20(R)-GINSENOSIDE RG3 POLYACYLATED DERIVATIVES, PREPARATION AND APPLICATION THEREOF

Abstract

The present invention provides 20(R)-ginsenoside Rg3 polyacylated derivatives of the formula (I) and preparation method and anti-tumor application thereof:

##STR00001##

wherein, R═CH.sub.3(CH.sub.2)nCO, n=0˜5.

Claims

1. A 20(R)-ginsenoside Rg3 polyacylated derivative of the formula (I), ##STR00008## wherein, R═CH.sub.3(CH.sub.2)nCO, n=0˜5.

2. A preparation method of the 20(R)-ginsenoside Rg3 polyacylated derivative of the formula (I) of claim 1, characterized by comprising the following steps of: 1) dissolving 20(R)-ginsenoside Rg3 in an organic solvent to make a solution of 20(R)-ginsenoside Rg3, 2) adding an acylation agent for esterification, 3) adding water to quench the reaction and adjusting the pH of the mixed solution to 7 with an alkali, and 4) filtering and re-crystallizing to obtain the final product.

3. The method of claim 2, characterized in that the organic solvent of step 1) is selected from the group consisting of triethylamine or anhydrous pyridine, preferably anhydrous pyridine.

4. The method of claim 2, characterized in that the acylation agent of step 2) is selected from the group consisting of acyl chloride or acid anhydride.

5. The method of claim 4, characterized in that the acyl chloride is selected from the group consisting of acetyl chloride, propionyl chloride, butyryl chloride, valeryl chloride or hexanoyl chloride; and the acid anhydride is selected from the group consisting of acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride or caproic anhydride.

6. The method of claim 2, characterized in that the reaction temperature of esterification is 80-100° C.

7. The method of claim 2, characterized in that the alkali in step 3) is inorganic alkalis.

8. The method of claim 7, characterized in that the inorganic alkali is selected from the group consisting of sodium carbonate, potassium carbonate, sodium bicarbonate or potassium hydrogen carbonate.

9. An anti-cancer drug comprising the 20(R)-ginsenoside Rg3 polyacylated derivative of claim 1.

10. A method of cancer treatment, the method comprising administering to a cancer patient the 20(R)-ginsenoside Rg3 polyacylated derivative of claim 1.

11. The anti-cancer drug of claim 9, wherein the cancer is a solid tumor.

12. The method of claim 10, wherein the cancer patient is treated for a solid tumor.

13. The method of claim 10, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, stomach cancer, intestinal cancer and liver cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be further descripted by the following embodiments, and the embodiments are to be considered in all respects illustrative rather than limiting of the disclosure described herein. Furthermore, the reagents and materials used in the embodiments are all commercially available, and if insufficient, please refer to Guidebook to Organic Synthesis, guidelines issued by drug regulators and manufacturer's instructions on relevant instruments and reagents.

EXAMPLE 1

[0034] 1) 20(R)-Ginsenoside Rg3 (4 g, 5.09 mmol) was added to 180 ml dry anhydrous pyridine and stirred for dissolving, to make a solution of 20(R)-ginsenoside Rg3.

[0035] 2) At room temperature (15-35° C.), acetyl chloride (0.71 ml, about 10 mmol) was added to the 20 (R)-ginsenoside Rg3 solution.

[0036] 3) The resulting mixture was heated under stirring and maintained at 80° C. for esterification.

[0037] 4) After the reaction was maintained at a temperature of 80° C. for 5 hours, the reaction solution was poured into ice water, and a saturated sodium bicarbonate aqueous solution was added to adjust pH to 7, resulting in the precipitation of a large amount of white solid.

[0038] 5) Filtration gave a white solid, washed with a large amount of water, and recrystallized from water/methanol to give a white powder.

[0039] 20 (R)-ginsenoside Rg3 octaacetate (3 g) as a white solid, soluble in water, ethanol. After the TLC plate was developed (the chromatographic solution was chloroform/methanol 50:1, Rf was 0.4) and sprayed with 10% H.sub.2SO.sub.4-ethanol reagent, then appeared purple. In ESI-MS spectrum, m/z [M+Na] is 1143.6, and molecular weight is 1121.

[0040] .sup.1H-NMR and .sup.13C-NMR of 20(R)-ginsenoside Rg3 octoacetate are as follows:

[0041] .sup.1H-NMR (400 MHz, DMSO-d6) δ (ppm): 5.301-5.255 (t, 1H, J=9.2 Hz), 5.192-5.146 (t, 1H, J=9.2 Hz), 5 .062 (s, 1H), 4.891-4.721 (m, 4H), 4.629 (s, 2H), 4.182-4.067 (m, 3H), 3.973-3.911 (m, 3H), 3.630-3.592 (t, 1H, J=1.2 Hz), 3.512 (s, 1H), 3.054-3.053 (d, 1H, J=7.6 Hz), 2.088 (s, 2H), 1.984-1.902 (m, 22H), 1.738 (s, 3H), 1.626-1.210 (m, 18H), 0.992-0.889 (m, 12H), 0.794 (s, 3H), 0.721 (s, 5H);

