β,26-diol 3- O -α-L-rhamnopyranosyl-(1→4)-β -D-glucopyranoside

White solid; [α] _D ^{2 7} -36.1° ( c 0.7, MeOH); IR v _{m a x} cm ^{- 1} (KBr): 3397, 2934, 1652, 1379, 1035, 668; ¹ H and ^{1 3} C- NMR: Table 1 and Table 2; ESI-MS [M+H] ⁺ m/z 917 (C _{4 6} H _{7 6} O _{1 8} +H).

Compound 7 : 26- O- β-D-glucopyranosyl-22α-hydroxy-(25 R )-furostane-3,26-diol 3- O -α-L-rhamnopyranosyl-(1→2)-[α-L- Rhamnopyranosyl-(1→4)]-β-D-glucopyranoside

White solid; [α] _D ^{2 7} -58.0° ( c 1.0, MeOH); IR v _{m a x} cm ^{- 1} (KBr): 3396, 2931, 1651, 1455, 1377, 1040, 910, 811, 668; ¹ H and ^{1 3} C-NMR: Tables 1 and 2; HMBC (CD ₃ OD, 400 MHz): H-6 to C-4, C-8; H-18 to C-12, C-13, C-14, C- 17; H-19 to C-1, C-5, C-9, C-10; H-20 to C-13, C-17, C-21; H-21 to C-17, C-20, C-22;H-26 to C-24,C-25,C-27;H-27 to C-24,C-25,C-26;H-1' to C-3;H-1"" To C-26.ESI-MS[M+K] ⁺ m/z 1090 (C _{5 1} H _{8 6} O _{2 2} +K).

Table I and Table II-based body as a non-sugar compound 5-7 (aglycone) (CD ₃ OD) ¹ H-NMR, respectively, of the ^{1 3} C-NMR data of the compound 5-7 and ribosomes (glycone) (CD ₃ OD) ¹ H-NMR and ^{1 3} C-NMR data.

(5) Oral feeding test of the extract of palmetto water of the present invention: 10 ⁶ or 3 × 10 ⁶ rat livers were subcutaneously injected into the back of three F344 rats (female rats, 8 weeks, weighing about 200 g). The cells of the tumor GP7TB cell line were orally administered with the potassium sulphate water extract and the phosphate buffer solution (PBS) of the negative control group. The dose of the pudding water extract was 0.1 gram per day (equivalent to 0.5 gram/kg), and the continuous tube was used. After feeding for 20 days and 40 days, the growth of tumor cells was observed, and the length, width and height of the tumor were measured and recorded. It was found that no tumor formation was observed after 40 days of tube feeding of the sunflower water extract group, as shown in Table 3.

(6) Single dose toxicity test of the present invention: Normal SCID/CB17 strain mice (female rats, 8 weeks, weighing about 20 g) were divided into 3 groups of 5 mice each, and the intraperitoneal injection amount of each mouse was 1 kg of PK-1-1 or PK-1-2, and phosphate buffer saline (PBS) as the negative control group. After 2 weeks, the growth was good, and the weight of each group increased normally. The liver, kidney, spleen, stomach, lung, brain tissue sections were normal.

(7) Apoptosis detection of PK-1-1 and PK-1-2 of the present invention: using an in situ end labeling technique ( T αT-mediated d U TP N ick- E nd L abeling; TUNEL, Promega Detecting GP7TB in rat hepatoma cells to see if the extract of Phyllostachys praecox can cause apoptosis. The cells were first cultured on a microscope slide, and then treated with 150 μg/ml of sunflower extract PK-1-1 and PK-1-2 for 24 hours, after 4% formaldehyde and activator. After treatment with (Triton X-100), TdT enzyme action and propidium iodide (PI) staining were added, and if yellow-green fluorescence was observed, it was shown to be apoptotic cells. The experimental results of PK-1-1 and PK-1-2 are shown in Figure 2 (a) and (b), respectively. Figure 2 (a) shows obvious yellow-green fluorescence, showing PK-1- 1 can significantly promote the apoptosis of GP7TB cells.

(8) The test for inhibiting the growth of liver tumor cell lines (GP7TB, Huh7, HepG2) by the partially purified PK-1-1, PK-17-1 and pure compound dichotomin (PK-22-1) of the present invention: Cell viability assay (MTS, Promega) To determine the effects of PK-1-1 and PK-17-1 on the growth and toxicity of the cells. First, 10 ⁴ rat liver tumor cell lines GP7TB and human liver tumor cell lines Huh-7 and HepG2 were placed in 96-well culture dishes. After overnight culture, different concentrations of extracts were added for 24 hours. Pour the culture solution and replace it with 20 μl of MTS medium per ml for 1 hour, because MTS contains [3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2- (4-sulfophenyl)-2H-tetrazolium], this compound is metabolized into purple formazan products by dehydrogenase such as NADPH or NADH in living cells, and can be read through 490 nm visible light. Cell viability after treatment with extract. As shown in Tables 4 and 5, the extract PK-17-1 inhibited GP7TB cells at a concentration of 25 μg/ml, and also had consistent inhibition results for Huh-7 and HepG2 cell lines.

Experiments were performed in triplicate results are averaged, as shown in Table VI, the compound obtained dichotomin (PK-22-1) of growth inhibition IC ₅₀ of cell GP7TB 1.65 M (1.97 g / ml) , the positive control IC _{adriamycin. 5 0} is 2.66 M (1.54 g/ml).