[0042] .sup.13C-NMR (100 MHz, Pyridin-d5) δ (ppm): 171.88, 171.81, 171.72, 171.61, 171.51, 171.31, 171.19, 171.15 (C═O), 132.05 (C-25), 127.30 (C-24), 104.92 (C-1″), 102.35 (C-1′), 91.57 (C-3), 79.27 (C-2′), 76.78 (C-5″), 74.96 (C-5′), 74.71 (C-2″), 74.71 (C-20), 73.56 (C-3″), 73.25 (C-3′), 72.98 (C-12), 70.79 (C-4″), 70.39 (C-4′), 63.95 (C-6″&6′), 57.54 (C-5), 53.76 (C-14), 51.56 (C-17), 51.47 (C-9), 47.40 (C-13), 44.35 (C-22), 41.17 (C-4), 40.85 (C-8), 40.04 (C-1), 38.18 (C-10), 36.06 (C-7), 32.75 (C-11), 30.09 (C-15), 27.22 (C-28), 25.64 (C-16), 24.96 (C-2), 24.83 (C-26), 22.39 (C-21), 21.93 (C-23), 21.71-21.79 (CH.sub.3CO) 18.29 (C-6), 17.65 (C-30), 17.26 (C-27), 16.17 (C-29), 16.13 (C-19), 16.90 (C-18)

[0043] Based on the test data of ESI-MS, .sup.1H-NMR and .sup.13C-NMR, it can be determined that the structural formula of 20(R)-ginsenoside Rg3 octoacetate is as follows:

##STR00004##

EXAMPLE 2

[0044] 1) 20(R)-Ginsenoside Rg3 (4 g, 5.09 mmol) was added to 180 ml dry anhydrous pyridine and stirred for dissolving, to make a solution of 20(R)-ginsenoside Rg3.

[0045] 2) At room temperature (20-25° C.), N-butyric anhydride (3.07 ml, about 10 mmol) was added to the 20(R)-ginsenoside Rg3 solution condition.

[0046] 3) The resulting mixture was heated under stirring and maintained at 100° C. for esterification.

[0047] 4) After the reaction was maintained at a temperature of 100° C. for 5 h, the reactant solution was poured into ice water, and a saturated sodium bicarbonate aqueous solution was added to adjust pH to 7, resulting in the precipitation of a large amount of white solid.

[0048] 5) Filtration gave a white solid, washed with a large amount of water, and recrystallized from water/methanol to give a white powder.

[0049] 20(R)-Ginsenoside Rg3 octo-n-butyrate (3 g) is a while solid soluble in water and ethanol. After the TLC plate was developed (the chromatographic solution was petroleum ether/ethyl acetate 3:1, Rf was 0.5) and sprayed with 10% H.sub.2SO.sub.4-ethanol reagent, then appeared purple. In ESI-MS spectrum, m/z [M+Na] is 1368.27, and molecular weight is 1345.

[0050] .sup.1H-NMR and .sup.13C-NMR of 20(R)-Ginsenoside Rg3 octo-n-butyrate are as follows:

[0051] .sup.1H-NMR (400 MHz, DMSO-d6) δ (ppm): 5.301-5.255 (t, 1H, J=9.2 Hz), 5.192 - 5.146 (t, 1H, J=9.2 Hz), 5.062 (s, 1H), 4.891-4.721 (m, 4H), 4.629 (s, 2H), 4.182-4.067 (m, 3H), 3.973-3.911 (m, 3H), 3.630-3.592 (t, 1H, J=1.2 Hz), 3.512 (s, 1H), 3.054-3.053 (d, 1H, J=7.6 Hz), 2.35 (m, 12H), 2.32 (s, 2H), 1.984-1.902 (m, 22H), 1.801-1.793 (m, 16H), 1.738 (s, 3H), 0.992-0.889 (m, 16H), 0.794 (s, 3H), 0.721 (s, 5H).

[0052] .sup.13C-NMR (100 MHz, Pyridin-d5) δ (ppm): 172.96, 172.89, 172.62, 172.51, 172.47, 172.28, 172.26, 172.06 (C═O), 130.55 (C-25), 125.81 (C-24), 103.44 (C-1″), 100.82 (C-1′), 90.01 (C-3), 77.41 (C-2′), 77.11 (C-5″), 76.68 (C-5′), 75.47 (C-2″), 72.04 (C-20), 74.43 (C-3″), 73.91 (C-3′), 71.64 (C-12), 69.14 (C-4″), 68.74 (C-4′), 63.30 (C-6′), 63.35 (C-6″), 56.34 (C-5), 51.39 (C-14), 50.16 (C-17), 49.99 (C-9), 48.73 (C-13), 42.59 (C-22), 40.25 (C-4), 40.04 (C-8), 39.25 (C-1), 36.85 (C-10), 35.59-35.90 (CH.sub.3CH.sub.2 CH.sub.2CO), 35.01 (C-7), 31.59 (C-11), 31.16 (C-15), 27.22 (C-28), 25.64 (C-16), 24.96 (C-2), 24.83 (C-26), 22.39 (C-21), 21.93 (C-23), 18.42 (C-6), 18.17-18.34 (CH3CH.sub.2CH.sub.2CO), 17.65 (C-30), 17.26 (C-27), 16.12 (C-29), 16.02 (C-19), 15.40 (C-18),13.64 (CH.sub.3CH.sub.2CH.sub.2CO).

[0053] Based on the test data of ESI-MS, .sup.1H-NMR and .sup.13C-NMR, it can be determined that the structural formula of 20(R)-ginsenoside Rg3 octo-n-butyrate is as follows:

##STR00005##

EXAMPLE 3

[0054] 1) 20(R)-Ginsenoside Rg3 (4 g, 5.09 mmol) was added to 180 ml dry anhydrous pyridine and stirred for dissolving, to make a solution of 20(R)-ginsenoside Rg3.

[0055] 2) At room temperature (15-25° C.), N-propionic anhydride (3.07 ml, about 10 mmol) was added to the 20(R)-ginsenoside Rg3 solution.

[0056] 3) The resulting mixture was heated under stirring and maintained at 80° C. for esterification.

[0057] 4) After the reaction was maintained at a temperature of 80° C. for 5 h, the reactant solution was poured into ice water, and a saturated sodium bicarbonate aqueous solution was added to adjust pH to 7, resulting in the precipitation of a large amount of white solid.

[0058] 5) Filtration gave a white solid, washed with a large amount of water, and recrystallized from water/methanol to give a white powder.

[0059] 20(R)-Ginsenoside Rg3 octo-n-propionate (3 g) is a while solid soluble in water and ethanol.

[0060] After the TLC plate was developed (the chromatographic solution was petroleum ether/ethyl acetate 3:1, Rf was 0.4) and sprayed with 10% H.sub.2SO.sub.4-ethanol reagent, then appeared purple. In ESI-MS spectrum, m/z [M+Na] is 1255.71, and molecular weight is 1233.

[0061] .sup.1H-NMR and .sup.13C-NMR of 20(R)-ginsenoside Rg3 octo-propionate are as follows:

[0062] .sup.1H-NMR (400 MHz, DMSO-d6) δ (ppm): 5.301-5.255 (t, 1H, J=9.2 Hz), 5.192-5.146 (t, 1H, J=9.2 Hz), 5.062 (s, 1H), 4.891-4.721 (m, 4H), 4.629 (s, 2H), 4.182-4.067 (m, 3H), 3.973-3.911 (m, 3H), 3.630-3.592 (t, 1H, J=1.2 Hz), 3.512 (s, 1H), 3.054-3.053 (d, 1H, J=7.6 Hz), 2.29-2.27 (m, 16H), 2.088 (s, 2H), 1.14-1.12 (m, 24H), 1.738 (s, 3H),), 0.992-0.889 (m, 16H), 0.794 (s, 3H), 0.721 (s, 5H);

[0063] .sup.13C-NMR(100 MHz, Pyridin-d5) δ (ppm): 175.27, 175.20, 175.04, 174.97, 174.87, 174.70, 174.68, 174.52 (C═O), 132.06 (C-25), 127.31 (C-24), 104.96 (C-1″), 102.38 (C-1′), 91.53 (C-3), 79.14 (C-2′), 77.11 (C-5″), 76.68 (C-5′), 75.47 (C-2″), 72.46 (C-20), 74.43 (C-3″), 73.91 (C-3′), 70.18 (C-12), 70.86 (C-4″), 70.36 (C-4′), 63.81 (C-6″&6′), 57.57 (C-5), 53.86 (C-14), 51.53 (C-17), 51.49 (C-9), 7.40 (C-13), 44.29 (C-22), 41.20 (C-4), 40.87 (C-8), 40.07 (C-1), 38.20 (C-10), 36.09 (C-7), 32.87 (C-11), 30.13 (C-15), 29.54-29.98 (CH3CH2CO), 29.10 (CH3CH2CO), 28.82-28.98 (CH3CH2CO), 27.08 (C-28), 24.24 (C-16), 24.96 (C-2), 24.83 (C-26), 22.39 (C-21), 21.93 (C-23), 19.65 (C-6), 18.97 (C-30), 18.96 (C-27), 17.53 (C-29), 17.50 (C-19), 16.90 (C-18), 10.45-10.58 (CH3CH2CO).

[0064] Based on the test data of ESI-MS, .sup.1H-NMR and .sup.13C-NMR, it can be determined that the structural formula of 20(R)-ginsenoside Rg3 octo-n-propionate is as follows:

##STR00006##

EXAMPLE 4

[0065] 1) 20(R)-Ginsenoside Rg3 (4 g, 5.09 mmol) was added to 180 ml dry anhydrous pyridine and stirred for dissolving, to make a solution of 20(R)-ginsenoside Rg3.

[0066] 2) At room temperature, hexanoyl chloride (1.387 ml, about 10 mmol) was added to the 20(R)-ginsenoside Rg3 solution.

[0067] 3) The resulting mixture was heated under stirring and maintained at 80° C. for esterification.

[0068] 4) After the reaction was maintained at a temperature of 80° C. for 5 hours, the reactant solution was poured into ice water, and a saturated sodium bicarbonate aqueous solution was added to adjust pH to 7, resulting in the precipitation of a large amount of white solid.

[0069] 5) Filtration gave a white solid, washed with a large amount of water, and recrystallized from water/methanol to give a white powder.

[0070] 20(R)-Ginsenoside Rg3 octo-n-hexanoate (3 g) is a while solid soluble in water and ethanol. After the TLC plate was developed (the chromatographic solution was chloroform/methanol 5:1, Rf was 0.4) and sprayed with 10% H.sub.2SO.sub.4-ethanol reagent, then appeared purple. In ESI-MS spectrum, m/z [M+Na] is 1602.2, and molecular weight is 1570.