(9) Dichotomin (PK-22-1) of the present invention inhibits rat liver tumor growth in vivo test: each rat is administered a back epithelial injection of 3 × 10 ⁶ GP7TB cells, about 1 After the week, the tumor formed about 0.3 cm, and the dichotomin (PK-22-1, 2.0 mg/Kg) or phosphate buffer control group was injected directly at the tumor site. One dose per day was administered for 8 days, and the tumor size was directly measured. The effects of the control group and the injection of dichotomin on tumor growth were compared. The results are shown in Table 7. The tumor size formed by dichotomin injection was only about 1/2 of the control group, and was shown by Figure 3(b), after dichotomin injection. After the tumor cells appear apoptosis (apoptosis). The results of the pathological section shown in Fig. 3 (a) showed that the tumor cells of the phosphate buffer control group still maintained the normal growth state of the tumor cell pleomorphism.

(10) Dichotomin (PK-22-1) extracted from palmetto of the present invention, partially purified product containing dichotomin (PK-22-2, PK-22-4) and extract containing no dichotomin (PK-22) -3) Effect on Fragmentation of Nuclear DNA in GP7TB Cells: To understand the growth inhibition of GP7TB cells by active ingredients and some purified substances, a series of partial purified and dichotomin were fixed at a fixed concentration (12.5 μg/ml). After treatment of GP7TB cells, after 48 hours, the DNA of the cells was extracted and analyzed by electrophoresis. The results shown in Figure 4 showed that the extract containing dichotomin produced nuclear DNA fragmentation of GP7TB and HepG2, among which dichotomin ( PK-22-1) has the most significant effect, and it can be inferred that dichotomin can promote the apoptosis of the above cell lines.

(11) Ex-vivo anti-tumor test of dichotomin (PK-22-1) and dichotomin (PK-22-3) extract of the present invention: 3 x in several 10 cm culture dishes 10 ⁶ GP7TB cells were treated with dichotomin (PK-22-1) and dichotomin (PK-22-3) extracts at 12.5 μg/ml for 24 hours, respectively, and these cells were collected at each F344. Rats were injected subcutaneously into the 3 sites of 5×10 ⁶ GP7TB or GP7TB cells treated with PK-22-1 and PK-22-3. After 3 weeks of observation, the results are shown in Figure 5 (a). Six of the 9 mice were not treated with GP7TB after treatment with dichotomin (PK-22-1), and Figure 5 (b) showed additional The tumors formed by 3 treatments with dichotomin (PK-22-1) were only about 1/6 of the GP7TB control group, and the tumor size formed by the treatment of extract containing no dichotomin PK-22-3 was compared with GP7TB. The same as the group.

(12) Inhibition test of Dichotomin (PK-22-1) of the present invention against other cancer cells Each cancer cell strain suspension was placed in a 96-well culture dish, stored at 37 ° C and passed through 5% CO ₂ , and passed through 24 After the hour of culture, 100 μl of growth medium and 2 μl of different concentrations (100, 10, 1, 0.1, and 0.01 μM) of dichotomin (PK-22-1) were added, and cancer cell culture of the control drug mitomycin was carried out in the same manner. After 72 hours of incubation, 20 μl of alamarBlue reagent was added to the culture dish, and after 6 hours of culture, the cell density was measured by a fluorescence detector. The fluorescence detector was a GENios and micro reader at a wavelength of 530 nm. Excitation was performed and the divergence was received at a wavelength of 590 nm for measurement. To IC _{5 0} value indicates that after the end of the test, the test substances administered to reduce cell number caused by the concentration of 50%, in order to display the value of the inhibitory activity of cancer cells.

For other cancer cells, dichotomin also has inhibitory activity. As shown in Table 8, in addition to the confirmed inhibitory activity against liver cancer cells, dichotomin has a better inhibitory activity against colorectal cancer and lung cancer cells.

It can be seen from the above experimental results that the furost-5-ene-3,22,26-triol glycoside compound proposed by the present invention can promote apoptosis, so that all cancer cells can be derived from the compound. Promote apoptotic mechanisms to prevent and treat diseases that occur in mammals or human cancers.

The present invention has been modified by those skilled in the art, and is intended to be modified as described in the appended claims.

Figure 1 is a schematic view showing the process of preparing a glucosinolate (furost-5-ene-3, 22, 26-triol) glycoside compound according to the present invention; Figure 2 is a result of an apoptosis experiment of the sylvestre extract of the present invention, wherein The second (a) is the experimental result of the PK-1-1 extract; the second (b) is the experimental result of the PK-1-2 extract; and the third is the purified Dichotomin (PK-22-1) injection of the present invention. After the tumor cells showed apoptosis, Figure 3 (a) is the control group injected with phosphate buffer (PBS); Figure 3 (b) is the injection purified Dichotomin (PK-22-1); Figure 4 is the invention Schematic diagram of the nuclear DNA cleavage effect of Dichotomin (PK-22-1) on GP7TB and HepG2; Figure 5 is a schematic diagram of the experimental results of the in vitro test of F344 rat by Dichotomin (PK-22-1) and PK22-3 of the present invention; preparative liquid chromatography and isolating the compound of the present invention, FIG six chromatographic 5-7 of FIG.

Claims (5)

A pharmaceutical composition for preventing or treating cancer, comprising: An effective amount of a furano-5-ene-3,22,26-triol glycoside of formula I, wherein the R ₁ is R is a monohydrogen group; the steric configuration of the 25th position carbon atom is R type; R ₂ is a monohydro group; R ₃ is a glucose; and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to claim 1, which has an effect of promoting apoptosis. The pharmaceutical composition according to claim 1, wherein the cancer comprises at least one of liver cancer, lung cancer, and colorectal cancer. The pharmaceutical composition according to claim 1, wherein the sulphate glycoside system of the formula I is derived from one of the water extracts or a part of the purified material of the genus. The pharmaceutical composition according to claim 1, wherein the sulphate glycoside system of the formula I is derived from one of the water extracts or a part of the purified extract of the Asparagus plant.