[0071] .sup.1H-NMR and .sup.13C-NMR of 20(R)-ginsenoside Rg3 octo-hexanoate are as follows:

[0072] .sup.1H-NMR (400 MHz, DMSO-d6) δ (ppm): 5.301-5.255 (t, 1H, J=9.2 Hz), 5.192-5.146, (t, 1H, J=9.2 Hz), 5.062 (s, 1H), 4.891-4.721 (m, 4H), 4.629 (s, 2H), 4.182-4.067 (m, 3H), 3.973-3.911 (m, 3H), 3.630-3.592 (t, 1H, J=1.2 Hz), 3.512 (s, 1H), 3.054-3.053 (d, 1H, J=7.6 Hz), 2.29-2.27 (m, 16H), 2.088 (s, 2H), 1.14-1.12 (m, 24H), 1.738 (s, 3H),), 0.992-0.889 (m, 16H), 0.794 (s, 3H), 0.721 (s, 5H)

[0073] .sup.13C-NMR (100 MHz, Pyridin-d5) δ (ppm): 173.27, 173.20, 173.04, 172.98, 172.82, 172.70, 172.67, 172.52 (C═O), 132.06 (C-25), 127.31 (C-24), 104.96 (C-1″), 102.38 (C-1′), 91.53 (C-3), 79.14 (C-2′), 77.11 (C-5″), 76.68 (C-5′), 75.47 (C-2″), 72.46 (C-20), 74.43 (C-3″), 73.91 (C-3′), 70.18 (C-12), 70.86 (C-4″), 70.36 (C-4′), 63.81 (C-6″&6′), 57.57 (C-5), 53.86 (C-14), 51.53 (C-17), 51.49 (C-9), 7.40 (C-13), 44.29 (C-22), 41.20 (C-4), 40.87 (C-8), 40.07 (C-1), 38.20 (C-10), 36.09 (C-7), 33.14-34.28 (CH3CH2CH2CH2CH2CO), 32.87 (C-11), 28.82-28.98 (CH3CH2CH2CH2 CH2CO), 30.13 (C-15), 27.08 (C-28), 24.24 (C-16), 24.96 (C-2), 24.83 (C-26), 22.39 (C-21), 22.48-22.10 (CH3CH2CH2CH2CH2CO), 21.93 (C-23), 19.65 (C-6), 18.97 (C-30), 18.96 (C-27), 17.53 (C-29), 17.50 (C-19), 16.90 (C-18), 14.41-14.67 (CH3CH2CH2CH2CH2CO).

[0074] Based on the test data of ESI-MS, .sup.1H-NMR and .sup.13C-NMR, it can be determined that the structural formula of 20(R)-Ginsenoside Rg3 octo-n-hexanoate is as follows:

##STR00007##

[0075] Experiment example Effect of 20(R)-ginsenoside Rg3 polyacylated derivatives on inhibiting tumor growth

[0076] 1. Tested Drug

[0077] 20(R)-ginsenoside Rg3 polyacylated derivatives:

[0078] 20(R)-ginsenoside Rg3 octo-acetate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-n-propionate, provided by Dalian Fusheng natural drug development Co. Ltd., were detected by area normalization method using HPLC UV-detector and evaporative light scattering detector, indicating that the purity were 99.6%, 99.2% and 99.1% respectively.

[0079] Preparation Method

[0080] Accurately measure a quantity of 20 (R)-ginsenoside Rg3 polyacylated derivatives, add 5% CMC-Na to make the desired concentration of the suspension. Administration volume is 0.5 ml/mouse.

[0081] 2. Experiment Materials

[0082] Positive drugs:

[0083] cyclophosphamide (CTX) for injection, provided by Shanghai Hualian Pharmaceuticals Group, administered once daily, for seven consecutive days;

[0084] 5 Fu injection solution, provided by Shanghai Xudonghaipu Pharmaceuticals Co., ltd.;

[0085] mitomycin (MMC) for injection, provided by Kyowa Hakko Kogyo Co. Ltd.

[0086] Tumor sources:

[0087] human breast cancer Bcap-37 model, human lung cancer A549 model, human intestinal cancer LOVO model, human stomach cancer MGC model and human liver cancer QGY model, with tumor cell lines being of the second generation or more, mouse B16 melanoma cell line, mouse Lewis lung cancer model, mouse sarcoma s180 model, all passaged and preserved in Pharmacological Division of Shanghai Institute of Pharmaceutical Industry.

[0088] 3. Experiment Animals

[0089] Origins:

[0090] nude mice, 6-week old, 18-22 g/mouse, provided by Laboratory Animal Center of Chinese Academy of Sciences, Shanghai Branch, with certification No. 2001A032.

[0091] C57BL/6 mice and Kunming mice, 18-22 g/mouse, provided by Laboratory Animal Center of Chinese Academy of Sciences, Shanghai Branch, with certification SYXK (Shanghai) 2004-0015.

[0092] Gender: male or female, the same gender for each batch in experiment

[0093] Animal numbers: 10 mice and 10 nude mice for each of positive control group and test group in experiment, and 20 for negative control group.

[0094] 4. Experiment Design

[0095] Dose settings:

[0096] doses for 20(R)-ginsenoside Rg3 octo-acetate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-n-propionate were set as high-dose group (0.3 mg/kg/d), medium-dose group (0.06 mg/kg/d), and low-dose group (0.012 mg/kg/d).

[0097] Dosage regimen: oral administration, twice daily.

[0098] human tumor model and cell-inoculated mouse model: po×14 bid;

[0099] mouse tumor-inoculated model: po×10 bid.

[0100] Control trial:

[0101] negative control group: given with the same volume of 5% CMC-Na as the test group, and the same dosage regimen as the test group.

[0102] positive control group: Cyclophosphamide CTX 30 mg/kg, MMC 2 mg/kg and 5Fu 30 mg/kg by intraperitoneal or intravenous administration, once daily, for seven consecutive days.

[0103] 5. Experiment Method and Key Steps

[0104] 5.1 Anti-Tumor Test

[0105] 5.1.1 Subcutaneous Inoculation Model

[0106] Tumor cells of vigorous growth stage were taken under sterile condition and made into cell suspensions of about 1-2×10.sup.7/ml by homogenization, the suspensions were inoculated subcutaneously at 0.2 ml/mouse in host mice and repeated on the next day according to the experiment design, the animals in each group were slaughtered after about three weeks, the tumors were collected and weighed, and the tumor inhibition rates were calculated according to the following equation:

tumor inhibition rate %=[(average tumor weight of control group-average tumor weight of treatment group)/average tumor weight of control group]×100%

[0107] The same operation was repeated in human tumor xenograft model, except that the feed, packing, cage and contact instruments were all sterilized under high pressure before use and the nude mice were fed in a laminar flow rack. The tumors and tumor-bearing animal weight gains were dynamically measured (tumor size measured using caliper: long diameter/a, short diameter/b, and tumor bulk=a×b.sup.2/2).

[0108] 5.1.2 Stomach In-Situ Inoculation Model

[0109] Two passages in vivo of MGC stomach cancer of vigorous growth stage were taken under sterile condition and made into cell suspensions of about 2×10.sup.7/ml by homogenization, the cell suspensions (0.05 ml) were surgically injected in greater curvature of stomach in nude mice and repeated on the next day according to the experiment design, and the tumor-bearing host life-extension rates were calculated according to the following equation:

life-extension rate %=average survival days of treatment group/average survival days of control group×100%

[0110] 5.2 Anti-Tumor Metastasis Test

[0111] 5.2.1 Tail Vein Inoculation Model

[0112] B16 mouse melanoma cells of logarithmic growth stage were taken under sterile condition and made into cell suspensions of about 2.5×10.sup.5/ml, the cell suspensions were injected 0.2 ml/mouse by tail vein in C57BL/6 mice and repeated on the next day according to the experiment design, the mice were slaughtered after three weeks, the lungs of mice in each group were collected, the colony of metastatic cancer cells in lung per mouse and average colony of tumor cells in each group were calculated, and the tumor inhibition rates were calculated according to the following equation:

tumor inhibition rate %=[(average colony of control group-average colony of treatment group)/average colony of control group]×100%

[0113] 5.2.2 Spleen Inoculation-Liver Metastasis Model

[0114] LOVO intestinal cancer cells (cultured in vitro) of logarithmic growth stage were taken under sterile condition and prepared into cell suspensions of about 1.8×10.sup.7/ml using RPMI1640 culture medium as diluent. The nude mice were subjected to general anesthesia and cut in belly after routine disinfection to belly skin, the spleens were taken out and inoculated with LOVO intestinal cancer cell suspensions (0.05 ml/mouse) and then restored, the incisions were sutured, and the mice were placed in the cages and then put in the laminar flow rack. The mice were treated on the next day according to the experiment design, observed and recorded for mortality rate of mice in each group, and the life-extension rates were calculated and compared to the negative control group.

[0115] 5.2.3 Liver In-Situ Inoculation Model

[0116] Under sterile condition, the in vivo passage of the second generation of QGY tumor source of vigorous growth stage was prepared into cell suspensions of about 1-2×10.sup.7/ml by homogenization (1:6), and the homogenates were filtered using 100-mesh stainless steel screen, getting ready for use. The nude mice were disinfected routinely and anaesthetized and cut at the xiphoid in the middle of the abdominal cavity to expose the liver, the cell suspensions (0.05 ml) were injected into liver parenchyma using imported 28ga ½ ml syringes, the abdominal cavity was closed and then sutured layer by layer. The nude mice were fed in the laminar flow rack, with all the feed, packing, cage and contact instruments being sterilized under high pressure before use The mice were treated on the next day according to the experiment design, observed and recorded for survival time within 45 days in each group, and the survival time was compared to the negative control group, and the life-prolonging rates were calculated.

[0117] 6. Results

[0118] 6.1 Results of Anti-Tumor Test of Rg3 Polyacylated Derivatives in Tumor Models

[0119] The results of anti-tumor test of 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate in mouse and human tumor models were shown in Table 1 and Table 2.

TABLE-US-00001 TABLE 1 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on mouse Lewis lung cancer (toe subcutaneous inoculation) (n = 3) Animal Tumor numbers Animal body Tumor-bearing inhibition Dose Administration (mice) weight (g) toe weight (g) rate Sample mg/kg/d Regimen Start/end Start/end X ± SD % Rg3-octo-n-propionate 0.3 po × 14bid 10/10 20.1/24.5  0.191 ± 0.105*** 78.50 high-dose group   Rg3-octo-n-propionate 0.06 po × 14bid 10/10 20.6/25.1  0.548 ± 0.09*** 61.58 medium-dose group   Rg3-octo-n-propionate 0.012 po × 14bid 10/10 20.6/24.2  0.508 ± 0.07*** 50.12 low-dose group   Rg3-octo-n-butyrate 0.3 po × 14bid 10/10 20.6/23.9  0.208 ± 0.05*** 76.66 high-dose group   Rg3-octo-n-butyrate 0.06 po × 14bid 10/10 20.9/25.0  0.347 ± 0.06*** 61.00 medium-dose group   Rg3-octo-n-butyrate 0.012 po × 14bid 10/10 20.1/25.2  0.440 ± 0.05*** 50.55 low-dose group   Rg3-octo-acetate 0.3 po × 14bid 10/10 20.5/23.6  0.633 ± 0.08*** 71.09 high-dose group   Rg3-octo-acetate 0.06 po × 14bid 10/10 20.5/24.4  0.366 ± 0.09*** 58.89 medium-dose group   Rg3-octo-acetate 0.012 po × 14bid 10/10 20.1/24.5  0.444 ± 0.09*** 50.15 low-dose group CTX GROUP 100 ip × 14qod 10/10 20.2/21.9 0.0541 ± 0.02*** 93.92 negative control group 0.5% CMC- po × 14bid 20/20 20.3/28.8  0.89 ± 0.11 Na Compared to the negative control group, ***p value <0.01

TABLE-US-00002 TABLE 2 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on human breast cancer Bcap-37 cells (subcutaneous inoculation) (n = 3) Animal Tumor numbers Animal body Tumor inhibition Dose Administration (mice) weight weight (g) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD % Rg3-octo-n-propionate 0.3 po × 14bid 10/10 20.9/24.2 0.360 ± 0.10*** 72.09 high-dose group Rg3-octo-n-propionate 0.06 po × 14bid 10/10 20.3/25.1 0.502 ± 0.07*** 61.11 medium-dose group Rg3-octo-n-propionate 0.012 po × 14bid 10/10 20.7/24.4 0.612 ± 0.09*** 52.58 low-dose group Rg3-octo-n-butyrate 0.3 po × 14bid 10/10 20.1/24.2 0.380 ± 0.06*** 70.52 high-dose group Rg3-octo-n-butyrate 0.06 po × 14bid 10/10 20.8/25.1 0.610 ± 0.08*** 60.95 medium-dose group Rg3-octo-n-butyrate 0.012 po × 14bid 10/10 20.1/24.0 0.627 ± 0.07*** 51.41 low-dose group Rg3-octo-acetate 0.3 po × 14bid 10/10 20.3/23.6 0.400 ± 0.09*** 68.98 high-dose group Rg3-octo-acetate 0.06 po × 14bid 10/10 20.2/24.5 0.520 ± 0.08*** 59.69 medium-dose group Rg3-octo-acetate 0.012 po × 14bid 10/10 20.0/25.7 0.672 ± 0.07*** 47.93 low-dose group CTX GROUP 100 ip × 2qod 10/10 20.4/22.0 0.130 ± 0.06*** 89.90 negative control group 0.5% po × 14bid 20/20 20.2/25.5  1.29 ± 0.09*** CMC-Na Compared to the negative control group, ***p <0.01

[0120] It can be seen from Table 1 and Table 2 that 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate have inhibitory effect on tumor growth in mouse and human solid tumor models, and the optimal tumor inhibition rate of the treatment group exceed 70%, with a significant difference as compared to the negative control group (p<0.01).

[0121] 6.2 Results of Anti-Tumor Metastasis Test of Rg3 Polyacylated Derivatives (Namely Rg3 Multi-Acylated Derivatives) in Tumor Metastasis Models

[0122] The results of anti-tumor metastasis test of 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate in mouse melanoma B16, S180 and Lewis lung cancer metastasis models and human lung cancer metastasis model were shown in Table 3-6.

TABLE-US-00003 TABLE 3 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on mouse melanoma B16 cells (n = 3) Animal Anti- numbers Animal body Lung clone metastasis Dose Administration (mice) weight numbers (mice) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD % Rg3-octo-n-propionate 0.3 po × 14bid 10/10 20.1/24.8  7.21 ± 7.3*** 85.01 high-dose group Rg3-octo-n-propionate 0.06 po × 14bid 10/10 19.3/24.3 14.82 ± 7.6*** 69.19 medium-dose group Rg3-octo-n-propionate 0.012 po × 14bid 10/10 19.6/25.7 21.44 ± 7.1*** 55.42 low-dose group Rg3-octo-n-butyrate 0.3 po × 14bid 10/10 20.1/21.1  8.07 ± 7.0*** 83.22 high-dose group Rg3-octo-n-butyrate 0.06 po × 14bid 10/10 19.3/25.3 16.29 ± 7.8*** 66.13 medium-dose group Rg3-octo-n-butyrate 0.012 po × 14bid 10/10 19.1/25.2 23.15 ± 7.9*** 51.88 low-dose group Rg3-octo-acetate 0.3 po × 14bid 10/10 20.0/24.0  9.63 ± 7.9*** 79.98 high-dose group Rg3-octo-acetate 0.06 po × 14bid 10/10 19.4/24.7 17.38 ± 7.1*** 63.87 medium-dose group Rg3-octo-acetate 0.012 po × 14bid 10/10 19.8/25.2 23.62 ± 7.0*** 50.90 low-dose group CTX GROUP 100 ip × 2qod 10/10 20.1/21.4  2.26 ± 2.8*** 95.30 negative control group 0.5% po × 14bid 20/20 19.2/25.5 48.10 ± 11.2 CMC-Na Compared to the negative control group, ***p value <0.01

TABLE-US-00004 TABLE 4 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on mouse sarcoma S180 cells (n = 3) Animal Anti- numbers Animal body Lung clone metastasis Dose Administration (mice) weight (g) numbers (mice) rate Sample mg/kg/d Regimen Start/end Start/end X ± SD % Rg3-octo-n- 0.3 po × 14bid 10/10  20.8/24.1 0.47 ± 7.4*** 84.00 propionate 0.06 po × 14bid 10/10  19.8/24.3 0.88 ± 7.4*** 70.12 high-dose group 0.012 po × 14bid 10/10  19.2/25.1 1.33 ± 7.2*** 54.86 Rg3-octo-n- 0.3 po × 14bid 10/10  20.1/21.2 0.55 ± 7.3*** 81.38 butyrate group 0.06 po × 14bid 10/10  19.1/25.5 0.96 ± 7.8*** 67.51 0.012 po × 14bid 10/10 19.91/25.4 1.40 ± 7.7*** 52.55 Rg3-octo-acetate 0.3 po × 14bid 10/10  20.9/24.1 0.62 ± 7.3*** 78.94 group 0.06 po × 14bid 10/10  19.2/24.7 1.01 ± 7.2*** 65.80 0.012 po × 14bid 10/10  19.6/25.3 1.43 ± 7.1*** 51.50 CTX GROUP 100 ip × 2qod 10/10  20.2/21.0 0.18 ± 2.1*** 93.84 negative control 0.5% po × 14bid 20/20  19.1/25.0 2.95 ± 10.5 group CMC-Na Compared to the negative control group, ***p value <0.01

TABLE-US-00005 TABLE 5 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on mouse Lewis lung cancer (subcutaneous inoculation) (n = 3) Animal Anti- numbers Animal body Lung clone metastasis Dose Administration (mice) weight numbers (mice) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD % Rg3- 0.3 po × 14bid 10/10 20.1/24.3 0.77 ± 0.11*** 72.15 octo-n-propionate 0.06 po × 14bid 10/10 19.2/24.4 1.09 ± 0.15*** 60.97 high-dose 0.012 po × 14bid 10/10 19.6/25.1 1.35 ± 0.12*** 51.55 group Rg3-octo-n-butyrate 0.3 po × 14bid 10/10 20.0/21.3 0.78 ± 0.11*** 71.85 group 0.06 po × 14bid 10/10 19.1/25.4 1.08 ± 0.11*** 61.23 0.012 po × 14bid 10/10 19.5/25.1 1.37 ± 0.17*** 50.87 Rg3-octo-acetate 0.3 po × 14bid 10/10 19.3/24.3 0.84 ± 0.15*** 69.96 group 0.06 po × 14bid 10/10 19.2/24.7 1.14 ± 0.12*** 58.90 0.012 po × 14bid 10/10 19.1/25.0 1.38 ± 0.13*** 50.30 CTX GROUP 100 ip × 2qod 10/10 19.4/21.0 0.50 ± 0.09*** 82.12 negative 0.5% CMC- po × 14bid 20/20 19.3/25.5 2.78 ± 0.26 control group Na Compared to the negative control group, ***p value <0.01

TABLE-US-00006 TABLE 6 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on human lung cancer A549 cells (subcutaneous inoculation) (n = 3) Animal Anti- numbers Animal body Lung clone metastasis Dose Administration (mice) weight numbers (mice) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD % Rg3 0.3 po × 14bid 10/10  20.8/24.9 0.77 ± 0.13*** 70.72 octo-n-propionate 0.06 po × 14bid 10/10  19.8/24.0 1.09 ± 0.10*** 61.09 high-dose 0.012 po × 14bid 10/10  19.2/25.9 1.35 ± 0.13*** 50.58 group Rg3-octo-n- 0.3 po × 14bid 10/10  20.1/21.7 0.78 ± 0.13*** 70.55 butyrate group 0.06 po × 14bid 10/10  19.3/25.3 1.08 ± 0.14*** 60.89 0.012 po × 14bid 10/10 19.91/25.0 1.37 ± 0.11*** 50.01 Rg3-octo-acetate 0.3 po × 14bid 10/10  19.9/24.0 0.84 ± 0.13*** 68.52 group 0.06 po × 14bid 10/10  19.4/24.5 0.81 ± 0.15*** 57.90 0.012 po × 14bid 10/10  19.7/25.2 0.96 ± 0.12*** 49.91 CTX GROUP 100 ip × 2qod 10/10  19.6/21.3 0.29 ± 0.12*** 85.10 negative control 0.5% CMC- po × 14bid 20/20  19.2/25.5 1.92 ± 0.15 group Na Compared to the negative control group, ***p value <0.01

[0123] It can be seen from Table 3-6 that the cancer metastasis inhibition rates of 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate in mouse melanoma B16, S180 and Lewis lung cancer metastasis models and human lung cancer metastasis model reach up to 70-80%.

[0124] 6.3 Results of Life-Extension Rate from Anti-Tumor Test of Rg3 Polyacylated Derivatives in Tumor Metastasis Models

[0125] The results of life-extension rate from anti-tumor test of ginsenoside Rg3 octo-n-propionate, ginsenoside Rg3 octo-n-butyrate and ginsenoside Rg3 octo-acetate in human stomach cancer metastasis, human intestinal cancer metastasis, human liver cancer metastasis models were shown in Table 7-9.

TABLE-US-00007 TABLE 7 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on human stomach cancer MGC cells (in-situ inoculation) (n = 3) Animal Average Life numbers Animal body survival extension Dose Administration (mice) weight (g) (d) rate Sample mg/kg/d Regimen Start/end Start/end X ± SD T/C × % Rg3 0.3 po × 14bid 10/10 20.1/24.5 48.30 ± 0.15*** 212.11 octo-n-propionate 0.06 po × 14bid 10/10 19.6/24.3 43.15 ± 0.15*** 189.52 high-dose group 0.012 po × 14bid 10/10 19.4/25.8 39.23 ± 0.18*** 172.30 Rg3-octo-n- 0.3 po × 14bid 10/10 20.3/21.2 47.86 ± 0.15*** 210.19 butyrate group 0.06 po × 14bid 10/10 19.1/25.4 42.22 ± 0.12*** 185.44 0.012 po × 14bid 10/10 19.1/25.4 39.35 ± 0.15*** 172.83 Rg3-octo-acetate 0.3 po × 14bid 10/10 19.7/24.1 46.04 ± 0.12*** 202.19 group 0.06 po × 14bid 10/10 19.6/24.6 41.21 ± 0.11*** 180.98 0.012 po × 14bid 10/10 19.8/25.4 38.84 ± 0.14*** 170.56 CTX GROUP 100 ip × 2qod 10/10 19.5/21.1 41.22 ± 3.5*** 181.01 negative control 0.5% CMC- po × 14bid 20/20 19.5/25.2 22.77 ± 2.14 group Na Compared to the negative control group, ***p value <0.01

TABLE-US-00008 TABLE 8 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on human intestinal cancer LOVO cells (spleen inoculation) Animal Average Life numbers Animal body survival extension Dose Administration (mice) weight (d) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD T/C × % Rg3- 0.3 po × 14bid 10/10 20.1/24.5 33.21 ± 5.5*** 243.11 octo-n-propionate 0.06 po × 14bid 10/10 19.6/24.3 26.08 ± 4.2*** 190.89 high-dose 0.012 po × 14bid 10/10 19.4/25.8 24.63 ± 3.9*** 180.33 group Rg3-octo-n- 0.3 po × 14bid 10/10 20.3/21.2 32.77 ± 4.1*** 239.88 butyrate group .06 po × 14bid 10/10 19.1/25.4 25.67 ± 3.9*** 187.94 0.012 po × 14bid 10/10 19.1/25.4 24.60 ± 5.8*** 180.12 Rg3-octo-acetate 0.3 po × 14bid 10/10 19.7/24.1 31.50 ± 5.1*** 230.59 group 0.06 po × 14bid 10/10 19.6/24.6 25.80 ± 4.8*** 188.90 0.012 po × 14bid 10/10 19.8/25.4 24.59 ± 4.5*** 179.99 CTX GROUP 100 ip × 2qod 10/10 19.5/21.1 29.36 ± 3.3*** 214.91 negative 0.5% CMC- po × 14bid 20/20 19.5/25.2 13.66 ± 2.31 control group Na Compared to the negative control group, ***p value <0.01

TABLE-US-00009 TABLE 9 Efficacy of 20(R)-ginsenoside Rg3 polyacylated derivatives on human liver cancer QGY cells (in-situ inoculation) Animal Average Life numbers Animal body survival extension Dose Administration (mice) weight (d) rate Sample mg/kg/d Regimen Start/end (g)Start/end X ± SD T/C × % Rg3-octo-n- 0.3 po × 14bid 10/10 20.2/24.2 44.19 ± 5.3*** 189.23 propionate 0.06 po × 14bid 10/10 19.5/24.1 39.72 ± 4.9*** 170.10 high-dose 0.012 po × 14bid 10/10 19.8/25.3 37.49 ± 3.5*** 160.55 group Rg3-octo-n- 0.3 po × 14bid 10/10 20.1/21.8 43.40 ± 4.8*** 185.85 butyrate group 0.06 po × 14bid 10/10 19.3/25.6 39.51 ± 3.9*** 169.22 0.012 po × 14bid 10/10 19.2/25.6 37.36 ± 5.3*** 160.01 Rg3-octo- 0.3 po × 14bid 10/10 19.3/24.8 42.49 ± 5.0*** 181.98 acetate group 0.06 po × 14bid 10/10 19.1/24.3 38.90 ± 4.1*** 166.59 0.012 po × 14bid 10/10 19.1/25.2 37.26 ± 4.5*** 159.58 CTX GROUP 100 ip × 2qod 10/10 19.1/21.0 37.23 ± 3.4*** 159.44 negative 0.5% CMC- po × 14bid 20/20 19.6/25.1 23.35 ± 2.3 control group Na Compared to the negative control group, ***p value <0.01

[0126] It can be seen from Table 7-9 that 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate can significantly increase the survival time in human stomach cancer, human intestinal cancer and human liver cancer models, with obvious life-prolonging effect. Among the groups, the high-dose groups of 20(R)-ginsenoside Rg3 octo-n-propionate, 20(R)-ginsenoside Rg3 octo-n-butyrate and 20(R)-ginsenoside Rg3 octo-acetate showed obvious life-prolonging effect on human stomach, human intestinal cancer and human liver cancer, all being better than the positive control group; and the low-dose groups showed the similar life-prolonging effect as the positive control group.